Different sensitivities to acid denaturation within a family of proteins: implications for acid unfolding and membrane translocation.
Colicins A, B, and N form a family of membrane pore-forming toxins with > 50% sequence identity in their toxic C-terminal domains. The colicin A C-terminal domain has been shown to insert into model membranes via an acidic molten-globule insertion intermediate, and thus this family provides a means to compare acid unfolding of related proteins. Unlike the domains of colicins A and B which are acidic, that of colicin N is very basic with fewer Asp and Glu residues. If surface positive charge density is the crucial factor in acidic molten globule formation, colicin N should begin to unfold at higher pH values than colicins A or B. However, comparison of their CD spectra reveals that colicins A and B both form acidic molten globules but colicin N does not. None of the proteins forms a denaturant-induced molten globule at neutral pH where the proteins exhibit very similar stabilities. The acidic unfolding cannot therefore be due to excess positive surface charge and may be caused by a subset of acidic residues as has been predicted for myoglobin. The difference between the colicins is confirmed by their in vivo membrane insertion, with colicins A and B inserting much faster than colicin N. Stopped-flow circular dichroism measurements of colicin A insertion into vesicles confirmed that a molten globule insertion intermediate occurs at the membrane surface.
Evans
L J
LJ
Department of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle-upon-Tyne, England.
Goble
M L
ML
Hales
K A
KA
Lakey
J H
JH
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature
D003087:Colicins