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Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins.(More)
The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain.(More)
Conformational properties of the OT-16 peptide, the C-terminal 20 amino acids of RNase A, were examined by nonradiative energy transfer. A modified OT-16 peptide was prepared by solid-phase synthesis with the inclusion of diaminobutyric acid (DABA) at the C-terminus. The OT-16-DABA peptide was labeled with a fluorescent 1,5-dimethylaminonaphthalene sulfonyl(More)
A variety of biophysical methods used to study proteins requires protein modification using conjugated molecular probes. Cysteine is the main residue that can be modified without the risk of altering other residues in the protein chain. It is possible to label several cysteines in a protein using highly selective labeling reactions, if the cysteines react(More)
The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of(More)
To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution(More)
Investigation of conformational transitions and, in particular, the folding/unfolding transitions of globular proteins by means of excitation energy transfer measurements depends on the availability of protein derivatives carrying donor and acceptor probes at well-defined pairs of sites. A series of bovine pancreatic trypsin inhibitor (BPTI) derivatives,(More)
When transfer of electronic excitation energy occurs between a donor-acceptor pair by the Förster mechanism, the decay of fluorescence of the donor follows first-order kinetics, with a rate constant that depends on the distance from donor to acceptor. In a system that contains donor-acceptor pairs of different separations, the fluorescence decay of the(More)
Sex-reversal mutations in human SRY cluster within its high-mobility group box, a conserved motif of DNA bending. A classical substitution at the crux of this angular domain (M64I) has been reported to impair DNA bending but not DNA binding, implying that sharp bending is required for transcriptional activation and testis determination. Surprisingly, we(More)