SUBMILLISECOND LASER T JUMP INDUCED PROTEIN FOLDING

University Of Illinois Urbana-Champaign, Champaign IL

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Abstract

It has recently become clear from NMR, circular dichroism, fluorescence and other spectroscopic studies that upon folding, many small proteins can form secondary and tertiary structure elements in less than a millisecond. Early and rapidly formed structure may turn out to be universally important for the foldability of natural and designed polypeptide sequences. It is the long term goal of this research to probe these fast dynamics experimentally and to use them as direct tests of analytical and computational (e.g. molecular dynamics) folding models. Eventually, these experiments will provide average temporal maps of the early spatial evolution of protein ensembles during the folding process. The specific aims are: 1) To further develop a fast laser-induced temperature-jump apparatus which can probe protein backbone motions during refolding on a 14 ns - 50 ms time scale. In particular, we will add the capability of detecting secondary structure formation directly, in addition to our present capability for detecting tertiary contact formation (collapse). 2) To measure the time scales of secondary and tertiary structure formation of proteins with very fast kinetic phases at different locations within the proteins. 3) To use such time- resolved structural information, as well as energetic information (e.g. reaction barriers as determined by the temperature dependence of the kinetics) to compare experimental results with theoretical and computational model predictions. A series of specific systems will be studied to sample structures ranging from all alpha to mostly beta motifs. Our initially proposed systems include apomyoglobin, cytochrome c, ubiquitin, and phosphoglycerate kinase, all of which have known sub-ms folding phases. The expression systems for these proteins are available to us, allowing us to probe specific tertiary contacts and secondary structure by site- directed mutagenesis and isotopic labeling.