Transmembrane region of wild-type and mutant M13 coat proteins. Conformational role of beta-branched residues.
Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.
Deber
C M
CM
Division of Biochemistry Research, Hospital for Sick Children, Toronto, Canada.
Li
Z
Z
Joensson
C
C
Glibowicka
M
M
Xu
G Y
GY
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003090:Coliphages / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation