CONTRIBUTION OF INCREASED LENGTH AND INTACT CAPPING SEQUENCES TO THE CONFORMATIONAL PREFERENCE FOR HELIX IN A 31-RESIDUE PEPTIDE FROM THE C-TERMINUS OF MYOHEMERYTHRIN

In order to examine the effects of chain length on the propensity of s hort peptides to form helix-like structures in aqueous solution, we ha ve studied a peptide of 31 residues consisting of the C-terminal seque nce (residues 88-118) of the four-helix bundle protein myohemerythrin from Themiste zostericola. This peptide, termed MDC, represents the fi nal two elements of secondary structure in the protein, the D-helix an d the C-terminal loop sequence, together with a five-residue sequence at the N terminus corresponding to the linker between the C- and D-hel ices. An N-capping sequence, VDAKNV, immediately precedes the D-helix sequence, and a C-capping sequence, VNHIKGT, corresponding to the alph a(L) termination motif, occurs at the C-terminal end. The effect of re placement of a cysteine residue in the middle of the sequence with an alanine was explored by the comparison of the MDC peptide and a 16-res idue peptide representing the sequence of the D-helix alone, both cont aining the change Cys99Ala. Significant changes in the NMR and CD spec tra were seen for both peptides compared to the wild-type sequence. A comparison of the fluorescence spectra of the wild-type and Cys99Ala p eptides indicated that a specific interaction between the side chains of Cys 99 and Trp 102 acts to quench the fluorescence of the tryptopha n ring and probably contributes a component that distorts the CD spect rum of the wildtype peptide at similar to 220-235 nm. The effect of an increase in the length of the peptide, with the incorporation of capp ing sequences derived from the native sequence, was explored by NMR an d CD spectroscopy of the 31-residue and 16-residue peptides in aqueous solution and in TFE/water mixtures. Evidence for the formation of a s ignificant population of helical conformers in the region of the MDC p eptide corresponding to the D-helix was observed in aqueous solution u sing CD and NMR spectroscopy. The C-terminal 10 residues of the MDC pe ptide behave in solution in a manner identical to that of a 10-residue peptide with the same sequence; a highly specific local interaction b etween an aromatic ring and a glycine amide proton appears to be retai ned in the longer peptide. Upon addition of trifluoroethanol (TFE), si gnificant shifts are observed in a number of resonances in the NMR spe ctrum, and both chemical shifts and NOEs provide evidence for a higher population of helix in the D-helix region of the peptide in TFE. Howe ver, TFE is unable to promote the propagation of helix beyond the N-ca p or alpha(L) termination motifs, and the specific local interaction o bserved in the C-terminal sequence is retained in TFE. The CD spectrum in TFE shows an increase in the proportion of helix, to an overall ma ximum of approximately 55% helix at 50% v/v TFE, corresponding to appr oximately 100% helix in the D-helix sequence of the peptide, since the N and C termini of the MDC peptide are not helical according to the N MR spectra. The high proportion of helix observed in the D-helix seque nce of the longer MDC peptide demonstrates that the presence of intact capping sequences can constrain the peptide conformational ensemble t o resemble that seen in the native protein. A compendium of results fr om this and previous peptide studies has also led to a novel observati on, the existence of a correlation between the amide proton chemical s hift and temperature coefficient.