Journal ArticleDOI
Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.
TLDR
The value of this probing technique lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.Abstract:
Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.read more
Citations
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Spectroscopic studies on the interaction of a water soluble porphyrin and two drug carrier proteins.
TL;DR: The large reduction of the effective binding constant from pH 2 to 7 suggests that electrostatic interactions are a major contribution to the binding of TSPP to the aforementioned proteins.
Journal ArticleDOI
DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.
TL;DR: The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA‐binding on or off by controlling the DNA‐induced conformational change in R2, and enhanced protease resistance dramatically.
Journal ArticleDOI
Rapid formation of a molten globule intermediate in refolding of α-lactalbumin
Munehito Arai,Kunihiro Kuwajima +1 more
TL;DR: In this article, the authors investigated the refolding kinetics of α-lactalbumin using stopped-flow CD and fluorescence, acrylamide quenching and pulsed hydrogen exchange NMR techniques.
Journal ArticleDOI
Conformational analysis of the androgen receptor amino-terminal domain involved in transactivation. Influence of structure-stabilizing solutes and protein-protein interactions.
TL;DR: It is shown that a 344-amino acid polypeptide representing the main determinants for transactivation has the propensity to form α-helical structure and that mutations which disrupt putative helical regions alter conformation.
Journal Article
Unfolding of acrylodan-labeled human serum albumin probed by steady- state and time-resolved fluorescence methods
TL;DR: Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34.
References
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Journal ArticleDOI
The interpretation of protein structures: estimation of static accessibility.
B. Lee,Frederic M. Richards +1 more
TL;DR: The accessibility of atoms in the twenty common amino acids in model tripeptides of the type Ala-X-Ala are given for defined conformation and the larger non-polar amino acids tend to be more “buried” in the native form of all three proteins.
Journal ArticleDOI
Removal of Fatty Acids from Serum Albumin by Charcoal Treatment
TL;DR: Fluorescence spectra of human serum albumin samples indicated that impurities are sometimes present which can be removed by charcoal at neutral pH, and acid-charcoal treatment is a much more rapid method of removing lipid impurities than other methods previously described.
Journal ArticleDOI
Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion.
TL;DR: The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity.