Reversible thermal unfolding of ribonuclease T1 in reverse micelles.
The reverse micellar system formed by the negatively charged surfactant AOT and the organic solvent isooctane is used to solubilize the protein RNase T1. The physicochemical properties of the entrapped protein have been studied using intrinsic tryptophan fluorescence and far-and near-UV CD. These studies indicate a similar structure for the protein in reverse micelles and in pH 7.0 buffer. Thermal unfolding has been studied as a function of W0, the molar ratio of water to AOT, in the solution. Measuring the change in fluorescence intensity as a function of temperature, we observe a reversible transition for W0 in the range 5-12. Heating rate dependencies carried out on these transitions (0.6-3.0 degrees C/min) indicate that the transition temperature and the apparent van't Hoff enthalpy change depend on the scanning rate as well as on W0. The values of the transition temperature, T(m) and the enthalpy change, delta H degrees(un), extrapolated to an infinitely slow scanning rate, are analyzed considering the electrostatic interaction of the charged residues of the protein with the charges of the surfactant molecules forming reverse micelles, the variation of the size of the reverse micelles, and the relative rates of unfolding, refolding, and irreversible denaturation.
Shastry
M C
MC
Department of Chemistry, University of Mississippi, 38677, USA.
Eftink
M R
MR
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004143:Dioctyl Sulfosuccinic Acid; D006358:Hot Temperature; D007700:Kinetics; D008823:Micelles; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan
D008823:Micelles; D012996:Solutions; D013501:Surface-Active Agents; D004143:Dioctyl Sulfosuccinic Acid; D014364:Tryptophan; D006163:Ribonuclease T1