This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The structure, stability and dynamics of peptides and proteins depend on both the folded and unfolded states. These states are linked both thermodynamically and kinetically. The unfolded state of proteins used to be characterized as a random coil; however, recent studies have shown the presence of significant interactions including residual local secondary structure (such as short alpha-helices) in addition to native and non-native tertiary contacts. Despite the importance of the unfolded state, relatively few studies have characterized this state under native-like conditions, since the equilibrium constant strongly favors the folded state under these conditions. Therefore, here we propose to use isotope-edited infrared spectroscopy coupled with density functional theory calculations to investigate the spectral dependence on the length of short ?-helices found in unfolded states of proteins. This method provides residue-specificity through the use of non-perturbing isotopic labels to probe the peptide backbone conformation and hydration with sufficient temporal resolution to study the molecular dynamics of interest. Specifically, helical peptides containing 4, 8 or 11 helical amino acids stabilized by a La3+ binding loop to overcome the typical instability of short helical peptides will be investigated. Density functional theory as implemented in Gaussian 03 will be used to calculate the infrared spectra of these peptides following geometric optimization of structures resulting from molecular dynamic simulations at multiple temperatures utilizing CHARMM. These calculations will be critical in the examination of the dependence of the IR spectra on helix length and the analysis of the experimental IR spectra corresponding to the unfolding of these peptides. This information will then be used as the basis for the study of unfolded state structure in larger peptides and proteins. The computations will start with the 8 helical residue peptide, since its NMR structure is known. The other peptide structures will be initially generated by the removal or addition of residues to the C-terminus of this peptide. The calculations will focus on the effect of isotopic labels on the vibrational spectra of the model peptides in addition to peptide backbone conformation and hydration. Similar to previous DFT calculations of helical systems, the computational cost of these calculations will be minimized by fixing the position of the peptide backbone. This strategy will permit the vibrational spectra of the peptides to be calculated with and without explicit water molecules aiding in the analysis of the experimentally IR results. The DFT explicit water calculations will involve the placement of water molecules in key positions to model H-bonding to the peptide backbone. These computational studies will also investigate the potential of using un-natural amino acids such as nitrile-derivatized alanine and phenylalanine residues as probes of local secondary structure in peptides and proteins.
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