Peptide Mimics of the M13 Coat Protein Transmembrane Segment

Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced α-helix → β-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24–42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight “two in-two out” dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis. Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced α-helix → β-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24–42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight “two in-two out” dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis. transmembrane wild type tricarboxyethylphosphine hydrochloride lysophosphatidylcholine deoxycholate Observations that excised or co-expressed fragments of the transmembrane (TM)1 segments of multi-spanning membrane proteins can reconstitute to a functional form (1.Kahn T.W. Engelman D.M. Biochemistry. 1992; 31: 6144-6151Crossref PubMed Scopus (144) Google Scholar, 2.Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 3.Groves J.D. Tanner M.J.A. J. Biol. Chem. 1995; 270: 9097-9105Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) have led to the hypothesis that such segments constitute independent folding domains that can be elaborated from the properties of individual TM helices (4.Popot J.L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (818) Google Scholar, 5.Deber C.M. Goto N.K. Nat. Struct. Biol. 1996; 3: 815-818Crossref PubMed Scopus (66) Google Scholar). The task of their structural analysis can thus be essentially reduced to the examination of tertiary contacts between membrane-embedded helices. Therefore, it appears valid to chose a hairpin (helix-loop-helix) structure as the minimal tertiary contact unit of analysis for determination of the extent and nature of helix-helix interactions that may arise in polytopic membrane proteins. For such analyses, studies with de novo designed peptides have demonstrated the feasibility of generating milligram amounts of synthetic TM segments using peptide constructs carrying terminal positive charges (6.Liu L.P. Li S.C. Goto N.K. Deber C.M. Biopolymers. 1996; 39: 465-470Crossref PubMed Scopus (68) Google Scholar, 7.Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (101) Google Scholar). As well, synthetic peptides allow for wide mutation possibilities without complications from requirements for cell viability.To explore the properties of isolated TM segments in a systematic manner, we have chosen M13 major coat protein for a model study. This 50-residue protein (8.Nakashima Y. Frangione B. Wiseman R.L. Konigsberg W.H. J. Biol. Chem. 1981; 256: 5792-5797Abstract Full Text PDF PubMed Google Scholar) is found in the capsid of filamentous coliphage M13 (9.Russel M. Mol. Microbiol. 1991; 5: 1607-1613Crossref PubMed Scopus (155) Google Scholar). During phage assembly, the α-helical M13 coat proteins participate in an oligomerization process within Escherichia coli plasma membrane, which ultimately leads to the encapsulation of viral DNA (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 11.Spruijt R.B. Hemminga M.A. Biochemistry. 1991; 30: 11147-11154Crossref PubMed Scopus (38) Google Scholar). In vivo, the coat protein is expected to be oligomeric during the life cycle of M13, with the minimal structure of such oligomers being a dimer (12.Haigh N.G. Webster R.E. J. Mol. Biol. 1998; 279: 19-29Crossref PubMed Scopus (30) Google Scholar). However, in vitro the oligomerization state of the isolated WT M13 coat protein (and its relative, IKe coat protein) on SDS-polyacrylamide gel electrophoresis and other gels have been variously reported as monomers (13.McDonnell P.A. Shon K. Kim Y. Opella S.J. J. Mol. Biol. 1993; 233: 447-463Crossref PubMed Scopus (145) Google Scholar, 14.Williams K.A. Farrow N.A. Deber C.M. Kay L.E. Biochemistry. 1996; 35: 5145-5157Crossref PubMed Scopus (69) Google Scholar, 15.Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar) or dimers (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 12.Haigh N.G. Webster R.E. J. Mol. Biol. 1998; 279: 19-29Crossref PubMed Scopus (30) Google Scholar, 16.Makino S. Woolford Jr., J.L. Tanford C. Webster R.E. J. Biol. Chem. 1975; 250: 4327-4332Abstract Full Text PDF PubMed Google Scholar, 17.Cavalieri S.J. Goldthwait D.A. Neet K.E. J. Mol. Biol. 1976; 102: 713-722Crossref PubMed Scopus (27) Google Scholar), with the observed state a sensitive function of micelle concentrations, ionic strength, and pH (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 18.Stopar D. Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1997; 36: 12268-12275Crossref PubMed Scopus (18) Google Scholar). That the wild type oligomeric state is condition-dependent suggests the likelihood of equilibration between various states but generally in the direction away from tight dimer formation to allow further propagation of oligomers.Previous mutagenesis studies on the M13 coat protein demonstrated that various Val → Ala mutations in the TM region have contrasting position-dependent effects on its helical stabilities. In particular, mutant V31A acquired an unusually large amount of thermal stability from this mutation, whereas the V29A mutant behaved very similarly to the WT (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar). Based on the mutagenesis results, the oligomerization-promoting helical interface of M13 coat protein was postulated to have a “two in-two out” motif24YAWAMVVVIVGATIGIKLF42(residues in the helix-helix interface are underlined) (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar) conceptually analogous to that of the glycophorin A dimer (20.Lemmon M.A. Treutlein H.R. Adams P.D. Brünger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (295) Google Scholar, 21.MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (868) Google Scholar).As indicated above, M13 coat protein presents the advantage as a model system that helix-helix interactions can be assessed by the propensity of the micelle-embedded M13 protein to undergo a thermally induced α-helix → β-sheet transition (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar). In such experiments, heating disrupts both helix-membrane and helix-helix interactions, eventually allowing the TM segments to escape the micelle into the aqueous environment, where they aggregate rapidly to form β-sheets because of their extreme hydrophobic nature. Although the specific type and aggregation state of this β-form of M13 coat protein are not precisely known, Hemminga and colleagues (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar) have reported that the β-aggregate consists of 440 strands upon heating WT M13 coat protein to 55 °C in 10 mm cholate. Thus, the clear β-sheet diagnostic CD spectra provide a useful assay for aggregation of the nascent water-based form of the protein as driven by the hydrophobic effect. Because helix-helix interactions become important only when relatively strong noncovalent dimers/oligomers pre-exist in the micelle, thermal stability can be used as a parameter to assess the sequence dependence of the intrinsic strength of inter-helical interactions.Based upon this overall knowledge of WT and mutant M13 coat proteins, we have de novo designed and synthesized a series of peptides which correspond to the presumed transmembrane segment (residues 24–42) of the M13 coat protein. If TM helices act as autonomous folding domains, with self-contained recognition faces that guide helix-helix packing, we are in a position to test the hypothesis that peptides comprised of isolated TM sequences should exhibit properties similar to those characteristic of the corresponding segments within the intact proteins. Observations that excised or co-expressed fragments of the transmembrane (TM)1 segments of multi-spanning membrane proteins can reconstitute to a functional form (1.Kahn T.W. Engelman D.M. Biochemistry. 1992; 31: 6144-6151Crossref PubMed Scopus (144) Google Scholar, 2.Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 3.Groves J.D. Tanner M.J.A. J. Biol. Chem. 1995; 270: 9097-9105Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) have led to the hypothesis that such segments constitute independent folding domains that can be elaborated from the properties of individual TM helices (4.Popot J.L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (818) Google Scholar, 5.Deber C.M. Goto N.K. Nat. Struct. Biol. 1996; 3: 815-818Crossref PubMed Scopus (66) Google Scholar). The task of their structural analysis can thus be essentially reduced to the examination of tertiary contacts between membrane-embedded helices. Therefore, it appears valid to chose a hairpin (helix-loop-helix) structure as the minimal tertiary contact unit of analysis for determination of the extent and nature of helix-helix interactions that may arise in polytopic membrane proteins. For such analyses, studies with de novo designed peptides have demonstrated the feasibility of generating milligram amounts of synthetic TM segments using peptide constructs carrying terminal positive charges (6.Liu L.P. Li S.C. Goto N.K. Deber C.M. Biopolymers. 1996; 39: 465-470Crossref PubMed Scopus (68) Google Scholar, 7.Liu L.P. Deber C.M. Biopolymers. 1998; 47: 41-62Crossref PubMed Scopus (101) Google Scholar). As well, synthetic peptides allow for wide mutation possibilities without complications from requirements for cell viability. To explore the properties of isolated TM segments in a systematic manner, we have chosen M13 major coat protein for a model study. This 50-residue protein (8.Nakashima Y. Frangione B. Wiseman R.L. Konigsberg W.H. J. Biol. Chem. 1981; 256: 5792-5797Abstract Full Text PDF PubMed Google Scholar) is found in the capsid of filamentous coliphage M13 (9.Russel M. Mol. Microbiol. 1991; 5: 1607-1613Crossref PubMed Scopus (155) Google Scholar). During phage assembly, the α-helical M13 coat proteins participate in an oligomerization process within Escherichia coli plasma membrane, which ultimately leads to the encapsulation of viral DNA (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 11.Spruijt R.B. Hemminga M.A. Biochemistry. 