ࡱ > { P; R; : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : ; ; ; ; ; ; ; ; ; ;
; ; ;
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; !; "; #; $; %; &; '; (; ); *; +; ,; -; .; /; 0; 1; 2; 3; 4; 5; 6; 7; 8; 9; :; ;; <; =; >; ?; @; A; B; C; D; E; !` g bjbj\\ Ru > > R W P P P P ( ( ( d "1 X " " " Ҁ ނ ։ $
R gb Ez l $ h h N 5 i ( l ? " gb l l 5 P P > l P | ( l ! ( Ͱ S " 2 A L H $ , 2 Ͱ ( Ͱ t X ; O 9 D 5 5 $ l l l l ։ ։ $ l d ։ ։ l ( - $ 1 P P P P P P
Copyright
by
Keerthi Gottipati
2008
HIDDEN TEXT: OptionalIf you do not include a copyright page, delete entire page and the following page break.
HIDDEN TEXT: NOTE: this page in hard copy with all original signatures must be submitted with the dissertation to the Graduate School; this is required whether the document is in electronic format or on paper. Whereas, the page included in the electronic document will be unsigned unless it is scanned in.
The Dissertation Committee for Keerthi Gottipati Certifies that this is the approved version of the following dissertation:
CORRELATED LONG-DISTANCE DYNAMICS MODULATE MONOCLONAL ANTIBODY BINDING RESISTANCE IN FLAVIVIRAL ENVELOPE PROTEIN DOMAIN-3: A MOLECULAR DYNAMICS SIMULATIONS STUDY
Committee:
Dr. James C. Lee, Supervisor
Dr. Stanley J. Watowich
Dr. Marion L. Dodson
Dr. Alan D. Barrett
__________________
Dean, Graduate School
HIDDEN TEXT: The top line is for the Supervisors signature. There should be as many lines as there are members on the committee. Lines must be solid, not dotted. To delete signature lines, select the line you want to delete, go to the Table menu, select Table Properties, click on the Table tab, and click on the Borders and Shading button, then remove the bottom border of the table. Use the professor's name without titles or degrees
CORRELATED LONG-DISTANCE DYNAMICS MODULATE MONOCLONAL ANTIBODY BINDING RESISTANCE IN FLAVIVIRAL ENVELOPE PROTEIN DOMAIN-3: A MOLECULAR DYNAMICS SIMULATIONS STUDY
by
Keerthi Gottipati, B.Tech
HIDDEN TEXT: Given first name, and previous academic degrees (B.A. or higher) B.A., B.S., etc. Your official name is the name which appears on your UT transcript.
Dissertation
Presented to the Faculty of the Graduate School of
The University of Texas Medical Branch
in Partial Fulfillment
of the Requirements
for the Degree of
Master in Biochemistry and Molecular Biology
HIDDEN TEXT: The degree sought must be worded in the form given in the Graduate Catalog, such as Doctor of Philosophy, Doctor of Musical Arts, Doctor of Education.
The University of Texas Medical Branch
December, 2008
CORRELATED LONG-DISTANCE DYNAMICS MODULATE MONOCLONAL ANTIBODY BINDING RESISTANCE IN FLAVIVIRAL ENVELOPE PROTEIN DOMAIN-3: A MOLECULAR DYNAMICS SIMULATIONS STUDY
Publication No._____________
Keerthi Gottipati, M.S.
The University of Texas Medical Branch, 2008
Supervisor: Dr. James C. Lee
Numerous monoclonal antibody (MAb) binding resistant mutations have been localized to the envelope protein domain-3 (ED3) of flaviviruses. Previously it was shown that regions constituting antibody binding sites of dengue-2 (DEN2) and West Nile virus (WNV) ED3 were energetically coupled with the interior of the protein. Protein-protein interactions are characterized by perturbation of residue dynamics at the binding interface and at regions physically far from the binding site. Based on above I hypothesized that mutations leading to resistance from antibody binding would perturb dynamics associated with binding at the interface and therefore also the correlation in motions with the interior of the protein. I investigated this hypothesis by analyzing the dynamics of wild type and mutant ED3s of DEN2 and WNV using molecular dynamics (MD) simulations and principal components analysis (PCA). I found that residues constituting binding sites for the MAbs, when mutated, drastically perturb the organized motions in the peptide. Cross-correlation analysis of the dynamics established that the MAb binding resistant mutations perturbed correlated long distance dynamics globally. Computationally this analysis aids in locating the MAb binding regions in flaviviral ED3s through simple analysis wild type and mutant MD trajectories.
Table of Contents
TOC \o "1-5" \h \z \u HYPERLINK \l "_Toc215459989" List of Tables PAGEREF _Toc215459989 \h viii
HYPERLINK \l "_Toc215459990" List of Figures PAGEREF _Toc215459990 \h ix
HYPERLINK \l "_Toc215459991" List of Abbreviations PAGEREF _Toc215459991 \h x
HYPERLINK \l "_Toc215459992" Chapter 1: Introduction PAGEREF _Toc215459992 \h 11
HYPERLINK \l "_Toc215459993" Chapter 2: Methods: PAGEREF _Toc215459993 \h 16
HYPERLINK \l "_Toc215459994" 2.2 Principal Components Analysis: PAGEREF _Toc215459994 \h 17
HYPERLINK \l "_Toc215459995" 2.3 Cross-correlation Analysis PAGEREF _Toc215459995 \h 18
HYPERLINK \l "_Toc215459996" Chapter 3: Molecular dynamics simulations of wild type and mutant Envelope protein Domain-3 of dengue-2 and West Nile virus PAGEREF _Toc215459996 \h 20
HYPERLINK \l "_Toc215459999" 3.1 Principal Components Analysis (PCA): PAGEREF _Toc215459999 \h 22
HYPERLINK \l "_Toc215460000" 3.1.1 Dynamic perturbations caused by mutations of DEN2 ED3: PAGEREF _Toc215460000 \h 24
HYPERLINK \l "_Toc215460001" 3.1.2 Dynamic perturbations caused by mutations of West Nile virus ED3: PAGEREF _Toc215460001 \h 29
HYPERLINK \l "_Toc215460002" 3.2 Cross-Correlation Analysis. PAGEREF _Toc215460002 \h 33
HYPERLINK \l "_Toc215460003" Chapter 4: Discussion PAGEREF _Toc215460003 \h 39
HYPERLINK \l "_Toc215460004" 4.1 Differences in the conformational freedom of wild type Dengue-2 and West Nile virus envelope ED3s: PAGEREF _Toc215460004 \h 39
HYPERLINK \l "_Toc215460005" 4.2 Perturbation in dynamics caused by resistant mutations of dengue-2 envelope prottein ED3: PAGEREF _Toc215460005 \h 40
HYPERLINK \l "_Toc215460006" 4.3 Perturbation in dynamics caused by resistant mutations of West Nile Virus envelope ED3: PAGEREF _Toc215460006 \h 41
HYPERLINK \l "_Toc215460007" 4.4 Role of long-distance dynamics in modulating binding resistance in ED3 of flaviviruses: PAGEREF _Toc215460007 \h 42
HYPERLINK \l "_Toc215460008" Appendix A PAGEREF _Toc215460008 \h 44
HYPERLINK \l "_Toc215460009" Supplementary Figures: PAGEREF _Toc215460009 \h 44
HYPERLINK \l "_Toc215460017" Appendix B PAGEREF _Toc215460017 \h 73
HYPERLINK \l "_Toc215460018" C.1 Amber-9 Name List for Energy Minimization: PAGEREF _Toc215460018 \h 73
HYPERLINK \l "_Toc215460019" C.2 Amber-9 Name List for Equilibration: PAGEREF _Toc215460019 \h 73
HYPERLINK \l "_Toc215460020" C.3 Amber-9 Name List for MD Production Run: PAGEREF _Toc215460020 \h 74
HYPERLINK \l "_Toc215460021" REFERENCES PAGEREF _Toc215460021 \h 75
HYPERLINK \l "_Toc215460022" Vita PAGEREF _Toc215460022 \h 80
HIDDEN TEXT: If you choose to place the chapter number (Chapter 1) and the chapter title (Introduction) on different lines, the automatically generated table of contents will reflect that format. After creating a new table of contents, set them on the same line by deleting the page number and paragraph marker at the end of each chapter number line.
List of Tables
TOC \h \z \t "Heading 7,h7" \c HYPERLINK \l "_Toc215459543" Table 1: Cumulative proportion of variance: DEN2 ED3 PAGEREF _Toc215459543 \h 28
HYPERLINK \l "_Toc215459544" Table 2: Cumulative proportion of variance: WNV ED3. PAGEREF _Toc215459544 \h 32
List of Figures
TOC \h \z \t "Heading 8,h8" \c HYPERLINK \l "_Toc215459549" Figure 1: ED3 of WNV and DEN2. PAGEREF _Toc215459549 \h 14
HYPERLINK \l "_Toc215459550" Figure 2: RMSD Vs Time plot PAGEREF _Toc215459550 \h 22
HYPERLINK \l "_Toc215459553" Figure 3: Eigenvalue Profiles PAGEREF _Toc215459553 \h 24
HYPERLINK \l "_Toc215459554" Figure 4: Projection Plots of DEN2 ED3. PAGEREF _Toc215459554 \h 27
HYPERLINK \l "_Toc215459555" Figure 5: Projection Plots of WNV ED3. PAGEREF _Toc215459555 \h 31
HYPERLINK \l "_Toc215459558" Figure 6: Cross-correlation plots PAGEREF _Toc215459558 \h 34
HYPERLINK \l "_Toc215459560" Figure 7: Difference cross-correlation plots: DEN2 ED3 PAGEREF _Toc215459560 \h 37
HYPERLINK \l "_Toc215459563" Figure 8: Difference cross-correlation plots: WNV ED3. PAGEREF _Toc215459563 \h 38
HYPERLINK \l "_Toc215459564" Supplementary Figure 1: DEN2 ED3 mutant projection plots PAGEREF _Toc215459564 \h 44
HYPERLINK \l "_Toc215459565" Supplementary Figure 2: WNV ED3 mutant projection plots PAGEREF _Toc215459565 \h 51
HYPERLINK \l "_Toc215459568" Supplementary Figure 3: Difference cross-correlations plots: DEN2 ED3 PAGEREF _Toc215459568 \h 56
HYPERLINK \l "_Toc215459570" Supplementary Figure 4: Difference cross-correlations plots: WNV ED3 PAGEREF _Toc215459570 \h 66
List of Abbreviations
WNV... West Nile Virus
DEN2 ...Dengue virus type -2
MAbMonoclonal Antibody
ED3... Envelope protein Domain-3
WTWild Type
MD...Molecular Dynamics
CAC-Alpha
RMSD.Root Mean Square Deviation
PC....Principal Component
NMR ....Nuclear Magnetic Resonance spectroscopy
HIDDEN TEXT: Using Major Sections are option, and most dissertations do not use major sections. If your dissertation is not divided into major sections, then you do not need use the style HEADING 1,H1, start chapters using Heading 2,h2.
