9408284 Millhauser The goal of the proposed research is the development of new electron spin resonance (ESR) methodologies for the study of peptides and proteins. The research plan contains four projects. I. The first project focuses on the interactions between helical peptides as a means for probing early events in protein folding. Modern theory suggests that protein folding begins with the formation of isolated regions of local secondary structure. Tertiary structure then follows from adhesion events between these localized regions. However, little is known about these adhesion events. To probe this process, 2D electron-electron double resonance (2D ELDOR) will be used to monitor interactions between structured helical peptides in solution. 2D ELDOR is a new ESR technique that has just been shown to be extremely useful for studying peptide-peptide interactions. The methodology will be further developed for mixed isotope experiments. In doing so, the critical relationship between helical peptide structure and the strength of peptide-peptide interactions will be determined. II. The second project is the synthesis of a rigid side chain nitroxide amino acid for incorporation into FMOC automated peptide synthesis. Recent research has shown that nitroxide spin labeled peptides can provide wealth of information on peptide structure and dynamics. However, almost all published experiments have used spin label modifications of natural amino acid side chains. Linkages of this type are necessarily flexible and therefore less than ideal for certain types of experiments. The specific label proposed here is rigid, follows L amino acid stereochemistry, and contains the necessary design features for a "helix-neutral" amino acid. III. The third project is an exploration of the use of neutral networks for determining motional parameters from slow motional ESR spectra. Slow motional spectra in ESR and solid state NMR result when the rotational cor relation time is too long to satisfy the motional narrowing conditions. Although more difficult to interpret, slow motional spectra are extremely sensitive to dynamic details of molecular motion. In the past, such spectra were stimulated and motional parameters were determined by estimating a best fit. This is a very laborious process that uses considerable computer and human resources. The proposal outlines and approach for tackling this problem form an entirely new perspective. Neural networks will be used to quickly extract dynamic details from slow motional spectra. IV. The last project is the development of a new spectroscopy called Biradical Electron-Electron Correlated Spectroscopy (BEE- COSY). This technique will be used for determining distances of up to 15A in doubly spin labeled peptides. COSY spectroscopy has long been used in NMR to reveal through bond scalar couplings between nuclear spins. In biradical peptides, electron-electron couplings results from a through space interaction. BEE-COSY will both detect and help quantify these couplings. The detectable length scale is between that of NMR (< 4 ) and fluorescence energy transfer (> 20 ) and is therefore extremely important for biomolecules. % % % Electron spin resonance (ESR) is a modern biophysical technique used to explore structure and motion in biologically important molecules. The main goal of the proposed research is to develop new ESR methods to help biochemists refine their ideas of protein structure and activity. In turn, these methods will serve as indispensable aids in basic and applied research. As an example, in our last funding period, we developed the use of double label ESR to reveal previously unseen structures in protein fragments. Our ideas were then implemented by another lab to solve the protein coat structure of the Pf1 virus. This critical problem had remained unsolved for nearly 10 years! For the new funding period we propose to explo re methods to probe protein assembly. In particular, our 2D ELDOR project may allow us to observe events that correspond to the earliest protein assembly processes. The insights gained from this research may prove critical for predicting structure and stability from protein sequences. Such information will help in drug design, understanding of diseases and the human genome project. * * *
