The luminescence of tryptophan and phenylalanine derivatives

Weinryb, Ira; Steiner, Robert F.

doi:10.1021/bi00847a007

Cited by 68 publications

(51 citation statements)

References 10 publications

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“…Both kq and r are functions of temperature. An estimation of r at any temperature was made by assuming its proportionality to the fluorescence yield, 4) (22,23). The yield (and hence T) decreases slightly with increasing temperature, as illustrated by a plot of (4+' -1) against 1/T (26).…”

Section: And Discussionmentioning

confidence: 99%

Dynamics of a protein matrix revealed by fluorescence quenching.

Eftink¹,

Ghiron²

1975

Proc. Natl. Acad. Sci. U.S.A.

152

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ABSPIACT The fluorescence of the su posedly buried trtophan in ribonuclease T1 has been found to be collisionally quenched by acrylamide with a rate constant of 3 X 108 M-1 sec-1. Only a slight decrease in the quenching rate is observed upon a 5-fold increase in the viscosity of the solution. For this to be the case, the diffusion of the quencher must be limited by the protein matrix. To explain the process of diffusion through this complex material, the formation of "holes" in the lattice of a protein due to nanosecond fluctuations must be invoked. ThIus, the dynamic character of a protein molecule is revealed. The quenching rate constant has an activtion energy of 9 kcal/mol which can be used to charactedte the nature of the cohesive forces in the microenvironment about the indole ring. The mechanical properties of a portion of a protein matrix can, therefore, be described as one would for a fluid.Perhaps the most pressing problem facing the protein chemist today is to adequately describe the dynamic properties of proteins in solution, now that their static structure can be determined crystallographically. Evidence for some type of motion occurring at a time interval as small as a fraction of a nanosecond has been obtained from nuclear magnetic resonance (1), nitroxide spin labeling (2), dielectric relaxation (3), and various fluorescence studies (4-6). Depending on the time domain and experimental approach, descriptive terms such as "breathing" (7), "relaxation," "segmental motion," and "mobile defect" (8) have been advanced to portray the conformational "motility" (9) of proteins.Although it is recognized that fluctuations on the nanosecond time scale are allowed at the periphery of macromolecules, experimental evidence for such rapid motion of residues buried in "core" regions is not as available. In fact, the observation made by Longworth (10), that the lone tryptophan in ribonuclease T1 fluoresces with fine structure at room temperature, even suggests the absence of certain dynamic events in this protein. The enzyme was assayed by measuring the absorbance of trichloroacetic-acid-soluble material formed by enzymic digestion of the tRNA for 15 min at 30' at pH 7.5 (Tris buffer).Fluorescence-lifetime measurements were performed on an instrument resembling the model 9200 Nanosecond Fluorescence Spectrometer (ORTEC) constructed by S. Stevens and J. W. Longworth. Lifetimes were assigned by comparison of the raw decay data to simulated patterns that were synthesized by adding a theoretical single exponential decay curve to a measured system response. This was done using a computer program (also provided by S. Stevens and J. W. Longworth) that was designed to minimize the error between the raw and simulated curves in order to seek the best fit. The decays could be excellently described as single exponentials. The single component fits were at least as good as those obtained for pure samples of indole (4.3 nsec) and Nacetyltryptophanamide (2.8 nsec). During the 12-hr time period required to make the lifetime measu...

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Section: And Discussionmentioning

confidence: 99%

Dynamics of a protein matrix revealed by fluorescence quenching.

Eftink¹,

Ghiron²

1975

Proc. Natl. Acad. Sci. U.S.A.

152

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“…2A) presumably corresponded to the presence of tryptophanyladenylate. Because the fluorescence of tryptophan was likely to have been quenched upon adenylation, as in the general case of reactions at the carboxyl group of tryptophan, this would have represented a quenching of the free tryptophan signal [21]. Thus, notwithstanding the slight shift in the direction of tryptophan seen in the difference curve, given the difference maximum around 330 nm, the cause of the fast 8 pM L-tryptophan, 100 pM ATP-Mg, 0.6 pM tryptophanyl-tRNA synthetase and 1 U/ml inorganic pyrophosphatase in 0.1 M Tris/ HCI pH 8.0.…”

Section: Fluorescence Change Linked To Each Processmentioning

confidence: 99%

“…Because a reaction at the carboxyl group of tryptophan would be expected to quench the fluorescence of tryptophan [21], and because the opposite effect was observed, the fluorescence emission of tryptophan and commercially obtained tryptophanamide were measured as a function of pH. Fig.…”

Section: Kinetics Of Tryptophanamide Formationmentioning

confidence: 99%

Tryptophanamide formation by Escherichia coli tryptophanyl‐tRNA synthetase

Andrews

Trezeguet

Merle

et al. 1985

European Journal of Biochemistry

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When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATPMg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001 -1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-48791. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by highperformance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a K , of 1 mM for ammonia and a rate constant of 5.7 min-' at pH 8.3,25"C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.The aminoacylation of tRNA proceeds via the production of a high-energy intermediate formed between the carboxyl group of the amino acid and the phosphate group of AMP, derived from ATP by the release of pyrophosphate. The resulting mixed anhydride bond between the amino acid and AMP provides both species with a high chemical transfer potential, which can lead to several reactions deviating from the regular charging of tRNA. Depending on the synthetase studied, reactions of varying specificity and ...

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“…(48,69,79,80) This is the case irrespective of the state of ionization or substitution of the a-carboxyl group. …”

Section: Tryptophan Derivativesmentioning

confidence: 99%