Chapter 1: Introduction
Rapid globalization, poor surveillance of diseases in tropical third world countries and the lack of a human vaccine have caused a global spread of dengue and West Nile viruses with tens of millions of reported cases every year ADDIN EN.CITE ADDIN EN.CITE.DATA [1-4]. These viruses belong to a family of positive strand RNA viruses (Flaviviridae) ADDIN EN.CITE Chambers19905517Chambers, T.J.Hahn, C.S.Galler, R.Rice, C.M.Flavivirus genome organization, expression, and replication.Annu Rev MicrobiolAnnu Rev Microbiol649-88441990[5], members of which include Japanese encephalitis, tick borne encephalitis and Langat virus. These viruses cause a febrile condition which may advance into fatal secondary stages (dengue Hemorrhagic Fever in case of dengue virus or encephalitis in case of West Nile virus) ADDIN EN.CITE Halstead19882217Halstead, S. B.,Pathogenesis of dengue: challenges to molecular biologyScienceScience476-8123948391988Monath19943317Monath, T.P.Dengue: the risk to developed and developing countries.Proc. Nat. Acad. Sci.Proc. Nat. Acad. Sci.2395-4009171994Murgue20026617Murgue, B.Zeller, H.Deubel, V. The ecology and epidemiology of West Nile virus in Africa, Europe and Asia.Curr Top Microbiol ImmunolCurr Top Microbiol Immunol195-2212672002[2, 3, 6]. The treatment for either disease is symptomatic ADDIN EN.CITE Barrett20017717Barrett, A. D.Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston 77555-0609, USA. abarrett@utmb.eduCurrent status of flavivirus vaccinesAnn N Y Acad SciAnnals of the New York Academy of SciencesAnn N Y Acad SciAnnals of the New York Academy of SciencesAnn N Y Acad SciAnnals of the New York Academy of Sciences262-71951AnimalsEncephalitis, Japanese/prevention & controlEncephalitis, Tick-Borne/prevention & controlFlavivirus/*immunologyFlavivirus Infections/*prevention & controlHumans*Viral VaccinesYellow Fever/prevention & control2001Dec0077-8923 (Print)11797783http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11797783 eng[7]. No human vaccines are currently available to facilitate acquired immunity against these viruses ADDIN EN.CITE Barrett20017717Barrett, A. D.Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston 77555-0609, USA. abarrett@utmb.eduCurrent status of flavivirus vaccinesAnn N Y Acad SciAnnals of the New York Academy of SciencesAnn N Y Acad SciAnnals of the New York Academy of SciencesAnn N Y Acad SciAnnals of the New York Academy of Sciences262-71951AnimalsEncephalitis, Japanese/prevention & controlEncephalitis, Tick-Borne/prevention & controlFlavivirus/*immunologyFlavivirus Infections/*prevention & controlHumans*Viral VaccinesYellow Fever/prevention & control2001Dec0077-8923 (Print)11797783http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11797783 eng[7]. Antibody based prophylaxis and the design of chimeric viral proteins expressing the antigenic sites of the candidate viruses has triggered the identification and characterization of epitopes and peptide binding sites on these viruses ADDIN EN.CITE Chambers20038817Chambers, T.J. Liang, Y.Droll, D.A.Schlesinger, J.J.Davidson, A.D.Wright, P.J.Jiang, X.Yellow Fever Virus/Dengue-2 Virus and Yellow Fever Virus/Dengue-4 Virus Chimeras: Biological Characterization, Immunogenicity, and Protection against Dengue Encephalitis in the Mouse ModelJ. Virol.J. Virol.3655-687762003[8].
The major antigenic protein component in both flaviviruses is constituted by the envelope glycoprotein E, embedded on the surface of the viral membrane ADDIN EN.CITE Modis2003353517Modis, Y.Ogata, S.Clements, D.Harrison, S. C.Howard Hughes Medical Institute, Children's Hospital and Harvard Medical School, 320 Longwood Avenue, Boston, MA 02115, USA.A ligand-binding pocket in the dengue virus envelope glycoproteinProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S A6986-9110012Binding SitesCrystallography, X-RayDengue Virus/genetics/*metabolism/pathogenicity/physiologyDimerizationHumansLigandsMembrane FusionModels, MolecularPeptide Fragments/chemistry/genetics/metabolismProtein Structure, QuaternaryProtein SubunitsRecombinant Proteins/chemistry/genetics/metabolismViral Envelope Proteins/*chemistry/genetics/*metabolismVirus Assembly2003Jun 100027-8424 (Print)12759475http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12759475 engRey1995595917Rey, F. A.Heinz, F. X.Mandl, C.Kunz, C.Harrison, S. C.Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolutionNatureNature291-83756529Amino Acid SequenceAntigens, Viral/chemistryComputer GraphicsCrystallography, X-RayEncephalitis Viruses, Tick-Borne/*chemistry/pathogenicityHydrogen-Ion ConcentrationMolecular Sequence DataProtein ConformationSequence Homology, Amino AcidViral Envelope Proteins/*chemistryVirulence1995May 257753193http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7753193 [9, 10]. The E Glycoprotein comprising of three structurally distinct domains (ED1-3) plays a central role in the interaction of the virus with the host by mediating viral attachment to the cell surface receptors and undergoing a conformational rearrangement in the endosome to facilitate fusion of viral and host cell membranes ADDIN EN.CITE Rey2003363617Rey, F. A.Virologie Moleculaire et Structurale, Unite Mixte de Recherche 2472/1157, Centre National de la Recherche Scientifique et Institut National de la Recherche Agronomique, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. rey@gv.cnrs-gif.frDengue virus envelope glycoprotein structure: new insight into its interactions during viral entryProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S A6899-90110012Carbohydrates/chemistryCrystallography, X-RayDengue Virus/chemistry/*pathogenicity/physiologyDimerizationHumansModels, MolecularProtein Structure, QuaternaryProtein SubunitsViral Envelope Proteins/*chemistry/physiologyVirulence2003Jun 100027-8424 (Print)12782795http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12782795 eng[11]. In vivo, domain-3 of the E protein (ED3) is the predominant virus type-specific antigenic domain ADDIN EN.CITE Roehrig2003404017Roehrig, J. T.Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado 80521, USA.Antigenic structure of flavivirus proteinsAdv Virus ResAdvances in virus research141-7559AnimalsAntigens, Viral/*chemistry/immunologyEpitopes, B-Lymphocyte/chemistryEpitopes, T-Lymphocyte/chemistryFlavivirus/*immunologyHumansMiceModels, MolecularViral Nonstructural Proteins/*chemistry/immunologyViral Structural Proteins/*chemistry/immunology20030065-3527 (Print)14696329http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14696329 engRoehrig2003404017Roehrig, J. T.Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado 80521, USA.Antigenic structure of flavivirus proteinsAdv Virus ResAdvances in virus research141-7559AnimalsAntigens, Viral/*chemistry/immunologyEpitopes, B-Lymphocyte/chemistryEpitopes, T-Lymphocyte/chemistryFlavivirus/*immunologyHumansMiceModels, MolecularViral Nonstructural Proteins/*chemistry/immunologyViral Structural Proteins/*chemistry/immunology20030065-3527 (Print)14696329http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14696329 engCrill2001464617Crill, W. D.Roehrig, J. T.Arbovirus Disease Branch, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado 80522, USA. wfc3@cdc.govMonoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cellsJ VirolJournal of virologyJ Virol7769-737516AnimalsAntibodies, Monoclonal/immunology/pharmacologyAntibodies, Viral/immunology/pharmacologyCercopithecus aethiopsDengue Virus/*physiologyVero CellsViral Envelope Proteins/*immunologyVirus Replication/drug effects/immunology2001Aug0022-538X (Print)11462053http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11462053 eng[12, 13]. Several antibody binding sites have been localized to this region owing to its orientation on the surface of the virus, which exposes a major portion of the domain to the solution ADDIN EN.CITE Beasley20024417Beasley, D. W.Barrett, A. D.WHO Collaborating Center for Tropical Diseases, Sealy Center for Vaccine Development, Department of Pathology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA.Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope proteinJ VirolJournal of virologyJ Virol13097-1007624AnimalsAntibodies, Monoclonal/immunologyCell Membrane/metabolism*Epitope MappingMiceNeutralization TestsRecombinant Proteins/immunologyViral Envelope Proteins/*immunology/physiologyVirulenceWest Nile virus/*immunology/pathogenicity2002Dec0022-538X (Print)12438639http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12438639 engGromowski2007121217Gromowski, G. D.Barrett, A. D.Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virusVirologyVirology349-603662Amino Acid SequenceAmino Acid Substitution/genetics/immunologyAntibodies, Monoclonal/immunologyAntibodies, Viral/immunologyAntibody AffinityAntigens, Viral/chemistry/genetics/*immunologyDengue Virus/*immunologyEnzyme-Linked Immunosorbent Assay*Epitope MappingEpitopes, B-Lymphocyte/genetics/*immunologyImmunodominant Epitopes/genetics/immunologyImmunoglobulin G/immunologyModels, MolecularMolecular Sequence DataMutagenesis, Site-DirectedNeutralization TestsProtein Structure, Tertiary/geneticsViral Envelope Proteins/chemistry/genetics/*immunology2007Sep 300042-6822 (Print)17719070http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17719070 engNybakken20053317Nybakken, G. E.Oliphant, T.Johnson, S.Burke, S.Diamond, M. S.Fremont, D. H.Department of Pathology, Washington University School of Medicine, St Louis, Missouri 63110, USA.Structural basis of West Nile virus neutralization by a therapeutic antibodyNatureNatureNatureNature764-94377059Adsorption/drug effectsAmino Acid SequenceAnimalsAntibodies, Monoclonal/immunology/pharmacology/therapeutic useAntibodies, Viral/*immunology/pharmacology/*therapeutic useBinding SitesBinding Sites, Antibody/drug effectsCercopithecus aethiopsConserved SequenceEpitope MappingGlycoproteins/chemistry/immunologyImmunodominant Epitopes/chemistry/immunologyModels, MolecularMolecular Sequence DataNeutralization TestsProtein ConformationVero CellsViral Envelope Proteins/chemistry/immunologyViral Vaccines/immunologyWest Nile virus/*chemistry/drug effects/*immunology2005Sep 291476-4687 (Electronic)16193056http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16193056 engVolk20045517Volk, D. E.Beasley, D. W.Kallick, D. A.Holbrook, M. R.Barrett, A. D.Gorenstein, D. G.Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555-1147, USA.Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virusJ Biol ChemThe Journal of biological chemistry38755-6127937Antibodies, Monoclonal/chemistryMagnetic Resonance SpectroscopyModels, MolecularMutationProtein BindingProtein ConformationProtein Structure, SecondaryProtein Structure, TertiaryViral Envelope Proteins/*chemistryWest Nile virus/*metabolism/*pathogenicity2004Sep 100021-9258 (Print)15190071http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15190071 eng[14-17]. ED3 is hence a primary target for antibody based prophylactic measures. DEN2 and WNV ED3s are encoded by approximately hundred amino acids and share 40% sequence identity (Figure 1a). Alignment of the two domain structures shows homologous secondary structural elements (Figure 1b); an Immunoglobulin-like fold formed by two beta-sheets (ABE and CFG sheets) and stabilized by an intramolecular disulfide bond (residues 302-333 in DEN2 and 305-335 in WNV ED3). However, in spite of homologous sequences, structures and functions, these two domains do not share similar binding ligands or cell surface receptors; they display high variability in their antibody binding characteristics and differ greatly in their thermodynamic stabilities ADDIN EN.CITE Yu2004262617Yu, S.Wuu, A.Basu, R.Holbrook, M. R.Barrett, A. D.Lee, J. C.Department of Human Biological Chemistry & Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1055, USA.Solution structure and structural dynamics of envelope protein domain III of mosquito- and tick-borne flavivirusesBiochemistryBiochemistryBiochemistryBiochemistryBiochemistryBiochemistry9168-764328Amino Acid SequenceAnimalsBinding SitesCulicidae/*virologyFlaviviridae/*chemistryKineticsProtein ConformationReceptors, VirusSequence AlignmentSolubilitySolutionsTicks/*virologyViral Envelope Proteins/*chemistry2004Jul 200006-2960 (Print)15248774http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15248774 eng[18].
