Abstract
Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.
Highlights
T 1 (RNase T1) were determinedby measuring several for studiesof the folded conformation
A single kinetic phase was observed for both the disulfide bonds, and, second, it allows an investigation of the unfolded conformations of RNase T1 under physiological conditions
The slow phases probably ing the disulfide bonds [4].theconformational stability result from cis-trans isomerization at the 2 proline of this small globular protein can be varied over a range of residues that have acis configuration infolded RNase about 15 kcal/mol
Summary
The unfolding of RNase T1 was monitored using several spectroscopic techniques in order to gain information about the mechanismof folding and to obtain independent estimates of the conformational stability. There is a 7-fold and the larger change at 287 nm shows that several of the decrease in the optical rotation at 295 nm when the protein Tyr residues have changed their environment on unfolding. It is clear from the spectra that this change inopthiceal by heating, and Fig. 2 shows typicalthermal unfolding curves rotation depends mainly on the secondary structure of the monitored by following the absorbance differences measured protein rather than on changes in the contribouftitohne side at 286 and 292 nm. The circular dichroism changes at 284 nm depend shows typical urea unfolding curves followed using fluoresmainly on the environmeonft the side chains of the aromatic cence measurements at 320 nm after excitation at either 278 amino acids and, on the tertiary structure, but the or 295 nm. The difference in free energy between the folded and unfolded conformations, AG, can be calculated using the equation
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