1991; 30: 11147-11154Crossref PubMed Scopus (38) Google Scholar). In vivo, the coat protein is expected to be oligomeric during the life cycle of M13, with the minimal structure of such oligomers being a dimer (12.Haigh N.G. Webster R.E. J. Mol. Biol. 1998; 279: 19-29Crossref PubMed Scopus (30) Google Scholar). However, in vitro the oligomerization state of the isolated WT M13 coat protein (and its relative, IKe coat protein) on SDS-polyacrylamide gel electrophoresis and other gels have been variously reported as monomers (13.McDonnell P.A. Shon K. Kim Y. Opella S.J. J. Mol. Biol. 1993; 233: 447-463Crossref PubMed Scopus (145) Google Scholar, 14.Williams K.A. Farrow N.A. Deber C.M. Kay L.E. Biochemistry. 1996; 35: 5145-5157Crossref PubMed Scopus (69) Google Scholar, 15.Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar) or dimers (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 12.Haigh N.G. Webster R.E. J. Mol. Biol. 1998; 279: 19-29Crossref PubMed Scopus (30) Google Scholar, 16.Makino S. Woolford Jr., J.L. Tanford C. Webster R.E. J. Biol. Chem. 1975; 250: 4327-4332Abstract Full Text PDF PubMed Google Scholar, 17.Cavalieri S.J. Goldthwait D.A. Neet K.E. J. Mol. Biol. 1976; 102: 713-722Crossref PubMed Scopus (27) Google Scholar), with the observed state a sensitive function of micelle concentrations, ionic strength, and pH (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar, 18.Stopar D. Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1997; 36: 12268-12275Crossref PubMed Scopus (18) Google Scholar). That the wild type oligomeric state is condition-dependent suggests the likelihood of equilibration between various states but generally in the direction away from tight dimer formation to allow further propagation of oligomers. Previous mutagenesis studies on the M13 coat protein demonstrated that various Val → Ala mutations in the TM region have contrasting position-dependent effects on its helical stabilities. In particular, mutant V31A acquired an unusually large amount of thermal stability from this mutation, whereas the V29A mutant behaved very similarly to the WT (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar). Based on the mutagenesis results, the oligomerization-promoting helical interface of M13 coat protein was postulated to have a “two in-two out” motif24YAWAMVVVIVGATIGIKLF42(residues in the helix-helix interface are underlined) (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar) conceptually analogous to that of the glycophorin A dimer (20.Lemmon M.A. Treutlein H.R. Adams P.D. Brünger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (295) Google Scholar, 21.MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (868) Google Scholar). As indicated above, M13 coat protein presents the advantage as a model system that helix-helix interactions can be assessed by the propensity of the micelle-embedded M13 protein to undergo a thermally induced α-helix → β-sheet transition (19.Deber C.M. Khan A.R. Li Z. Joensson C. Glibowicka M. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11648-11652Crossref PubMed Scopus (89) Google Scholar). In such experiments, heating disrupts both helix-membrane and helix-helix interactions, eventually allowing the TM segments to escape the micelle into the aqueous environment, where they aggregate rapidly to form β-sheets because of their extreme hydrophobic nature. Although the specific type and aggregation state of this β-form of M13 coat protein are not precisely known, Hemminga and colleagues (10.Spruijt R.B. Wolfs C.J. Hemminga M.A. Biochemistry. 1989; 28: 9158-9165Crossref PubMed Scopus (73) Google Scholar) have reported that the β-aggregate consists of 440 strands upon heating WT M13 coat protein to 55 °C in 10 mm cholate. Thus, the clear β-sheet diagnostic CD spectra provide a useful assay for aggregation of the nascent water-based form of the protein as driven by the hydrophobic effect. Because helix-helix interactions become important only when relatively strong noncovalent dimers/oligomers pre-exist in the micelle, thermal stability can be used as a parameter to assess the sequence dependence of the intrinsic strength of inter-helical interactions. Based upon this overall knowledge of WT and mutant M13 coat proteins, we have de novo designed and synthesized a series of peptides which correspond to the presumed transmembrane segment (residues 24–42) of the M13 coat protein. If TM helices act as autonomous folding domains, with self-contained recognition faces that guide helix-helix packing, we are in a position to test the hypothesis that peptides comprised of isolated TM sequences should exhibit properties similar to those characteristic of the corresponding segments within the intact proteins. We are grateful to Alex Brünger and Paul Adams (Yale University) for providing us with the global conformation search program for the simulation of M13 dimers. We acknowledge Nam Wang and Rey Interior at the Hospital for Sick Children Biotechnology Service Center for participation in peptide synthesis and amino acid analysis. We thank Avi Chakrabartty, Alan Davidson, Li-Ping Liu, and Chris Yuen for helpful discussions.

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