Two critical antigenic epitopes were identified on ED3 of DEN2 from MAb binding studies. Site-directed mutagenesis of recombinant ED3 ADDIN EN.CITE Gromowski2007121217Gromowski, G. D.Barrett, A. D.Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, and Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX 77555-0609, USA.Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virusVirologyVirology349-603662Amino Acid SequenceAmino Acid Substitution/genetics/immunologyAntibodies, Monoclonal/immunologyAntibodies, Viral/immunologyAntibody AffinityAntigens, Viral/chemistry/genetics/*immunologyDengue Virus/*immunologyEnzyme-Linked Immunosorbent Assay*Epitope MappingEpitopes, B-Lymphocyte/genetics/*immunologyImmunodominant Epitopes/genetics/immunologyImmunoglobulin G/immunologyModels, MolecularMolecular Sequence DataMutagenesis, Site-DirectedNeutralization TestsProtein Structure, Tertiary/geneticsViral Envelope Proteins/chemistry/genetics/*immunology2007Sep 300042-6822 (Print)17719070http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17719070 engGromowski2008111117Gromowski, G. D.Barrett, N. D.Barrett, A. D.Department of Pathology, Sealy Center for Vaccine Development, Center for Biodefense and Emerging Infectious Diseases, Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA.Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virusJ VirolJournal of virologyJ Virol8828-3782172008Sep1098-5514 (Electronic)18562544http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18562544 engLisova2007181817Lisova, O.Hardy, F.Petit, V.Bedouelle, H.Unit of Molecular Prevention and Therapy of Human Diseases (CNRS-URA3012), Institut Pasteur, 28 rue Docteur Roux, F-75724 Paris Cedex 15, France.Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virusJ Gen VirolThe Journal of general virology2387-9788Pt 9Amino Acid SequenceAnimalsAntibodies, Viral/immunologyCulicidae/virologyDengue Virus/*geneticsEnzyme-Linked Immunosorbent AssayEpitopes/chemistryEscherichia coli/geneticsFlavivirus/classification/geneticsImmunoglobulin Fab Fragments/immunologyModels, MolecularMutagenesisNeutralization TestsPlasmidsProtein ConformationTransfectionViral Envelope Proteins/*genetics2007Sep0022-1317 (Print)17698647http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17698647 eng[15, 19, 20] followed by MAb binding was used to isolate mutants resistant to antibody binding. These mutations were primarily localized on the lateral CFG sheet, the N-terminus loop and beta strand A of the domain (Figure 1d and 1e) which were shown to constitute binding epitopes for the respective MAbs. In West Nile virus, screening viral variants for MAb resistance and subsequent sequencing of the mutants identified several MAb binding resistant mutations on the surface of ED3, located on the BC loop and the N-terminus ADDIN EN.CITE Beasley20024417Beasley, D. W.Barrett, A. D.WHO Collaborating Center for Tropical Diseases, Sealy Center for Vaccine Development, Department of Pathology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA.Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope proteinJ VirolJournal of virologyJ Virol13097-1007624AnimalsAntibodies, Monoclonal/immunologyCell Membrane/metabolism*Epitope MappingMiceNeutralization TestsRecombinant Proteins/immunologyViral Envelope Proteins/*immunology/physiologyVirulenceWest Nile virus/*immunology/pathogenicity2002Dec0022-538X (Print)12438639http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12438639 engLi20059917Li, L.Barrett, A. D.Beasley, D. W.Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, and Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609, USA.Differential expression of domain III neutralizing epitopes on the envelope proteins of West Nile virus strainsVirologyVirology99-1053351Amino Acid SequenceAnimalsAntibodies, Monoclonal/immunologyAntibodies, Viral/*immunologyAntigenic VariationCercopithecus aethiopsEpitopes/chemistry/genetics/*immunology/metabolismHumansModels, MolecularMolecular Sequence DataMutagenesis, Site-DirectedMutationNeutralization TestsRecombinant Fusion Proteins/metabolismVero CellsViral Envelope Proteins/chemistry/genetics/*immunology/*metabolismWest Nile virus/genetics/*immunology/metabolism2005Apr 250042-6822 (Print)15823609http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15823609 eng[14, 21] (Figure 1c).
It is a well established fact that peptide motions over a wide range of time scales from picoseconds to milliseconds and seconds and more modulate a gamut of functions involving subtle (less than 1 ) to major structural rearrangements. Several instances of changes in the global distribution of dynamics in a peptide upon ligand binding have been reported. The changes in dynamics associated with the formation of the Barnase and Barstar complex ADDIN EN.CITE Zhuravleva2007444417Zhuravleva, A.Korzhnev, D. M.Nolde, S. B.Kay, L. E.Arseniev, A. S.Billeter, M.Orekhov, V. Y.Swedish NMR Centre at Goteborg University, Box 465, 405 30 Goteborg, Sweden.Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational studyJ Mol BiolJournal of molecular biologyJ Mol Biol1079-923674Bacterial Proteins/*metabolism*Computer SimulationModels, Molecular*Nuclear Magnetic Resonance, BiomolecularProtein ConformationProtein Structure, TertiaryRibonucleases/*chemistry/*metabolism2007Apr 60022-2836 (Print)17306298http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17306298 eng[22], fluctuations in dynamics of PDZ domain upon ligand binding ADDIN EN.CITE De Los Rios2005262617De Los Rios, P.Cecconi, F.Pretre, A.Dietler, G.Michielin, O.Piazza, F.Juanico, B.Laboratoire de Biophysique Statistique, ITP, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland. paolo.delosrios@epfl.chFunctional dynamics of PDZ binding domains: a normal-mode analysisBiophys JBiophysical journal14-21891Adenosine Triphosphate/chemistryAnisotropyBinding SitesBiophysics/*methodsCarbon/chemistryCrystallography, X-RayGuanosine Triphosphate/chemistryHeatLigandsMagnetic Resonance SpectroscopyModels, MolecularModels, StatisticalPeptides/chemistryProtein BindingProtein ConformationProtein Structure, TertiarySignal TransductionSoftwareTemperatureThermodynamics2005Jul0006-3495 (Print)15821164http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15821164 engDhulesia2008252517Dhulesia, A.Gsponer, J.Vendruscolo, M.Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.Mapping of two networks of residues that exhibit structural and dynamical changes upon binding in a PDZ domain proteinJ Am Chem SocJournal of the American Chemical SocietyJ Am Chem Soc8931-913028Binding SitesCluster AnalysisHumansModels, ChemicalModels, MolecularNuclear Magnetic Resonance, BiomolecularPDZ DomainsPeptide MappingProtein ConformationProtein Tyrosine Phosphatase, Non-Receptor Type 13/*chemistry/metabolismThermodynamics2008Jul 161520-5126 (Electronic)18558679http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18558679 eng[23, 24] and upon phosphorylation of the Shc adaptor protein ADDIN EN.CITE Suenaga2004272717Suenaga, A.Kiyatkin, A. B.Hatakeyama, M.Futatsugi, N.Okimoto, N.Hirano, Y.Narumi, T.Kawai, A.Susukita, R.Koishi, T.Furusawa, H.Yasuoka, K.Takada, N.Ohno, Y.Taiji, M.Ebisuzaki, T.Hoek, J. B.Konagaya, A.Kholodenko, B. N.Bioinformatics Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.Tyr-317 phosphorylation increases Shc structural rigidity and reduces coupling of domain motions remote from the phosphorylation site as revealed by molecular dynamics simulationsJ Biol ChemThe Journal of biological chemistry4657-622796*Adaptor Proteins, Signal TransducingAdaptor Proteins, Vesicular Transport/*chemistry/metabolismBinding SitesModels, MolecularPhosphorylationProtein ConformationThermodynamicsTyrosine/chemistrysrc Homology Domains2004Feb 60021-9258 (Print)14613932http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14613932 eng[25] etc. provide various examples of ligand induced changes in local and long-distance dynamics. This phenomenon of long-distance dynamic perturbations is of utmost importance to me, since in the case of ED3 of WNV it was observed that the MAb binding resistant mutations significantly altered the dynamics of residues at regions physically distant (> 10 ) from the site of mutation in the peptide ADDIN EN.CITE Maillard20088817Maillard, R. A.Jordan, M.Beasley, D. W.Barrett, A. D.Lee, J. C.Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1055, USA.Long range communication in the envelope protein domain III and its effect on the resistance of West Nile virus to antibody-mediated neutralizationJ Biol ChemThe Journal of biological chemistry613-222831Antibodies, Monoclonal/*immunologyMagnetic Resonance SpectroscopyModels, MolecularNeutralization TestsProtein Structure, SecondaryProtein Structure, TertiaryViral Envelope Proteins/*chemistry/*immunology/metabolismWest Nile virus/*metabolism2008Jan 40021-9258 (Print)17986445http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17986445 eng[26]. Hence based on the above two observations, I hypothesize that the dynamics of distant residues in ED3 could be coupled to each other and a mutation altering the domains activity (here binding to MAb) would perturb these coupled dynamics between different structural elements of the domain. In other words, a resistant mutation interferes with a conformational rearrangement that would normally occur on MAb binding through these coupled motions.
Here I simulate the dynamics of the wild type ED3 of DEN2 and WNV together with their respective experimentally characterized mutants in order to understand the relevance of coupled dynamics to MAb binding resistance. I used principal components analysis to determine the changes in the distribution of dynamics caused by the mutations. Mutations resistant to MAb binding were found to perturb the global dynamics of the domains to a greater extent than non-resistant mutations as was evident from their eigenvalue profiles. I tested my central hypothesis by determining the long-range effects of mutations on coupled dynamics through cross-correlation analysis. Indeed, significant perturbations in large-scale motions (seen in the MAb resistant mutations) were accompanied by global changes in the correlated dynamics. Based on these results, I propose that coupled dynamics enables conformational rearrangement upon antibody binding in the native ED3s of flaviviruses. A perturbation of these motions by specific mutations of residues belonging to this coupled network, resulting in non-native like dynamics, is a plausible mechanism of resistance to antibody binding. As a result of the above analysis I were able to identify most of the binding resistant mutations in the DEN2 and WNV ED3s. In addition, I may have identified a prior speculated, but as yet unidentified putative MAb binding region in the ED3 of DEN2.
Figure 1: ED3 of WNV and DEN2.
(a) Sequence alignment of DEN2 ED3 (PDB: 1TG8) and WNV ED3 (PDB: 1S6N) sequences. (b) Structural alignment of the two domains. Secondary structural elements are depicted in grey in both the al i g n m e n t s . N o t i c e t h e a b s e n c e o f s t r a n d D i n b o t h d o m a i n s . T h e n o m e n c l a t u r e i s a c c o r d i n g t o t h e I g G f o l d o f f l a v i v i r a l E D 3 s w h i c h t y p i c a l l y h a d 4 - s t r a n d s o n o n e s h e e t a n d 3 o n t h e o t h e r . T h e C E l o o p i n s t e a d s p a n s t h e l e n g t h o f t h e a b s e n t D s t r a n d . ( c ) E x perimentally determined epitopes (spheres) of WNV specific antibodies ADDIN EN.CITE ADDIN EN.CITE.DATA [14, 21] (d) DEN2 virus epitope I ADDIN EN.CITE ADDIN EN.CITE.DATA [15] and (e) DEN2 virus epitope II ADDIN EN.CITE ADDIN EN.CITE.DATA [19, 20] mapped on to the respective secondary structures. Residues forming the core of the MAb binding region, mutations in which cause the greatest resistance to MAb binding, are labeled and colored in magenta. Residues in yellow cause partial resistance to MAb binding when mutated. The mapped epitopes of both domains are highlighted in the sequence. Core residues in the epitope are highlighted in magenta, peripheral residues are in yellow.
