This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The beta-sheet is one of the two common structural motifs in proteins.n Understanding its folding mechanism is therefore important to understand to what extend large proteins fold. The objective of this application is to develop new knowledge at the single residue level of beta-sheet folding pathway using beta-hairpin as model. The hairpins proposed in this work are chosen to probe the factors that contribute to its stability like the intrinsic amino acid turn tendency, the stability of the hydrophobic cluster, the interstrand hydrogen bonding, and interstrand side-chain-side-chain interactions. Thus, our long term goal is to understand the physical and chemical factors that regulate the beta-sheet formation. The rationale for the proposed research is that knowledge of folding mechanism at the single residue level will provide better information to performed site-directed mutagenesis, help in the synthesis of new peptides and proteins with specific functions, and will allow taking control of the folding/unfolding process. The proposed work is innovative because higher structural information at the single residue level of the folding/unfolding of beta-hairpins will be available. With respect to expected outcomes, the combination of the work proposed in specific aims is collectively expected to identify and quantify the factors that are responsible for beta hairpin folding/unfolding mechanism. During recent years, great improvements in the time and spectral resolution of protein folding experiments have been achieved;resulting in new physical and chemical knowledge about the correlation of the unfolded and folded states. Also, an increase in theoretical calculations with increased computer capacity brings the protein folding dilemma to a new level of complexity. However, there are still many fundamental questions on the nature of the folding pathways. Basically, these questions are related to where and how the folding initiation step takes place and on what range of timescale does it occur. Furthermore, it is important to establish how the primary amino acid sequence encodes the folding pathway. This knowledge serves to predict the folding mechanism for a particular protein from its sequence and structure. Moreover, since the genome era is finally mature, new de novo designed peptides and proteins with certain unique functions such as therapeutic agents and drug delivery agents could be developed. The beta-sheet is one of the two common structural motifs in proteins. Understanding its folding mechanism is therefore important to understand to what extend large proteins fold. Thus, our long term goal is to understand the physical and chemical factors that regulate the beta-sheet formation. The objective of this application is to develop new knowledge at the single residue level of p-sheet folding pathway using p-hairpin as model. Beta-hairpin is the p-sheet basic building block consisting in two parallel strands joined by a twisted turn. Our working hypothesis is that higher structural detailed information of the beta hairpin folding pathway, important for the understanding of the extend of protein folding, can be obtained by site directed amide carbonyl isotope labeling with IR spectroscopy and laser induced temperature jump using IR detection. The first type of experiments will provide information on the thermal stability and the second on the formation of the folding-unfolding dynamics. A systematic study of a series of p-hairpin analogues with the same strands and different turns or a common turn with strand variation are proposed to study the local conformational change along the primary amino acid sequence. The rationale for the proposed research is that knowledge of folding mechanism at the single residue level will provide better information to performed site-directed mutagenesis, help in the synthesis of new peptides and proteins with specific functions, and will allow taking control of the folding/unfolding process. Therefore, the specific aims of this work are to identify and quantify the: (1) effect of moving the hydrophobic cluster position across the strand relative to the turn to the thermal stability and folding kinetics; (2) the contribution and position dependence of side-chain-side chain interactions to the conformational stability and dynamic properties for the folding/unfolding mechanism; (3) the contribution and position dependence of cross-strand electrostatic and aromatic side-chain interactions to the folding/unfolding mechanism; (4) the contribution of the turn sequence to the overall stability and dynamics.
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