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Description:
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A thermodynamic study of five homologous HPr proteins derived from organisms
inhabiting diverse environments has been undertaken . The aim of this study was to further
our understanding of protein stabilization in extremes of environment . Two of the proteins
were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus
staerothermophilus ) and two from haloalkaliphilic organisms (Bacillus halodurans and
Oceanobacillus iheyensis ) ; these proteins were compared with HPr from the mesophile Bacillus
subtilus . Genes for three of these homologous HPr proteins were for the first time cloned
from their respective organisms into expression vectors and they were over -expressed and
purified in Escherichia coli . Stability measurements were performed on these proteins under a
variety of solution conditions (varying pH , salinity and temperature ) by thermal and solvent
induced denaturation experiments . Stability curves were determined for every homologue
and these reveal very similar conformational stability for these homologues at their
habitat temperatures . The BstHPr homologue is the most thermostable and also has the
highest G25 ; the stability of other homologues was ranked as Bst >Bh >St >Bs >OiHPr .
Other key thermodynamic parameters , like Cp , have been estimated for all the homologues and it was found that these values are identical within errors of estimation . Also , it was found that the values of TS are very similar for these homologues . Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm . The crystal structures of the BstHPr and a single tryptophan -containing variant (BstF29W ) of this homologue are also reported here . Also reported is a domain -swapped dimeric structure for the BstF29W variant , together with a detailed investigation into the
solution oligomeric nature of this protein . The crystal structure of BstHPr is analyzed to
enumerate various stabilizating interactions like hydrogen bonds and salt -bridges and these were compared with those for the mesophilic homologue BsHPr . Finally , an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr . A detailed thermodynamic study of
these variants is presented in an attempt to understand the origins of the differences in
stability of the HPr homologues . |