DEN2
WNV
301
304
MCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFE-IMDLEKRHVLGRLITVNPIVT
VCSKAFKFLGTPADTGHGTVVLELQYTGTDGPCKVPISSVASLNDLTPVGRLVTVNPFVS
DEN2
WNV
360
364
--EKDSPVNIEAEPPFGDSYIIIGVEPGQLKLDWFK 393
VATANAKVLIELEPPFGDSYIVVGRGEQQINHHWHK 399
HIDDEN TEXT: Using Major Sections are option, and most dissertations do not use major sections. If your dissertation is not divided into major sections, then you do not need use the style HEADING 1,H1, start chapters using Heading 2,h2.
Chapter 2: Methods:
2.1 Molecular Dynamics Simulations:
Molecular dynamics simulations on the wild type and mutant ED3s of DEN2 and WNV were performed using the Amber 9 simulations package ADDIN EN.CITE Case2005636317Case, D. A.Cheatham, T. E., 3rdDarden, T.Gohlke, H.Luo, R.Merz, K. M., Jr.Onufriev, A.Simmerling, C.Wang, B.Woods, R. J.Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Raod, TPC15, La Jolla, CA 92037, USA. case@scripps.eduThe Amber biomolecular simulation programsJ Comput ChemJ Comput Chem1668-882616*AlgorithmsCarbohydrates/chemistryComputer Simulation/*trends*Models, Biological*Models, ChemicalModels, MolecularNucleic Acids/chemistryProteins/chemistrySoftware/*trendsThermodynamics2005Dec16200636http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16200636 [27] for 2.5 ns. Topologies were generated using the ff99SB ADDIN EN.CITE Simmerling2002636317Simmerling, C.Strockbine, B.Roitberg, A. E.Center for Structural Biology and Department of Chemistry, State University of New York - Stony Brook, Stony Brook, New York 11794, USA. carlos.simmerling@sunysb.eduAll-atom structure prediction and folding simulations of a stable proteinJ Am Chem SocJ Am Chem Soc11258-912438Amino Acid SequenceComputer SimulationModels, ChemicalModels, MolecularMolecular Sequence DataNuclear Magnetic Resonance, BiomolecularOligopeptides/*chemistry*Protein FoldingThermodynamics2002Sep 2512236726http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12236726 [28] force field on LEaP. Coordinates for wild type DEN2 ED3 were obtained in the form of a truncated peptide from the crystal structure of the E protein (PDB code: 1TG8) whereas the coordinates for WNV ED3 were NMR derived (PDB code: 1S6N). Since the domains secondary structure and function is retained in solution as in the intact virus ADDIN EN.CITE Maillard2008252517Maillard, R. A.Jordan, M.Beasley, D. W.Barrett, A. D.Lee, J. C.Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555-1055, USA.Long range communication in the envelope protein domain III and its effect on the resistance of West Nile virus to antibody-mediated neutralizationJ Biol ChemThe Journal of biological chemistryJ Biol ChemThe Journal of biological chemistryJ Biol ChemThe Journal of biological chemistry613-222831Antibodies, Monoclonal/*immunologyMagnetic Resonance SpectroscopyModels, MolecularNeutralization TestsProtein Structure, SecondaryProtein Structure, TertiaryViral Envelope Proteins/*chemistry/*immunology/metabolismWest Nile virus/*metabolism2008Jan 40021-9258 (Print)17986445http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17986445 eng[26] I could use the individual ED3s of DEN2 and WNV ED3 structures for MD as a representative model for the behavior of the domain in the complete E protein. The simulations were carried out without periodic boundary conditions in which aqueous solvation effects are represented implicitly by a generalized Born (GB)/ surface area model ADDIN EN.CITE Still1990646417Still, W. C., Tempczyk, A., Hawly, R. C., Hendrickson, T.Semianalytical Treatment of Solvation for Molecular Mechanics and DynamicsJournal of American Chemical SocietyJournal of American Chemical Society6127-291121990[29]. A salt concentration of 0.2M was set using a modified generalized Born theory based on the Debye-Hckel limiting law for ion screening of interactions ADDIN EN.CITE Srinivasan1999656517Srinivasan, J., Trevathan, M. W., Beroza, P., Case, D. A.Application of a Pairwise Generalized Born Model to Proteins and Nucleic Acids: Inclusion of Salt Effects Theoretical Chemical AccountsTheoretical Chemical Accounts426-341011999[30]. The maximum distance set for calculation of pairwise interactions and the maximum distance for the electrostatic, van der Waals and other energy terms of the generalized Born interaction in the GB model was set at 16 which was greater than the largest dimension of the protein. Langevin dynamics ADDIN EN.CITE Wu2003666617Wu, X., Brooks, B. R.Self-Guided Langevin Dynamics Simulation MethodChemical Physical Letters Chemical Physical Letters512-183812003[31] was used for temperature regulation which was maintained at 300K throughout the time of the simulation. Bond lengths between hydrogens and heavy atoms were constrained using SHAKE algorithm. The integration step size was set at 1.5 fs and the coordinates were recorded after every 500 steps (0.75 ps). Unrestrained dynamics to relieve bad contacts was run for 22.5 ps before equilibration and production runs. Least squares fit of backbone CA atoms with reference to the energy minimized structure over all frames in the trajectory was done to remove rotational and translational motions in the frames before further analysis. On average, from the potential energy profile and root mean square deviations (RMSD) of the CA atoms (Figure 2) I determined that all DEN2 mutants were well equilibrated within 500 ps. The RMSD profile for wild type WNV ED3 did not reach a steady value throughout the simulation. Based on the results for all mutants, a putative average equilibration time for the WNV mutants was chosen to be 1000 ps. I hence excluded the first 700 frames (525 ps) in DEN2 ED3 and the initial 1600 frames (1200 ps) from WNV ED3 trajectories in all data analysis.
2.2 Principal Components Analysis:
4.4.1 Generation of Principal Components:
The dynamics of one of N atoms (here CA) may be represented by its 3-dimensional co-ordinates as a vector function (r) of time (t) as shown below:
EMBED Equation.3 EMBED Equation.3 (1)
The data matrix of these vectors obtained from all the frames in the trajectory would be of the order 3N X F where F is the number of frames. PCA involves the eigen decomposition of the covariance of this data matrix ADDIN EN.CITE Garcia1992676717Garcia, A. E.Large-amplitude nonlinear motions in proteinsPhys Rev LettPhys Rev Lett2696-269968171992Apr 2710045464http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10045464 [32]. The ijth entry of the covariance matrix Cov (3N x 3N) is given by:
EMBED Equation.3 (2)
where EMBED Equation.3 is the time average over the whole trajectory.
PCA diagonalizes the covariance matrix Cov by solving EMBED Equation.3 , so as to obtain the diagonal matrix EMBED Equation.3 with the diagonal entries being the eigenvalues ranked by magnitude. The cth column of the transformation matrix T is the cth e i g e n v e c t o r c o f t h e c o v a r i a n c e m a t r i x t h a t i s T = [ v 1 , v 2 , v 3 . . . v f ] . T h i s i s a l s o t h e c t h p r i n c i p a l c o m p o n e n t o f t h e d a t a m a t r i x . W h i l e t h e e i g e n v e c t o r s d e s c r i b e t h e d i r e c t i o n o f t h e d y n a m i c s o f t h e r e s i d u e s ( i n t h e d e c r e a s i n g o r d e r o f v a r i a n c e ) , t h e corresponding eigenvalues give the proportion to which each principal component contributes to overall motions. Consequently, the first principal component corresponds to collective motions with largest amplitude followed by the second and so on. I obtained the principal components for the trajectories of all the wild type and mutant ED3 peptides. Only backbone CA atoms were used in the analysis. Several N and C terminal residues (297-300; 391-395 in DEN2 ED3 and 304-306; 397-401 in WNV ED3) which biased the variance in the data due to their high mobility were not incorporated in the analysis. Projection of trajectories on to the principal component space was obtained by calculating the mean positional fluctuation of the backbone CA atoms of all residues along the direction of a principal component over the time of the simulation. Ptraj module of the Amber 9 simulations package was used to generate the principal components.
2.3 Cross-correlation Analysis
The cross-correlation coefficient between CA atoms of residues i and j is given by ADDIN EN.CITE Swaminathan1991616117Swaminathan, S., Harte, W. E. Jr., Beveridge, D. L.Investigation of Domain Structure in Proteins via Molecular Dynamics Simulation: Application to HIV-1 Protease DimerJournal of American Chemical SocietyJournal of American Chemical Society2717-2111371991[33]:
EMBED Equation.3 (3)
where EMBED Equation.3 is the displacement vector for the ith residue CA atom in the peptide and EMBED Equation.3 is the time average over the whole trajectory. Two residues are said to be positively correlated in motion if the average displacement of their bond vectors over the simulation time is in the same direction with respect to one another. Similarly two residues are said to be negatively correlated in motion if their average displacements are opposite in direction to one another, i.e. residues have negative relative displacement. Consequently, no correlation is seen between two residues if their relative average displacement from mean position is zero. A difference cross-correlation matrix for a mutant ED3 was generated by subtracting the respective cross-correlation values of the wild type domain III from those of the mutant. Due to dependence on the starting structure, time of averaging used for calculation of the correlation values and a possible reversal of correlation values even within a single simulation, cross-correlation analysis is a problematic technique for comparison of trajectories from two individual simulations ADDIN EN.CITE Hunenberger1995696917Hunenberger, P. H.Mark, A. E.van Gunsteren, W. F.Laboratorium fur Physikalische Chemie ETH-Zentrum, Zurich, Switzerland.Fluctuation and cross-correlation analysis of protein motions observed in nanosecond molecular dynamics simulationsJ Mol BiolJ Mol Biol492-5032524AnimalsAprotinin/*chemistryCattle*Computer SimulationCrystallography, X-RayModels, MolecularMolecular StructureMuramidase/*chemistry1995Sep 297563068http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7563068 [34]. In order to avoid misinterpretations, all datasets have been collected from well equilibrated trajectories and contain identical number of frames from the trajectories. The difference plots were calculated from multiple simulations. Mean and standard deviations of the difference between individual instances were measured. The standard deviation thus calculated was my estimate of noise in the obtained difference matrices. Consequently, only those differences in correlations that were at least equal to or greater than twice the standard deviation of noise in the data were considered significant.
HIDDEN TEXT: Using Major Sections are option, and most dissertations do not use major sections. If your dissertation is not divided into major sections, then you do not need use the style HEADING 1,H1, start chapters using Heading 2,h2.
Chapter 3: Results And Discussion Chapter 3: Molecular dynamics simulations of wild type and mutant Envelope protein Domain-3 of dengue-2 and West Nile virus
MD simulations were performed on the wild type and single mutants of DEN2 and WNV ED3s. Binding affinities were determined for various mutations in surface exposed putative antigen binding regions of DEN2 ED3 ADDIN EN.CITE ADDIN EN.CITE.DATA [15, 19, 20] and for all the naturally occurring mutations of WNV ED3 ADDIN EN.CITE ADDIN EN.CITE.DATA [14, 21]. I report here the results of MD simulations of wild type DEN2 and WNV ED3s in comparison to two single mutants from each domain. These mutations have been chosen to illustrate the correlation between in vitro perturbations in MAb binding affinities and their internal dynamics. Mutations were considered to be resistant to the MAb if their binding affinities in vitro were less than 10 times that of the wild type. Hence by definition, these residues constituted the hotspots for MAb binding on the respective envelope ED3s. From the above criterion, mutant P384G of DEN2 ED3 was determined to be resistant to MAb binding while mutant K361G was non-resistant ADDIN EN.CITE ADDIN EN.CITE.DATA [15] (Figure 2a inset). In case of WNV, mutant T332K was resistant while the mutation H320A, which lies well outside the proximity of the mapped epitope of the MAb was presumed to be non-resistant ADDIN EN.CITE ADDIN EN.CITE.DATA [21] (Figure 2b inset). In vitro binding affinities of all studied mutations of DEN2 ED3 (Table-1) are known ADDIN EN.CITE ADDIN EN.CITE.DATA [15, 19, 20]. In WNV ED3, not all mutations have pre-determined binding affinities. However, from the location of the mapped epitopes I mutated the residues in the vicinity of the mapped epitopes (putative resistant mutations) and away from the epitopes (putative non-resistant mutations) to alanine (Table-2) for comparison.
