Picosecond time-resolved fluorescence of ribonuclease T1. A pH and substrate analogue binding study

Abstract

The tryptophyl fluorescence of ribonuclease T1 decays monoexponentially at pH 5.5, tau = 4.04 ns but on increasing pH, a second short-lived component of 1.5 ns appears with a midpoint between pH 6.5 and 7.0. Both components have the same fluorescence spectrum. Acrylamide quenches both fluorescence components, and the short-lived component is quenched fivefold faster than the predominant long component. Binding of the substrate analogue 2'-guanylic acid at pH 5.5 quenches the fluorescence by 20% and introduces a second decay component, tau = 1.16 ns. Acrylamide quenches both tryptophyl decay components, with similar quenching rates. The fluorescence anisotropy decay of ribonuclease T1 was consistent with a molecule the size of ribonuclease T1 surrounded by a single layer of water at pH 7.4, even though the anisotropy decay at pH 5.5 deviated from Stokes-Einstein behavior. The fluorescence data were interpreted with a model where the tryptophyl residue exists in two conformations, remaining in a hydrophobic pocket. The acrylamide quenching is interpreted with electron transfer theory and suggests that one conformer has the nearest atom approximately 3 A from the protein surface, and the other, approximately 2 A.

Structure of a rapidly formed intermediate in ribonuclease T1 folding

“…Table 2 (Table 2). They are, however, similar to the two lifetimes of 3.78 ns (amplitude = 89%) and 1.5 ns that are found in the native native state of the wild-type protein at pH 8 and 20 "C (Chen et al, 1987). The existence of two fluorescence lifetimes of Trp 59 at the stage of the intermediate may indicate that the structure of the intermediate is not as rigid as the native structure and thus the fluorophore can exist in two different conformations.…”

“…This is consistent with the molecular environment of Trp 59, which is completely buried in the hydrophobic core of native RNase T1. (Chen et al, 1987;Gryczynski et al, 1988) and probably originates from two distinct conformations of Trp 59 (Chen et al, 1987). Presumably, Trp 59 of (S54G,P55N)-RNase T1 can also exist in at least two distinct conformations at the stage of the folding intermediate.…”

“…Excited Trp 59 of RNase T1 is known to decay with two lifetimes in unfolded as well as in native wild-type RNase T1 at pH 8 and 20 "C, but with a single lifetime in the native protein at pH 5 and 20 "C. At lower temperatures (10 "C), only a single lifetime can be detected in native RNase T1 both at pH 8 and pH 5 . The two fluorescence lifetimes at pH 8 and 20 "C were suggested to originate from the existence of two conformations of the indole ring of Trp 59 (Chen et al, 1987). Only a single conformation is apparently available for Trp 59 at pH 5 and at lower temperatures.…”

Backbone dynamics of ribonuclease T1 and its complex with 2?GMP studied by two-dimensional heteronuclear NMR spectroscopy

“…The difference in the xo reflects different protein concentrations and hence different solution viscosities in both samples. The values obtained by the NMR approach are well within the range of correlation times of RNase T1 obtained from fluorescence decay measurements (Eftnik, 1983;Lakowicz et al, 1983;James et al, 1985;Chen et al, 1987).…”

How Do Rotameric Conformations Influence the Time-Resolved Fluorescence of Tryptophan in Proteins? A Perspective Based on Molecular Modeling and Quantum Chemistry

“…Long before however, the two lifetimes had been proposed to originate from two conformers of the copper binding site which interact differently with the single Trp rotamer [48]. The fluorescence decay of ribonuclease T1 changes from monoexponential at low pH to bi-exponential under slightly destabilizing conditions like high pH, high temperature or in the presence of denaturant [49,50]. Still, a molecular mechanics study showed the existence of quasi-one Trp59 rotamer [51].…”