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Description:
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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 . |