The average RMSD values of DEN2 and WNV ED3s differed roughly by 1 with WNV ED3 and its mutants typically having a higher average of 2.5 compared to wild type and mutants of DEN2 ED3 at 1.5 (Figure 2). The Molecular Dynamics trajectories of WNV ED3 and its mutants seem to indicate that the peptide shows high degree of variation throughout the time of simulation. The reason could be two fold, one, the protein takes a longer time to relax to the forcefield of the simulation from NMR coordinates; Two, the ED3 of WNV could be more flexible than DEN2 ED3, hence causing greater variations in RMSD within a short time of simulation. Unlike DEN2 ED3, the WNV system does not reach equilibrium even after allowing for a 1.5ns period (Methods). These differences in equilibration rates and RMSD values could be due to the different sources of coordinates for the starting structures of DEN2 (X-ray crystallography) ADDIN EN.CITE Rey2003111117Rey, F. A.Virologie Moleculaire et Structurale, Unite Mixte de Recherche 2472/1157, Centre National de la Recherche Scientifique et Institut National de la Recherche Agronomique, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. rey@gv.cnrs-gif.frDengue virus envelope glycoprotein structure: new insight into its interactions during viral entryProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of America6899-90110012Carbohydrates/chemistryCrystallography, X-RayDengue Virus/chemistry/*pathogenicity/physiologyDimerizationHumansModels, MolecularProtein Structure, QuaternaryProtein SubunitsViral Envelope Proteins/*chemistry/physiologyVirulence2003Jun 100027-8424 (Print)12782795http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12782795 eng[11] and WNV (NMR) ED3s ADDIN EN.CITE Volk2004171717Volk, D. E.Beasley, D. W.Kallick, D. A.Holbrook, M. R.Barrett, A. D.Gorenstein, D. G.Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas 77555-1147, USA.Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virusJ Biol ChemThe Journal of biological chemistryJ Biol ChemThe Journal of biological chemistryJ Biol ChemThe Journal of biological chemistry38755-6127937Antibodies, Monoclonal/chemistryMagnetic Resonance SpectroscopyModels, MolecularMutationProtein BindingProtein ConformationProtein Structure, SecondaryProtein Structure, TertiaryViral Envelope Proteins/*chemistryWest Nile virus/*metabolism/*pathogenicity2004Sep 100021-9258 (Print)15190071http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15190071 eng[17]. The trajectories for DEN2 ED3 show that all simulations were sufficiently equilibrated within the region used for analysis of dynamics. The equilibration time varied markedly for WNV ED3 wild type and mutants. However, for purpose of comparison, I chose a comprehensive time of simulation of 1.5 ns as my working equilibration period for the system. Only data after this time point were used in the analysis.
Figure 2: RMSD Vs Time plot
(a) RMSD of the backbone CA atoms of wild type DEN2 ED3 (black) compared to its mutants P384G (resistant mutation) in red and K361G (non-resistant mutation) in blue. All frames were referenced to the energy minimized structure and frames starting from 0.5 ns onwards were collected for data analysis as all trajectories reached a plateau in their RMSD values at this point. (b) RMSD of the backbone CA atoms of wild type WNV ED3 (black) compared to its mutants T332K (resistant mutation) in red and H320A (non-resistant mutation) in blue. All frames were referenced to the energy minimized structure and frames starting from 1.2 ns onwards were collected for data analysis. Insets show the location of the mutations mapped on to the ED3s of DEN2 and WNV respectively
3.1 Principal Components Analysis (PCA):
Data from Molecular dynamics trajectories is essentially a composite of all the motions sampled during the period of simulation by all atoms in the system. PCA was used to extract dominant large-scale motions from the multitude of sampled dynamics. Principal components are obtained by the diagonalization of the covariance matrix for the CA atoms over the entire simulation. Eigenvalues of the principal components give the proportional contribution of the corresponding eigenvectors to the total variance within the data. The number of principal components required to sufficiently describe the total variance in the data were determined from a profile of their eigenvalues (Figure 3). Dominant large-scale dynamics of a system would lead to high covariance in the residue coordinates; consequently resulting in higher eigenvalues for the dominant principal components; as in the case of wild type WNV ED3 (Figure 3b). The rather shallow gradient of the wild type DEN2 ED3 eigenvalues (Figure 3a) suggests that motions described by its highest principal components (PC1 and PC 2) were comparatively smaller in magnitude. The dynamics of wild type DEN2 ED3 were hence not dominated by large-scale motions during the time of simulation.
Figure 3: Eigenvalue Profiles
(a) Eigenvalue profiles of DEN2 ED3 wild type (blue), resistant mutant P384G (pink) and non-resustant mutant K361G (yellow). Wild type and non-resistant mutant of DEN2 ED3 have a shallow gradient of eigenvalues indicative of absence of large-scale dynamic motions. High eigenvalue of principal component-1 of P384G implies that dynamics described by PC 1 are rather large-scale and dominant in the domains internal motions.
(b) Eigenvalue profiles of WNV ED3 wild type (blue), resistant mutant T332K (pink) and non-resistant mutant H320A (yellow). Wild type WNV ED3 (unlike DEN2 ED3) has a higher eigenvalue for PC1 implying that more large-scale dynamic perturbations compared to those of DEN2 ED3. Resistant mutant T332K has altered distribution of dynamics, reducing the number of large-scale motions as evident from lower gradient in the eigenvalues. The non-resistant mutant H320A has a gradient similar to that of the wild type, possibly retaining native like dynamic motions.
3.1.1 Dynamic perturbations caused by mutations of DEN2 ED3:
Higher the eigenvalues, more dominant are the motions described by respective principal components. A high eigenvalue of PC1 is representative of high covariation in dynamics along that principal component and hence a lower contribution to variance from the remaining principal components. A steep drop in the eigenvalues of the resistant mutation P384G of DEN2 ED3 (Figure 3a) indicates that dominant large-scale dynamics are sampled by PC1 of P384G, unlike the wild type domain. The non-resistant mutant K361G, however had a profile similar to the wild type, meaning that its dynamics were similar in range to the wild type and unlike the resistant mutant. The existence of large-scale dynamic perturbations may be tested by projecting the molecular dynamics trajectories on to the 2D space defined by the first two principal components (Figure 4). The data points for the resistant mutant P384G show greater co-variation in the direction of PC 1 in the projection plot indicative of dominant motions sampled along this vector (Figure 4b). In case of absence of large-scale dynamics such as in the wild type and non-resistant mutant K361G of DEN2 ED3, there is no one dominant principal component and hence the projection plots are devoid of directionality along PC1 (Figure 4c).
I calculated the percentage contribution of the first two principal components to the total variance in the dynamics of DEN2 ED3 (Table-1). The data in table-1 are arranged in the increasing order of the cumulative contribution to variance from the first two principal components. In other words, the dynamics in the mutants at the head of the table are smaller in range compared to the mutants at the end of the table. All dynamics are relative to those sampled by the wild type DEN2 ED3 (highlighted in red). Upon comparison of the data in table-1 and the corresponding projection plots of the mutants (Supplementary Figure 1), I observe the following: 1) as the eigenvalue proportions increase (going down the table), the directionality along the respective principal components in the projections also increases. 2) All studied mutations of DEN2 ED3 had higher proportion of variance from the first two eigenvalues relative to the native peptide, thus increasing large-scale dynamics compared to the wild type. 3) Mutations resistant to MAb binding, in general, have a significantly higher cumulative percentage contribution to variance from E1 and E2 (grey entries). These mutations are K305G, K307G, V308A, K310G/E, I312F, P332G, E383G, P384G, L387A, L389A, and W391G. In vitro binding affinities of these mutants to specific MAbs are lower than 10 times that of the wild type ED3 ADDIN EN.CITE ADDIN EN.CITE.DATA [15, 19]. The residues forming these mutations have hence been deemed to constitute the epitopes for binding of specific MAbs (Figure 1). 4) Some residues that do not constitute the mapped epitopes of DEN2 ED3 also showed a drastic increase in the cumulative proportion of variance contributed by PC1 and PC2. These mutants are G381A, G385E, T303G, K334A/Q/G and R345A (See Discussion).
Figure 4: Projection Plots of DEN2 ED3.
(a) Projection of Molecular Dynamics trajectories of wild type DEN2 ED3, (b) resistant mutant P384G and (c) non-resistant mutant K361G onto the first two principal components. Data points represent the average position described by all backbone CA atoms in the space of the two PCs progressing through the simulation (red to violet). Variance in data points in plots (a) and (c) is distributed almost evenly between the two principal components indicating the absence of dominant large-scale dynamics. Variance in plot-b is higher in the direction of PC1 suggesting that large-scale internal motions were sampled during the time of simulation in the mutant P384G thereby increasing its conformational freedom.
E1E2TotalWT13.699.2138.43Q386G13.858.5940.82V309A11.4510.6741K388G14.679.6841.78K361G13.9810.1242.8K388A16.018.1843.1P384E16.588.0743.26K344A13.2411.4444.36E327G14.511.344.72E383A16.759.6544.89E311A14.0210.6545.74E338G16.0310.7946.83K310G(II)16.0512.0147.01D329G14.9211.5347.33P336G19.699.0647.93L389A(II)17.2213.6348.62K307G(I, II)21.589.6649.15G381A18.2812.6649.72W391G(II)16.5313.1949.92K334Q*23.29.5249.97L387A(II)25.429.8250.37K305G(I)21.0510.3150.6P384G(I)25.439.8450.76I312G(II)21.3211.4551.03T303G*23.179.9451.22G385E23.812.2852.99P332G(I,II)26.710.4453.51K334A*25.6212.2854.39K310E(II)27.6912.1456.2I312F(II) 24.7813.4357.97R345A38.187.0359.06E383G(I)36.047.3159.51K334G*50.325.8467.01V308A(II)41.6412.3767.7Table 1: Cumulative proportion of variance: DEN2 ED3
Percentage contribution of first two eigenvalues (E1, E2) to the variance in data for the wild type (red) and mutant DEN2 ED3s. Highlighted in grey are the mutations leading to MAb binding resistance in the domain. These residues have been shown to constitute the epitopes:
(I) DEN2 ED3 type specific epitope.
(II) dengue complex specific epitope.
* K334 and T303 are located adjacent to the disulfide bond C302-C333. Mutations in these residues may destabilize the secondary structure of ED3 leading to large scale dynamic motions and a high cumulative percentage eigenvalue.
R345 belongs to a putative MAb binding epitope (see discussion)
G385 has been implicated to form a part of the epitope of DEN2 ED3 binding MAbs ADDIN EN.CITE Hiramatsu1996383817Hiramatsu, K.Tadano, N.Men, R. Lai, C-J.Mutational Analysis of a Neutralization Epitope on the Dengue Type 2 Virus (DEN2) Envelope Protein: Monoclonal Antibody Resistant DEN2/DEN4 Chimeras Exhibit Reduced Mouse NeurovirulenceVirologyVirologyVirology437-4522421996[35]
3.1.2 Dynamic perturbations caused by mutations of West Nile virus ED3:
The gradient between the first and second eigenvalues of mutant H320A of WNV ED3 (Figure 5c) was similar to that of the wild type possibly indicating no change in the dynamics sampled by the non-resistant mutant. Consequently, the projection plot of H320A showed defined directionality along PC 1 as in the case of the wild type ED3. The eigenvalue profile of the resistant mutation T332K, however, varied significantly from its native peptide. The mutation had lower cumulative magnitude from the first two eigenvalues. Its projection plot showed uniform distribution of data points across the space described by PC 1 and 2 suggesting an absence of large-scale dynamics in any one direction (Figure 5b). In both viruses, therefore, the non-resistant mutants share similar dynamics as the wild type whereas the resistant mutations deviate from the wild type behavior.
Upon analysis, the projection plots of WNV ED3 mutants (Supplementary Figure 2) revealed the common motif of increase in directionality of the data points along the principal component vectors with a corresponding increase in their respective eigenvalue proportions. However, there were several differences between the nature of mutations in DEN2 ED3 and WNV ED3, viz., 1) Unlike in DEN2 ED3, there is no predominance of mutations in WNV ED3 along any one range of eigenvalues compared to the wild type. In other words while some mutations increased large-scale dynamics of the wild type, there were others which shifted the dynamics to smaller eigenvalues. 2) Several of the mutations that significantly decreased the dynamics compared to wild type such as K307A, K307E, T332K and T330I have been experimentally shown to be resistant to MAb binding ADDIN EN.CITE ADDIN EN.CITE.DATA [14, 21]. Their projection plots reflect this lower covariation in the dynamics, a result of absence of dominant large-scale dynamics. 3) Further, mutation T330A with higher cumulative value of E1 and E2 is resistant to MAb binding ADDIN EN.CITE ADDIN EN.CITE.DATA [21]. Its projection plot shows a concomitant increase in directionality along PC1 owing to larger first eigenvalue. Hence in general all experimentally determined MAb resistant mutations deviate from the native dynamics in both viruses (grey entries in the tables). Mutations in the vicinity of the mapped epitopes (putative resistant mutations) were expected to cause deviation in the dynamics of the domain. This was exactly the result for the mutants D333A, G331A G389A, and G334A which caused an increase in dominant large-scale dynamics and mutant Q391A, T366A and E390A which decreased these motions compared to the wild type. Experimentally determined non-resistant mutations T332A and A365S, as antipicated, had eigenvalues similar to the wild type ED3.
My results for WNV ED3 also show instances where a non-resistant mutation (K310T, K310G) showed altered large-scale dynamics of the domain which was against what was expected. Among the putative non-resistant mutations, H320A, P335A, L325A behaved similar to the wild type as was expected. The mutations P315A, G380A and L355A were my putative non-resistant mutations which deviated from the expected no-change in dynamics. Depending on the nature of the mutation at the site, there is an increase or decrease of dominant large-scale motions. For instance K307A/E/R has different effects on the dynamics of WNV ED3. T330I reduces large-scale dynamics of WNV ED3 whereas T330A increases these motions. These discrepancies in results for the WNV ED3 were anticipated since my MD trajectories were not well equilibrated to begin with and hence reliable interpretation of results would not be possible under such conditions
Figure 5: Projection Plots of WNV ED3.
Projection of Molecular Dynamics trajectories of (a) wild type WNV ED3 (b) resistant mutant T332K and (c) non-resistant mutant H320A onto the first two principal components. Data points represent the average position described by all backbone CA atoms in the space of the two PCs progressing through the simulation (red to blue). Wild type of WNV ED3 and its non-resistant mutant sample more than one major conformational state during the time of the simulation as suggested by the projection along PC 1. The resistant mutation T332K suppresses the large scale dynamics of the wild type as seen from an even distribution of its projection along both PCs.
E1E2TotalT366A11.868.4839.17K310T10.6610.0840.77K307E12.398.4242.18T332K14.399.3442.82P315A14.7311.0843.95L325A19.997.7444.12L355A17.249.9845.9Q391A13.2312.3346.86T330I18.9611.0648.15K307A16.7815.248.85E390A15.9112.0549.03K307R25.57.8550.22D333A28.059.4952.26P335A25.158.9552.9H320A23.5511.1653.02WT24.7811.2854.39G331A28.279.3954.53G334A32.917.6555.39T332A25.2114.6356.39G389A30.9311.0157.47A365S27.8913.657.58G380A36.1413.2661.43K310G36.511.8361.9T330A41.1511.2366.99Table 2: Cumulative proportion of variance: WNV ED3.
Percentage contribution of first two eigenvalues (E1, E2) to the variance in data for the wild type (red) and mutant WNV ED3s. Highlighted in grey are experimentally mapped mutations leading to MAb binding resistance in the domain. These residues have been shown to constitute the epitopes:
K310 in WNV ED3 is located within the MAb binding region. It has not been shown to harbor binding resistant mutations. However, the residue is critical for viral infectivity probably mediating cell curface receptor binding (See Discussion).
3.2 Cross-Correlation Analysis.
Cross-correlation coefficients give the magnitude of concerted motions between atoms ADDIN EN.CITE Swaminathan1991616117Swaminathan, S., Harte, W. E. Jr., Beveridge, D. L.Investigation of Domain Structure in Proteins via Molecular Dynamics Simulation: Application to HIV-1 Protease DimerJournal of American Chemical SocietyJournal of American Chemical Society2717-2111371991[33] during a simulation. The c r o s s - c o r r e l a t i o n m a t r i x f o r t h e E D 3 s o f D E N 2 a n d W N V p r o d u c e s a t y p i c a l s y m m e t r i c p l o t w i t h a c o r r e l a t i o n c o e f f i c i e n t o f 1 a l o n g t h e d i a g o n a l a n d p l u m e s o f p o s i t i v e l y c o r r e l a t e d o f f - d i a g o n a l v a l u e s c h a r a c t e r i s t i c o f a n t i p a r a l l e l - s h e e t r e g i o n s ( F i g u r e 6 ) . T h u s t h e c r o s s - c o r r e l a t i o n p l o t g i v e s a p i c t u r e o f a l l c o n c e r t e d m o t i o n s w i t h i n t h e d o m a i n . I n o r d e r t o s t u d y t h e e f f e c t s o f m u t a t i o n s o n t h e s e c o n c e r t e d m o t i o n s I d e t e r m i n e d t h e d i f f e r e n c e c r o s s - c o r r e l a t i o n v a l u e s b e t w e e n t h e m u t a n t s a n d t h e w i l d t y p e . Only those differences in correlations that were at least twice the standard deviation of noise in the data were considered as significant perturbations (See Chapter-3: Methods for details). Changes in dynamics of residues within a periphery of 10 from the site of mutation were considered to be local structural perturbations originating at the site of mutation and beyond that as long-distance effects.
Figure 6: Cross-correlation plots
Cross-correlation plots of (a) wild type DEN2 ED3 and (b) Wild type WNV ED3. The plots are symmetric about the diagonal which depicts intra-residue correlation (=1). Positive correlations are shown in magenta and negative correlations in cyan. Plumes of positively correlated off-diagonal elements represent the inter-strand correlated motions in ED3 which is typical of (-sheet proteins.
A common observation from all difference cross-correlation plots was that a mutation in ED3 not only disturbed the cross-correlation coefficients between proximal atoms but also of residues distant from the site of mutation. In the MAb binding resistant mutant of DEN2 ED3, P384G, significant changes in cross-correlation were observed in residues 341-352 of the CE loop (Figure 7a). These residues are approximately 18 away from P384 in the secondary structure of ED3. Hence it is implied that the changes in dynamics seen at the CE loop are not due to local perturbations caused by the mutation. These long-distance perturbations in concerted motions were less significant in the mutation K361G (Figure 7b). The perturbations seen were within 10 of the site of mutation. In most DEN2 ED3 mutants, the CE loop was the most perturbed dynamically. However, the differences in the magnitude of perturbation varied significantly between mutants (Supplementary Figure 3). Since a change in correlation between atoms is a direct consequence of variation in the dynamics of the participating atoms it followed that those mutants whose dynamics were significantly altered as a consequence of the mutation also showed a global increase of cross-correlations. Hence all mutants that had higher values for E1 and E2 also had increased global cross-correlation patterns.
Difference cross-correlation plots for WNV ED3 mutants also showed the presence of local and long-distance cross-correlation changes. Local changes in cross-correlation coefficients were seen in both mutants H320A and T332K (Figure 8). For instance, the resistant mutation T332K is on the BC loop of ED3 at a distance less than 10 from the N-terminal residues K307- K310, which in turn border the adjacent FG loop residues G389-Q391 (Figure 1) in the IgG like fold of WNV ED3. I hence anticipate that a substitution of the Threonine side chain to Lysine at position 332 would sterically hinder the packing of the residues at the interface of BC-loop and the N-terminus, thus indirectly perturbing the packing between the N-terminus and the FG loop (Figure 8, small inset). However, such local structural perturbations could not explain the changes in dynamics at the residues distant from the site of mutation (Figure 8, large inset). The extent and magnitude of long-distance perturbations differed with the nature of the mutation (Supplementary Figure 4); those with higher cumulative proportion of the first two eigenvalues had higher incidence of global dynamic perturbations compared to mutants with smaller cumulative values (Table-2).
In summary, mutants that rendered the ED3 peptide more dynamic (lower half of Table-1, 2) shared a common property of increase in long-distance perturbations in the cross-correlation coefficients and were resistant to MAb binding. These results supported the hypothesis that binding a MAb would result in perturbations in the dynamics of the active site (epitope) causing a concurrent change in dynamics of the interior of the peptide through long-range interactions. This phenomenon could enable the rearrangement of the peptides conformational subspace upon ligand (antibody) binding without drastically altering its topology. These data hence support the theory that the interaction of the domains with antibodies involves not just the physical binding region (epitope) but a network of residues within the domain.
Figure 7: Difference cross-correlation plots: DEN2 ED3
Difference cross-correlation plots of MAb binding resistant mutations of DEN2-ED3 with site of mutations marked in red. A negative difference less than -0.3 could mean either there is an increase in anti-correlated motions or a decrease in correlated motions in general. On the other hand a positive increase in cross-correlation value (greater than +0.3) means that those residues showed a significant increase in correlated motions in the mutant compared to the wild type. Notice that significant perturbations were seen in the global dynamics of the resistant mutant P384G which has higher incidence of large-scale dynamics (from Table-1) compared to the wild type and non-resistant mutant K361G. Secondary structure of DEN2-ED3 has been overlaid on the matrices to the show the spatial location of respective strands and loops.
Figure 8: Difference cross-correlation plots: WNV ED3.
Difference cross-correlation plots of (a) T332K, MAb binding resistant mutation of WNV ED3 and (b) non-resistant mutant H320A with site of mutation marked in red. Secondary structure of WNV-ED3 is overlaid on the plot. A difference of +/-0.4 relative to the cross-correlations in the wild type was considered significant perturbation (See Methods). Large Insets show the regions at farther away (> 10 ) from the site of mutation whose concerted motions are perturbed in the resistant mutation T332K. These perturbations are present to a reduced degree in the non-resistant mutation H320A. Small inset is the local perturbation caused by the T332K mutation between the N-terminus (< 10 from the site of mutation) and the adjacent FG loop.
HIDDEN TEXT: Using Major Sections are option, and most dissertations do not use major sections. If your dissertation is not divided into major sections, then you do not need use the style HEADING 1,H1, start chapters using Heading 2,h2.
