title: 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.
mesh_terms: D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism*; D005454:Fluorescence Polarization; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006157:Guanosine Monophosphate / Q000494:pharmacology*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan; D014961:X-Ray Diffraction
chemical_list: D000178:Acrylamides; D006150:Guanine Nucleotides; D020106:Acrylamide; D006157:Guanosine Monophosphate; D014364:Tryptophan; D004722:Endoribonucleases; D006163:Ribonuclease T1