Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor.
Biophysical study of the transmembrane (TM) domains of integral membrane proteins has traditionally been impeded by their hydrophobic nature. As a result, an understanding of the details of protein-protein interactions within membranes is often lacking. We have demonstrated previously that model TM segments with flanking cationic residues spontaneously fold into alpha-helices upon insertion into membrane-mimetic environments. Here, we extend these studies to investigate whether such constructs consisting of TM helices from biological systems retain their native secondary structures and oligomeric states. Single-spanning TM domains from the epidermal growth factor receptor (EGFR), glycophorin A (GPA), and the influenza A virus M2 ion channel (M2) were designed and synthesized with three to four lysine residues at both N- and C-termini. Each construct was shown to adopt an alpha-helical conformation upon insertion into sodium dodecyl sulfate micelles. Furthermore, micelle-inserted TM segments associated on SDS-PAGE gels according to their respective native-like oligomeric states: EGFR was monomeric, GPA was dimeric, and M2 was tetrameric. This approach was then used to investigate whether one or both of the TM segments (Tar-1 and Tar-2) from the Escherichia coli aspartate receptor were responsible for its homodimeric nature. Our results showed that Tar-1 formed SDS-resistant homodimers, while Tar-2 was monomeric. Furthermore, no heterooligomerization between Tar-1 and Tar-2 was detected, implicating the Tar-1 helix as the oligomeric determinant for the Tar protein. The overall results indicate that this approach can be used to elucidate the details of TM domain folding for both single-spanning and multispanning membrane proteins.
Melnyk
R A
RA
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Partridge
A W
AW
Deber
C M
CM
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D004926:Escherichia coli; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017459:Receptors, Amino Acid / Q000737:chemistry*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D017459:Receptors, Amino Acid; C031959:aspartic acid receptor