Viscosity dependence of acrylamide quenching of ribonuclease T1 fluorescence. The gating mechanism.
The structural regulation of the access of acrylamide molecules, as quenchers, to the buried tryptophans of a protein can be modelled by a simple gate concept. Such a gate, when open, allows transient exposure of the fluorophore to the quencher molecule in solution. We have previously shown that the observed viscosity dependence of acrylamide quenching process in ribonuclease T1 (RNAse T1) is not reconcilable with the gating mechanism. However, on that occasion, we neglected the effect of changes in the activity of the quencher molecule and the possible presence of static quenching. The experimental observation of a considerable contribution by static quenching and the realization that static quenching might produce dramatic effects in steady state measurements led us to reexamine the question. It is shown that in a gating model the static component can also influence the apparent dynamic quenching. In this paper, we present derived equations for the gated quenching mechanism including possible contributions from the static component. We also carefully remeasured the acrylamide quenching of RNAase T1 as a function of increasing glycerol concentration. Computer simulations were carried out to compare the experimental data set to the generalized model. We reach the conclusion that even the new, quite complex equations fail to predict the qualitative and quantitative features of the observed quenching experiments. We arrived at the conclusion that the fluorophore is never the target of the quencher molecules in solution.
Somogyi
B
B
Department of Biophysics, University Medical School of Debrecen, Hungary.
Norman
J A
JA
Punyiczki
M
M
Rosenberg
A
A
D020106:Acrylamide; D000178:Acrylamides; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014783:Viscosity
D000178:Acrylamides; D020106:Acrylamide; D006163:Ribonuclease T1