alphaA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30-40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for… Read More

The folding pattern of the alpha-crystallin domain, a conserved protein module encoding the molecular determinants of structure and function in the small heat-shock protein superfamily, was determined in the context of the lens protein alphaA-crystallin by systematic application of site-directed spin labeling. The sequence-specific secondary structure was assigned primarily from… Read More

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and… Read More

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were… Read More

Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact sites, buried sites, or loop sites. In addition, the sequence dependence of mobility can identify… Read More

Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the alpha-crystallin domain of alphaA-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was demonstrated that this sequence in alphaA-crystallin and HSP 27 forms a beta-strand involved in… Read More

The folding pattern of the segment of alphaA-crystallin encoded by exon 2 and containing putative substrate binding sites was explored using site-directed spin labeling (SDSL). For this purpose, a nitroxide scan was carried out between residues 60 and 108. At each site, structural constraints describing the local environment and topography… Read More

Site-directed spin-labeling (SDSL) was used to investigate the secondary structure, solvent accessibility, and tertiary and quaternary interactions along the sequence located between residues 133 and 144 in the alpha-crystallin domain of human heat-shock protein 27 (HSP 27). The sequence is conserved among mammalian sHSP and shows similarity to the region… Read More

Twelve sequential single cysteine mutants of alphaA-crystallin extending between amino acids Y109 and L120 were prepared and reacted with a sulfhydryl specific spin label in order to investigate the role of this sequence in the assembly of the alphaA-crystallin quaternary structure and its chaperone-like function. The sequence is located in… Read More

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component… Read More