Chapter 4: Discussion
4.1 Differences in the conformational freedom of wild type Dengue-2 and West Nile virus envelope ED3s:
A high eigenvalue implies large-scale dominant motions in the direction of the principal component and higher cumulative contribution of the two principal components to the variance in the data indicates higher degree of organization in the dynamics. The cumulative proportion of variance associated with the first two eigenvalues in wild type dengue-2 ED3 is lower compared to the wild type WNV ED3. This suggests that WNV ED3 samples larger dynamic motions; Also that its motions are more organized than those of wild type ED3 of DEN2. This was my evidence that wild type WNV ED3 sampled rather large-scale dynamics compared to the wild type dengue-2 ED3. Projection of the MD trajectories of the wild type DEN2 (Figure 4a) and WNV (Figure 5a) ED3s on to the two-dimensional space described by the two highest principal components supports this interpretation. Whereas the DEN2 ED3 samples a single region in the conformational space, WNV ED3, which has a higher variance along PC 1, is seen sampling multiple states in an equal time of simulation. It has been previously shown that the thermodynamic stability of wild type DEN2 ED3 (7.8 kcal/mol) is almost twice that of wild type WNV ED3 ADDIN EN.CITE Yu2004262617Yu, S.Wuu, A.Basu, R.Holbrook, M. R.Barrett, A. D.Lee, J. C.Department of Human Biological Chemistry & Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1055, USA.Solution structure and structural dynamics of envelope protein domain III of mosquito- and tick-borne flavivirusesBiochemistryBiochemistryBiochemistryBiochemistryBiochemistryBiochemistry9168-764328Amino Acid SequenceAnimalsBinding SitesCulicidae/*virologyFlaviviridae/*chemistryKineticsProtein ConformationReceptors, VirusSequence AlignmentSolubilitySolutionsTicks/*virologyViral Envelope Proteins/*chemistry2004Jul 200006-2960 (Print)15248774http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15248774 eng[18] (3.4 kcal/mol). I speculate that in general, DEN2 ED3 has a lower conformational freedom and hence presumably less dynamic than WNV ED3. Based on above, I infer that differences in binding properties of the homologous domains of DEN2 and WNV could be a manifestation of the differences in the large-scale dynamics sampled by them.
4.2 Perturbation in dynamics caused by resistant mutations of dengue-2 envelope prottein ED3:
Resistance to MAb binding in DEN2 ED3 seems to be a function of the extent of conformational freedom experienced by the mutant ED3. Since all mutations studied in ED3 seem to increase the large-scale dynamics sampled by the domain, I infer that mutations of DEN2 ED3, in general, are destabilizing. However, only those mutations that drastically increase the large-scale fluctuations when compared to the native peptide render the domain resistant to MAb binding. Literature shows that K310 along with the hydrophobic amino acids V308, I312, L387, L389 and W391 forms a MAb binding pocket of dengue virus binding monoclonal antibodies (Figure 1 and entries marked II in Table-1). The resistant mutations in these residues K310G/E, V308A, I312G/F, L387A, L389A and W391G had eigenvalues much higher than the native peptide. Similary residues constituting the virus specific MAb binding site, K305, K307, P332, E383 and P384 (Figure 1 and entries marked I in Table-1) also exhibit drastic increase in their cumulative eigenvalue proportion relative to the wild type.
Several mutations whose cumulative eigenvalue proportions differed significantly from those of the wild type ED3, however, have not been shown to be resistant to MAb binding. Even though this may be contrary to what is expected, the discrepancy is satisfactorily explained by considering the spatial location of the residues in question. For instance K334 in DEN2 ED3 lies adjacent to the disulfide bond C302-C333 ADDIN EN.CITE Rey2003111117Rey, F. A.Virologie Moleculaire et Structurale, Unite Mixte de Recherche 2472/1157, Centre National de la Recherche Scientifique et Institut National de la Recherche Agronomique, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. rey@gv.cnrs-gif.frDengue virus envelope glycoprotein structure: new insight into its interactions during viral entryProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of AmericaProc Natl Acad Sci U S AProceedings of the National Academy of Sciences of the United States of America6899-90110012Carbohydrates/chemistryCrystallography, X-RayDengue Virus/chemistry/*pathogenicity/physiologyDimerizationHumansModels, MolecularProtein Structure, QuaternaryProtein SubunitsViral Envelope Proteins/*chemistry/physiologyVirulence2003Jun 100027-8424 (Print)12782795http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12782795 eng[11]; a deletion of its side chain is hence more likely to cause a destabilization of this bond leading to a high conformational freedom. The residues T303 and P332 also flank this disulfide bond in the structure of DEN2-ED3. The higher value for G381A and G385E could be due to the fact that thes residues lie within the mapped epitope II. The mutations however, were not known to cause a significant change in binding affinity in one set of experiments. The role of G385 in MAb binding, however, has been previously implicated in literature ADDIN EN.CITE Hiramatsu1996383817Hiramatsu, K.Tadano, N.Men, R. Lai, C-J.Mutational Analysis of a Neutralization Epitope on the Dengue Type 2 Virus (DEN2) Envelope Protein: Monoclonal Antibody Resistant DEN2/DEN4 Chimeras Exhibit Reduced Mouse NeurovirulenceVirologyVirologyVirology437-4522421996[35]. Peculiarly the spatial location of the amino acid R345 is completely outside the two mapped MAb binding regions on DEN2 ED3. It is located in the DE loop, which is not exposed to the surface on a mature DEN2 virion. It was hence surprising that the mutation R345A, which did not belong to either epitope, had a relatively high gradient between the eigenvalues compared to the wild type. Interestingly, however, in 1998 Roehrig et. al. had shown that the peptide spanning residues 333 to 351 in DEN2 ED3 is bound by at least one MAb ADDIN EN.CITE ADDIN EN.CITE.DATA [36]. This region is exposed to solution under low pH conditions in the endosome following viral entry into the host cell hence rendering itself susceptible to MAb binding ADDIN EN.CITE Kuhn2002282817Kuhn, R. J.Zhang, W.Rossmann, M. G.Pletnev, S. V.Corver, J.Lenches, E.Jones, C. T.Mukhopadhyay, S.Chipman, P. R.Strauss, E. G.Baker, T. S.Strauss, J. H.Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. rjkuhn@bragg.bio.purdue.eduStructure of dengue virus: implications for flavivirus organization, maturation, and fusionCellCellCellCellCellCell717-251085Capsid/chemistryCryoelectron MicroscopyDengue Virus/*chemistry/genetics/*physiology/ultrastructureHumansHydrogen-Ion ConcentrationImage Processing, Computer-AssistedLipid Bilayers/chemistryMembrane Fusion/physiologyModels, MolecularViral Envelope Proteins/chemistry2002Mar 80092-8674 (Print)11893341http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11893341 eng[37]. I hence may have identified an additional binding hotspot for MAbs specific to this region on the ED3 of DEN2 ADDIN EN.CITE Goncalvez2004292917Goncalvez, A.P.Purcell, R.H.Lai, C-J.Epitope Determinants of a Chimpanzee Fab Antibody That Efficiently Cross-Neutralizes Dengue Type 1 and Type 2 Viruses Map to Inside and in Close Proximity to Fusion Loop of the Dengue Type 2 Virus Envelope GlycoproteinJ. Virol.J. Virol.12919-2878232004[38].
4.3 Perturbation in dynamics caused by resistant mutations of West Nile Virus envelope ED3:
As depicted above, the conformational freedom of the wild type ED3 of WNV is higher than that of DEN2 ED3; It could be possible that mutations in WNV are capable of either increasing or decreasing the magnitude of large-scale dynamic motions whereas in the case of DEN2, they mostly tend to increase these motions possibly destabilizing the domain. Wild type WNV ED3 showed a higher covariation along the first principal component compared to wild type DEN2 ED3 implying that WNV sampled dominant large-scale motions while the dynamics in ED3 of DEN2 were less organized. Though homologous in structure and function, the two domains thus express different dynamic behavior. This difference in dynamic behavior recognized by different MAbs could explain their distinct antibody binding and neutralization properties. However, in general, mutations that deviate significantly from the wild type in terms of the extent of large-scale dynamics seem to be largely resistant to MAb binding. These include the previously mapped epitope residues T332, K307, T366 and T330.
The mutations K310T/G, in spite of perturbing the dynamics of the native protein considerably, have not been shown to alter the binding affinity of WNV ED3 to its MAbs. However, the mutation was isolated in a screen for MAb resistant viruses and renders the virus incapable of infecting the host cell ADDIN EN.CITE ADDIN EN.CITE.DATA [21]. Since ED3 is also the putative cell receptor binding domain ADDIN EN.CITE ADDIN EN.CITE.DATA [9, 39], it may be probable that the mutation though does not interfere with antibody binding could disrupt the binding interface between the host cell surface receptor and the domain. I propose that this residue could indeed perturb the binding between ED3 and its binding ligand. However, the conclusions for WNV ED3 and its mutants can not be reliably substantiated with the present results since the experimental data available for comparison of WNV dynamics is incomplete.
In summary, MAb binding resistant mutations clearly altered the extent of large-scale dynamics sampled by the ED3s whereas the non-resistant mutations retained the distribution of dynamics as in the wild type. I do not know from the present varied data set what exact difference in percentage variance from the wild type would be significant enough to perturb its binding properties. However, using the above analysis, I was qualitatively able to locate the binding hotspots on the ED3s of both DEN2 and WNVes.
4.4 Role of long-distance dynamics in modulating binding resistance in ED3 of flaviviruses:
In the present study I showed that residues in ED3 of DEN2 and WNV are networked through long-distance concerted dynamic motions and hence affect the dynamics collectively and globally when mutated. It was also showed that resistant mutations either increase or decrease the magnitude of large-scale motions globally in the ED3 of flaviviruses. This perturbation in large-scale dynamics is concomitantly followed by a change in inter-residue communication across the peptide. Hence, it is implied that resistant mutations that lead to significant changes in covariation in dynamics also produce higher variations in correlated motions within the domains. I showed that residues constituting the binding sites for MAb binding are seemingly more effective in relaying changes in dynamics to the interior of the protein than other surface exposed residues. Since such residues are analogous to hotspots in any protein-protein interface, I believe that such long-distance communication underlies the conformational rearrangement of a peptide following binding. This property may be universal to all active binding sites.
Appendix A
Supplementary Figures:
Supplementary Figure 1: DEN2 ED3 mutant projection plots
Projection of MD trajectories of mutants of DEN2 ED3 onto the first two principal components. Data points represent the average position described by all backbone CA atoms in the space of the two PCs progressing through the simulation (red to blue). Variance of data in plots is given by the directionality along a principal component. Plots are arranged in the order of increasing value of cumulative percentage variance from E1 and E2 (Table-1). As one progresses down the table, there is an increase in the large-scale internal motions sampled during the time of simulation. Resistant mutations in DEN2 ED3 which alter the wild type large-scale dynamics considerably are hence found towards the end of the panel.
SHAPE \* MERGEFORMAT
SHAPE \* MERGEFORMAT
SHAPE \* MERGEFORMAT
Supplementary Figure 2: WNV ED3 mutant projection plots
Projection of MD trajectories of mutants of WNV ED3 onto the first two principal components. Data points represent the average position described by all backbone CA atoms in the space of the two PCs progressing through the simulation (red to blue). Variance of data in plots is given by the directionality along a principal component. Plots are arranged in the order of increasing value of cumulative percentage variance from E1 and E2 (Table-2). As one progresses down the table, there is an increase in the large-scale internal motions sampled during the time of simulation. Resistant mutations in WNV ED3 were found to be bi-variate. Unlike DEN2 ED3, there were mutations that reduced dominant large-scale motions in the domain as well.
Supplementary Figure 3: Difference cross-correlations plots: DEN2 ED3
Difference cross-correlation plots of mutants of DEN2-ED3 with site of mutations marked in red. A negative difference less than -0.3 could mean either there is an increase in anti-correlated motions or a decrease in correlated motions in general. On the other hand a positive increase in cross-correlation value (greater than +0.3) means that those residues showed a significant increase in correlated motions in the mutant compared to the wild type. Plots are arranged in the increasing order of cumulative percentage of variance from PC1 and PC2 (Table-1). Both local and long-distance perturbations are seen in most mutations. However, significant perturbations in the global long-distance dynamics are observed in mutations which sampled dominant large-scale motions during the simulations. A bulk of these mutations was in residues belonging to experimentally determined MAb binding regions (epitopes). Secondary structure of DEN2-ED3 has been overlaid on the matrices.
