On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins

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Title: On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins
Author: Miles, Jr., George Emmett
Abstract: Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides , one of class F and one of class S , making them unique in the family of & #946 ; -barrel pore -forming toxins ( & #946 ; -PFTs ) . By contrast , other & #946 ; -PFTs form homooligomeric pores . For example , the staphylococcal & #945 ; - hemolysin is a homoheptamer . Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore . In this work , biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB ) and LukS (HlgC ) components encoded by Staphylococcus aureus . I demonstrate that LukF and LukS assemble to form an SDS -stable pore on rabbit erythrocyte membranes . In addition , the pore -forming properties of recombinant leukocidin were investigated with planar lipid bilayers . Although leukocidins and staphylococcal & #945 ; -hemolysin share partial sequence identity and related folds , LukF and LukS produce a pore with a unitary conductance of 2 .5 nS (1 M KCl , 5 mM HEPES , pH 7 .4 ) , which is over three times greater than that of & #945 ; -hemolysin measured under the same conditions . The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods , gel shift electrophoresis and sitespecific chemical modification during single channel recording . Four LukF and four LukS subunits were shown to co -assemble into an octameric transmembrane structure . The existence of an additional subunit in part explains properties of the leukocidin pore , such as its high conductance . Additionally , this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein . It is also relevant to understanding the mechanism of assembly of & #946 ; -PFT pores , and suggests new possibilities for engineering these proteins . In additional studies , the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of & #946 ; -PFTs . HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology , such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials .
URI: http : / /hdl .handle .net /1969 .1 /3952
Date: 2006-08-16

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On the structure and assembly of staphylococcal leukocidin: a study of the molecular architecture of beta-barrel pore-forming toxins. Available electronically from http : / /hdl .handle .net /1969 .1 /3952 .

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