PEPTIDE VIBRATIONAL SPECTROSCOPIC STUDIES AS MODELS FOR PROTEIN FOLDING EVENTS

Silva, R. A. Gangani D.

ABSTRACT

Human Chorionic Gonadotropin (hCG) is a hormone important in maintaining early stages of pregnancy. This is also used as a marker for detecting certain types of cancers. Here we report folding studies of the three hairpin loops of the -subunit of hCG using complementary optical spectroscopic techniques: electronic circular dichroism (ECD), vibrational circular dichroism (VCD) and infrared absorbance (IR). Our studies, which exposed the peptides to different environments, brought out their hidden intrinsic propensities towards particular secondary structures. Accounting for the clear variance in propensities of these peptides, a novel folding mechanism was proposed for hCG subunit folding; according to which the formation of first hairpin (H1) hydrophobically collapses with the second sequence (H3), facilitating formation of that hairpin and finally locking the third sequence (H2) into a loop.

The thesis next focuses on the most common secondary structural type in proteins, the -helix. Segment specific stability of a selected -helix was analyzed by using five model peptides, Ac-(AAAAK)₃AAAAY-NH₂ with ¹³C labels introduced onto selected consecutive Ala residues at the backbone carbonyls. VCD together with IR spectra for the amide I region of these peptides in D₂O at 5 ^oC showed a difference in structure for the labeled segments of the peptide, particularly with regard to the ordering of the N-terminus compared to the disorder of the C-terminus. The temperature dependent VCD and IR spectra gave information about the melting behavior of different segments of the peptide. Spectral data analyses showed different transition temperatures for differently labeled peptides. Our study opened a new window for identifying segments in a secondary structure by promoting VCD into a true segment-specific conformational analysis tool.

Another study was carried out to identify the development of 3₁₀-helices, using model peptides containing high percentages of (-Me)Ala residues, which are known to promote 3₁₀-helices. Our results showed that in many cases it was not possible to distinguish 3₁₀- from - conformations by ECD data alone but that additional data from VCD is very useful for such interpretations. Further, we have shown that none of the factors as solvent, concentration, length, amphipathy can be considered as a unique factor for stabilizing a 3₁₀-helical conformation.