[10] Solute quenching of protein fluorescence

Lehrer, Sherwin S.; Leavis, Paul C.

doi:10.1016/s0076-6879(78)49012-3

Cited by 228 publications

(161 citation statements)

References 29 publications

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“…No upward curvature is noted, implying that the static quenching parameter is small. No significant downward curvature is noted, implying an absence of significantly different multiple emission components (31). The slope of the Stern-Volmer plots for native sct-PA and native tct-PA are similar, suggesting similar quencher access to fluorophores in these molecules.…”

Section: Resultsmentioning

confidence: 87%

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

Loscalzo¹

1988

J. Clin. Invest.

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We examined the similarities and differences in conformation between recombinant human single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA), and compared these structural data with measurements of enzymatic activity. The intrinsic protein fluorescence of native tct-PA was 54% that of sct-PA. Differences in steady state protein fluorescence were also noted with denaturation of these plasminogen activators, as well as in the quenching of intrinsic fluorescence of the reduced, alkylated species by iodide. Using the chromogenic substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), the catalytic efficiency of sct-PA was found to be 26% that of tct-PA, and this was primarily a reflection of the difference in K.,. On addition of soluble fibrin monomer prepared with the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the catalytic efficiency of both species increased by 13-fold for sct-PA and by 3.5-fold for tct-PA to approximately the same value. Using the fluorophore eosin iodoacetamide covalently coupled to the single free cysteine in the molecule, Cys 83, the microenvironment of the fibrin-binding site located near this residue was studied. On addition of soluble fibrin monomer to eosin-labeled tct-PA, no effect on eosin fluorescence was noted. Eosin-labeled tct-PA had 16% less eosin fluorescence than did sct-PA and on addition of soluble fibrin monomer to eosin-labeled sct-PA, a decrease in eosin fluorescence, approaching that of eosin coupled to tct-PA, was observed. Together, these structural and kinetic data suggest that sct-PA undergoes a conformational change on binding to fibrin monomer that leads to dramatic differences in catalytic efficiency of the single-chain species. In so doing, sct-PA bound to fibrin assumes the kinetic profile of tct-PA bound to fibrin.

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Section: Resultsmentioning

confidence: 87%

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

Loscalzo¹

1988

J. Clin. Invest.

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“…Small aliquots of concentrated acrylamide were added, and the fluorescence intensity was measured during constant stirring. The effects of acrylamide quenching can be described by the Stern-Volmer Equation 1 (15),…”

Section: Methodsmentioning

confidence: 99%

Long Range Allosteric Control of Cytoplasmic Dynein ATPase Activity by the Stalk and C-terminal Domains

Höök

Mikami

Shafer

et al. 2005

Journal of Biological Chemistry

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The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an ϳ100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated ϳ1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5-adenylyl-␤,␥-imidodiphosphate but was blocked by ADP-AlF 3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.Cytoplasmic dynein is a microtubule-based motor that utilizes energy from ATP hydrolysis to facilitate a wide variety of functions in eukaryotic cells such as spindle alignment, nucleus positioning and chromosome separation during mitosis, and retrograde axonal transport of subcellular components (1, 2). Dynein belongs to the functionally diverse family of AAAϩ proteins (3). Members of this family contain a conserved domain of 200 -250 residues termed AAA, or "ATPases associated with various cellular activities," that contains five signature motifs involved in ATP binding and hydrolysis. Most AAA proteins form homohexameric rings in which each subunit is composed of either one or two AAA domains. The ϳ380-kDa dynein motor, in contrast, consists of a ring of six dissimilar and covalently linked AAA domains and at the extreme C terminus an ϳ30-kDa region of unknown function. Fused to the motor domain is a substantial projection, referred to as the stem, which contains sites for dimerization and association with cargo binding subunits (4). Emerging from between AAA4 and AAA5 is another projection, the stalk, which is thought to consist of an antiparallel coiled-coil with a globular domain for microtubule binding at its tip (5).Sequence analysis of the dynein motor domain suggests that the ability to bind and hydrolyze nucleotides differs among the six AAA domains (6). Whereas the first AAA domain contains the entire set of nucleotide-interacting motifs, the probability of identifying these motifs in AAA2-AAA4 ...

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“…Fluorescence quenching titrations with acrylamide were performed as described by Lehrer and Leavis [15]. Quenching was analysed according to the Stern-Volmer equation:…”

Section: Materials Aflatoxin B1 Bsp 8-anilinonaphthalene 1-sulphonmentioning

confidence: 99%

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

Sluis‐Cremer

Wallace

Burke

et al. 1998

European Journal of Biochemistry

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The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (K d ϭ 8Ϯ 2 µM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 Å by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h ϭ 1.6 Ϯ0.1 ; K′ ϭ 14Ϯ0.6 µM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20→Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.Keywords : glutathione S-transferase ; aflatoxin B1; sulphobromophthalein; co-operative binding.Glutathione S-transferases (GSTs) are a family of multifunctional enzymes found in all vertebrates, plants, insects, nematodes, yeast and aerobic bacteria. They constitute a complex supergene family that collectively metabolises a broad variety of potentially toxic xenobiotics and reactive endogenous compounds resulting from oxidative metabolism. GSTs are an integral part of the phase-I detoxification mechanism and primarily catalyse the nucleophilic addition of the thiol of reduced glutathione (γ-glutamyl-cysteinyl-glycine) to electrophilic centres in a wide variety of hydrophobic electrophiles, including alkyl and aryl halides, epoxides, quinones and activated alkenes [1]. The dimeric cytosolic GSTs exhibit multiple enzyme forms that, according to primary structure and molecular recognition, can be grouped into one of six species independent gene classes (alpha, kappa, mu, pi, theta and sigma). In addition to their catalytic activities, GSTs also function as ligand binding proteins and thereby facilitate the intracellular storage of a variety of hydrophobic non-substrate compounds, including hormones, metabolites and drugs [2]. The binding of these compounds to GSTs prevents the build up of apolar molecules at lipophilic sites such as membranes...

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[10] Solute quenching of protein fluorescence

Cited by 228 publications

References 29 publications

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

Structural and kinetic comparison of recombinant human single- and two-chain tissue plasminogen activator.

Long Range Allosteric Control of Cytoplasmic Dynein ATPase Activity by the Stalk and C-terminal Domains

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

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