Dynamics of a protein matrix revealed by fluorescence quenching.

Abstract

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...

Hydrogen exchange and structural dynamics of proteins and nucleic acids

“…The possibility that other small molecules might similarly enter proteins was raised by the work of Eftink & Ghiron (1975, who found that acrylamide (C 3 H 5 ON, molecular weight 71) could also quench the fluorescence of a variety of protein tryptophans. These investigators put forward the idea that the fluorescence-quenching capability of acrylamide could provide a 'kinetic ruler' that would gauge the relative accessibility of particular tryptophan residues to solvent.…”

Principles of Protein Structure

“…The same technique has been used to demonstrate conformational mobility of apparently rigid residues in lysozyme (425), ribonuclease (426), and myoglobin (427). It has further been found that the fluorescence of a buried Trp in ribonuclease can be quenched by O2 (428,429), indicating that O2 can penetrate into the molecule. Similar results emerged from fluorescence relaxation (430), phosphorescence (431), and hydrogen exchange experiments (414).…”

Biologically Active Novel Conformational State of Botulinum, the Most Poisonous Poison

“…Thermally induced conformational changes in BoNT/A light chain protein unfolding were also investigated by quenching of Trp fluorescence. The degree of exposure of Trp residues in solution as a function of temperature, monitored by fluorescence quenching technique, provides valuable information concerning the dynamics of protein folding (33). Acrylamide is an efficient quencher that preferentially quenches the more solvent-accessible Trp residues in multi-tryptophan proteins (34,35).…”