TRANSIENT INFRARED PROBING OF PROTEIN FOLDNG AND CONFORMATIONAL DYNAMICS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protein folding problem is considered to be one of the fundamental questions in structural biology. Folding research is today a very active area, where the experimental and theoretical techniques for probing folding at the molecular level are becoming more and more refined. The goal is to provide an experimental and theoretical basis for understanding and predicting protein folding pathways, the stable structures, and thermally and kinetically accessible conformation substates, given the primary amino acid sequence. A quantitative understanding of protein folding is apparently important for protein engineering. Understanding how proteins fold can also help to interpret quantitatively the structure-function relationships and folding related diseases. Furthermore, a predictive understanding of protein folding will accelerate the discovery of information contained in the large number of gene sequences that are now becoming available. It was proposed to develop instruments that are capable of triggering and probing conformational changes in proteins (and other molecular systems as well) on various timescales. Time-resolved infrared (IR) spectroscopy offers great flexibility and power for monitoring kinetic events on the molecular level with structure specificity and will be used to generate detailed structure interpretations of the transient species and their dynamics over the time range of interest. Using these instruments, we propose to study primarily how proteins fold. A detailed set of experiments are planned to gain detailed insight into the formation of protein secondary and tertiary structures. We are further extending current instruments and developing new instruments that are capable of triggering and probing conformational changes in proteins on various timescales. The specific aim of developing a nanosecond temperature-jump (T-jump) infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times is being continued and extended. The microsecond FTIR coupled continuous-flow mixing and the millisecond FTIR coupled stopped-flow apparatus development is now available. The 2-dimensional (2D) correlation analysis has permitted site specific conformation studies and explorations of CN motions as a probe of dynamics. Studies of the helix-coil transition in alpha-helical peptides were performed, as well as studies of the stability and folding kinetics of beta-hairpin model peptides. We further work on the combination of the stop-flow apparatus with fluorescence detection and the incorporation of ATR spectroscopy into our IR capabilities to study membrane proteins. Another direction of this project will be the study of peptide/protein aggregation. Peptide and protein aggregation is the underlying cause of many diseases. This project is aimed to understand some fundamental aspects of peptide aggregation through a systematic approach. For example preliminary results on beta-hairpins suggest that the beta-turn plays a significant role in controlling the formation of beta-aggregates.