Time-resolved absorption and fluorescence spectroscopy are used to study the dynamics of protein structural changes subsequent to rapid mixing or excitation with short laser pulses. Kinetic models are used to fit and interpret the measured data. Our recent efforts have focussed on understanding the folding of the villin subdomain and the assembly of amylin. The 35-residue villin subdomain consists of three helices which interact to form a hydrophobic core. Amylin is an extremely hydrophobic 37-residue peptide which is coexpressed with insulin and fibrilized in the islet cells of diabetic patients.? ? To probe the folding mechanism of villin we previously carried out structural and physical-chemical studies of molecules with point mutations and other sequence modifications. These studies have included determination of the structures of some of the folded states by X-ray diffraction carried out in collaboration with Dr. Thang Chiu and David Davies in the LMB/NIDDK. Conservative mutations had no significant effects on the folding rates, suggesting that the transition state for folding of villin is close to the denatured ensemble of conformations. On the other hand, the K24Nle and K29Nle mutations both stabilize the folded structure and accelerate folding by factors of ~2.5, and the double mutant, with a folding time of ~ 700 ns, ranks as the fastest folding protein studied to this time.? ? To further explore the folding of villin we are engineering a variant which includes a pair of probes which undergo fluorescence resonance energy transfer. We have replaced the N-terminal leucine by a cysteine to which the dye Alexa-350 can be attached and the C-teminal phenylalanine at position 35 with naphthylalanine. In the native state these two residues are only about 1 nm apart so the naphthylalanine (donor) is more than 90% quenched by the Alexa-350. In the unfolded state this distance increases to ~4.0 nm and the donor is only ~40% quenched. This pair thus provides a probe of the global unfolding of villin and of the average end-to-end distance in the unfolded state. At this point we know that the folding of this doubly-labelled variant is very similar to that of the variant which we have previously studied. More detailed equilibrium and kinetic characterization is in progress.? ? We have also begun to study the dynamics of the unfolded states of human and rat amylin. The rat sequence, which contains three prolines at positions 25, 28 and 29, does not assemble into fibrils. To do so, we make use of the triplet quenching method developed in our laboratory (Lapidus et al 2000). The C-terminal tyrosine (Y37)is replaced by tryptophan, the triplet excited state of which is quenched by an internal disulfide formed between residues C2 and C7 in both sequences. By measuring loop formation dynamics as a function of the concentration of guanidinium chloride, which solubilizes the these hydrophobic peptides, and temperature we will be able to observe the effects of intramolecular interactions on their chain dynamics. These studies may provide insight into the mechanism of assembly of human amylin into fibrils.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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U.S. National Inst Diabetes/Digst/Kidney
United States
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