Time-resolved fluorescence studies of ribonuclease T1 in reversed micelles.

Time-resolved fluorescence intensity and anisotropy decay data were obtained for ribonuclease T1 entrapped in bis(2-ethylhexyl) sodium sulfosuccinate/heptane reverse micelles, as a function of the size of the inner water pool at neutral pH. Data have been presented previously to show that this protein retains its native structure and undergoes reversible thermal unfolding in these reverse micelles (Shastry and Eftink,Biochemistry 36, in press). The fluorescence decay of entrapped protein is similar to that for the protein in buffer. The rotational correlation time of entrapped ribonuclease T1 is found to be longer than that in buffer; this rotational correlation time decreases with increasing size of the water pool but is still over twice the value for the protein in buffer for the largest size of water pool investigated, indicating an increased microviscosity within the reverse micelle. Thermal unfolding of the protein results in a significant decrease in the rotational correlation time of the entrapped proteins, consistent with the protein being unfolded but not interacting with the inner surfactant wall of the reverse micelle.