SHAPE \* MERGEFORMAT
SHAPE \* MERGEFORMAT
Supplementary Figure 4: Difference cross-correlations plots: WNV ED3
Difference cross-correlation plots of mutants of WNV-ED3 with site of mutations marked in red. A negative difference less than -0.4 could mean either there is an increase in anti-correlated motions or a decrease in correlated motions in general. On the other hand a positive increase in cross-correlation value (greater than +0.4) means that those residues showed a significant increase in correlated motions in the mutant compared to the wild type. Plots are arranged in the increasing order of cumulative percentage of variance from PC1 and PC2 (Table-2). Once again, all mutants display local and long-distance perturbations in cross-correlations to some extent. An increase in large scale dynamics (higher E1 and E2 values in Table-2) was coincident with significant perturbations in long-distance cross-correlations. Secondary structure of WNV-ED3 is overlaid on the plot.
Appendix B
C.1 Amber-9 Name List for Energy Minimization:
Initial minimization w/ position restraints on system:
ntx = 1, irest = 0, ntrx = 1, ntxo = 1,
ntpr = 10, ntwx = 0, ntwv = 0, ntwe = 0,
ntf = 1, ntb = 0,
igb = 5, saltcon= 0.2, gbsa = 1,
cut = 999, rgbmax = 999, nsnb = 10,
imin = 1,
maxcyc = 1000,
ncyc = 5000,
ntmin = 1, dx0 = 0.1, drms = 0.0001,
nscm = 0,
t = 0.0, dt = 0.0015, nrespa = 1,
temp0 = 300.0, tempi = 300.0,
ig = 1190146579,
ntt = 0,
tautp = 0.5,
vlimit = 20.0,
ntc = 1, tol = 0.0005,
ntr = 1, RESTRAINT_WT=500.0, RESTRAINTMASK=":1-101",
/
C.2 Amber-9 Name List for Equilibration:
Initial ntt=3 dynamics on system:
ntx = 1, irest = 0, ntrx = 1, ntxo = 1,
ntpr = 100, ntwx = 500, ntwv = 0, ntwe = 0,
ntf = 2, ntb = 0,
igb = 5, saltcon= 0.2, gbsa = 1,
cut = 16, rgbmax = 16, nsnb = 25,
imin = 0,
nstlim = 15000,
nscm = 0,
t = 0.0, dt = 0.0015, nrespa = 2,
temp0 = 300.0, tempi = 300.0,
ig = 1190146579,
ntt = 3, gamma_ln = 1.0,
vlimit = 20.0,
ntp = 0,
ntc = 2, tol = 0.00001,
Relax the structures for 225 ps total:
ntx = 5, irest = 1, ntrx = 1, ntxo = 1,
ntpr = 100, ntwx = 500, ntwv = 0, ntwe = 0,
ntf = 2, ntb = 0,
igb = 5, saltcon= 0.2, gbsa = 1,
cut = 16, rgbmax = 16, nsnb = 25,
nstlim = 150000,
dt = 0.0015, nrespa = 2,
temp0 = 300.0, tempi = 300.0,
ig = 1190146579,
ntt = 3, gamma_ln = 1.0,
tautp = 0.5,
vlimit = 15.0,
ntc = 2,
/
C.3 Amber-9 Name List for MD Production Run:
Production dynamics
ntx = 5, irest = 1, ntrx = 1, ntxo = 1,
ntpr = 100, ntwx = 500, ntwv = 0, ntwe = 0,
ntf = 2, ntb = 0,
igb = 5, saltcon= 0.2, gbsa = 1,
cut = 16.0, rgbmax = 16.0, nsnb = 25,
nstlim = 1500000,
t = 0.0, dt = 0.0015, nrespa = 2,
temp0 = 300.0, tempi = 300.0,
ig = 1190208013,
ntt = 3, gamma_ln = 1.0,
vlimit = 15.0,
ntc = 2,
/
HIDDEN TEXT: Optionalmust be placed in this order if it is included in the dissertation. If you dont want to include a glossary, then delete the entire page and the following page break.
REFERENCES
ADDIN EN.REFLIST 1. J. G. Estrada-Franco et al., West Nile virus in Mexico: evidence of widespread circulation since July 2002. Emerg. Infect. Dis., 2003. 9(12),p. 1604-07.
2. S. B. Halstead. Pathogenesis of dengue: challenges to molecular biology. Science, 1988. 239(4839),p. 476-81.
3. T. P. Monath. Dengue: the risk to developed and developing countries. Proc. Nat. Acad. Sci., 1994. 91(7),p. 2395-400.
4. H. G. Zeller and I. Schuffenecker. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas. . Eur J Clin Microbiol Infect Dis, 2004. 23(3),p. 147-56.
5. T. J. Chambers et al., Flavivirus genome organization, expression, and replication. Annu Rev Microbiol, 1990. 44(649-88.
6. B. Murgue et al., The ecology and epidemiology of West Nile virus in Africa, Europe and Asia. Curr Top Microbiol Immunol, 2002. 267(195-221.
7. A. D. Barrett. Current status of flavivirus vaccines. Ann N Y Acad Sci, 2001. 951(262-71.
8. T. J. Chambers et al., Yellow Fever Virus/Dengue-2 Virus and Yellow Fever Virus/Dengue-4 Virus Chimeras: Biological Characterization, Immunogenicity, and Protection against Dengue Encephalitis in the Mouse Model. J. Virol., 2003. 77(6),p. 3655-68.
9. Y. Modis et al., A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci U S A, 2003. 100(12),p. 6986-91.
10. F. A. Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature, 1995. 375(6529),p. 291-8.
11. F. A. Rey. Dengue virus envelope glycoprotein structure: new insight into its interactions during viral entry. Proc Natl Acad Sci U S A, 2003. 100(12),p. 6899-901.
12. J. T. Roehrig. Antigenic structure of flavivirus proteins. Adv Virus Res, 2003. 59(141-75.
13. W. D. Crill and J. T. Roehrig. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol, 2001. 75(16),p. 7769-73.
14. D. W. Beasley and A. D. Barrett. Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. J Virol, 2002. 76(24),p. 13097-100.
15. G. D. Gromowski and A. D. Barrett. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology, 2007. 366(2),p. 349-60.
16. G. E. Nybakken et al., Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature, 2005. 437(7059),p. 764-9.
17. D. E. Volk et al., Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus. J Biol Chem, 2004. 279(37),p. 38755-61.
18. S. Yu et al., Solution structure and structural dynamics of envelope protein domain III of mosquito- and tick-borne flaviviruses. Biochemistry, 2004. 43(28),p. 9168-76.
19. G. D. Gromowski et al., Characterization of dengue virus complex-specific neutralizing epitopes on envelope protein domain III of dengue 2 virus. J Virol, 2008. 82(17),p. 8828-37.
20. O. Lisova et al., Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus. J Gen Virol, 2007. 88(Pt 9),p. 2387-97.
21. L. Li et al., Differential expression of domain III neutralizing epitopes on the envelope proteins of West Nile virus strains. Virology, 2005. 335(1),p. 99-105.
22. A. Zhuravleva et al., Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study. J Mol Biol, 2007. 367(4),p. 1079-92.
23. P. De Los Rios et al., Functional dynamics of PDZ binding domains: a normal-mode analysis. Biophys J, 2005. 89(1),p. 14-21.
24. A. Dhulesia et al., Mapping of two networks of residues that exhibit structural and dynamical changes upon binding in a PDZ domain protein. J Am Chem Soc, 2008. 130(28),p. 8931-9.
25. A. Suenaga et al., Tyr-317 phosphorylation increases Shc structural rigidity and reduces coupling of domain motions remote from the phosphorylation site as revealed by molecular dynamics simulations. J Biol Chem, 2004. 279(6),p. 4657-62.
26. R. A. Maillard et al., Long range communication in the envelope protein domain III and its effect on the resistance of West Nile virus to antibody-mediated neutralization. J Biol Chem, 2008. 283(1),p. 613-22.
27. D. A. Case et al., The Amber biomolecular simulation programs. J Comput Chem, 2005. 26(16),p. 1668-88.
28. C. Simmerling et al., All-atom structure prediction and folding simulations of a stable protein. J Am Chem Soc, 2002. 124(38),p. 11258-9.
29. W. C. Still, Tempczyk, A., Hawly, R. C., Hendrickson, T., Semianalytical Treatment of Solvation for Molecular Mechanics and Dynamics. Journal of American Chemical Society, 1990. 112(6127-29.
30. J. Srinivasan, Trevathan, M. W., Beroza, P., Case, D. A., Application of a Pairwise Generalized Born Model to Proteins and Nucleic Acids: Inclusion of Salt Effects Theoretical Chemical Accounts, 1999. 101(426-34.
31. X. Wu, Brooks, B. R., Self-Guided Langevin Dynamics Simulation Method. Chemical Physical Letters 2003. 381(512-18.
32. A. E. Garcia. Large-amplitude nonlinear motions in proteins. Phys Rev Lett, 1992. 68(17),p. 2696-2699.
33. S. Swaminathan, Harte, W. E. Jr., Beveridge, D. L., Investigation of Domain Structure in Proteins via Molecular Dynamics Simulation: Application to HIV-1 Protease Dimer. Journal of American Chemical Society, 1991. 113(7),p. 2717-21.
34. P. H. Hunenberger et al., Fluctuation and cross-correlation analysis of protein motions observed in nanosecond molecular dynamics simulations. J Mol Biol, 1995. 252(4),p. 492-503.
35. K. Hiramatsu et al., Mutational Analysis of a Neutralization Epitope on the Dengue Type 2 Virus (DEN2) Envelope Protein: Monoclonal Antibody Resistant DEN2/DEN4 Chimeras Exhibit Reduced Mouse Neurovirulence. Virology, 1996. 224(2),p. 437-45.
36. J. T. Roehrig et al., Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology, 1998. 246(2),p. 317-28.
37. R. J. Kuhn et al., Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell, 2002. 108(5),p. 717-25.
38. A. P. Goncalvez et al., Epitope Determinants of a Chimpanzee Fab Antibody That Efficiently Cross-Neutralizes Dengue Type 1 and Type 2 Viruses Map to Inside and in Close Proximity to Fusion Loop of the Dengue Type 2 Virus Envelope Glycoprotein. J. Virol., 2004. 78(23),p. 12919-28.
39. F. A. Rey et al., The envelope glycoprotein from tick-borne encephalitis virus at 2 resolution. Nature, 1995. 375( ),p. 291-98.
Vita
HIDDEN TEXT: Note that no page numbers appear on Vita pages, though they are included in counting.
Keerthi Gottipati is the youngest of three children born to Dr. Ranga Rao Gottipati and wife Dr. Raghava Rani Gottipati. She was born on the 22nd of August 1983 in the city of Hyderabad in India. St. Anns High School, Secunderabad was her Alma Mater for twelve years through Secondary School from where she graduated with distinction in 1999. She attended Junior college from 1999 to 2001 at St. Alphonsas Junior College. She graduated with Bachelors degree in Industrial Biotechnology from Anna University, Chennai, India in 2005 soon after which she joined UTMB for her Graduate studies.
Permanent address:
#408, Bungalow-41, Aakruti Nivas, Sanathnagar, Hyderabad, AP 500018
This dissertation was typed by Keerthi Gottipati.
N
P
! " # $ < = ? @ T U W X l n o p r Q
n
n t c d x ܹ h% h' h% huY h% h h% h0/h h% hD; h% hD; OJ QJ ]h% h* CJ OJ QJ h% hz h% h6z\ h% h6z\ 5h% h* 5h% h* OJ QJ ]h% h* CJ h% h* 6 ( - . N
O
P
x^x ' ' dh # $ a$ gd: f ! " # $ = > ? u <