THE REFOLDING OF CIS-PEPTIDYLPROLYL AND TRANS-PEPTIDYLPROLYL ISOMERS OF BARSTAR

Barstar, a small globular protein which undergoes reversible unfolding, is a good candidate for studies on protein folding. It possesses two cysteine residues that complicate folding studies by forming a variable mixture of disulfide-bridged forms. We have constructed and analyzed, therefore, a double mutant Cys40-->Ala,Cys82-->Ala. Equilibrium unfolding with urea follows a simple two-step mechanism. The midpoint for unfolding ([U]1/2) is 3.87 +/- 0.03 M urea, with m(partial derivative DELTAG/partial derivative [urea]) = 1.25 +/- 0.04 kcal/mol2. The free energy of unfolding, DELTAG(U-F)H2O, is 4.84 +/- 0.18 kcal/mol. Identical results were found on monitoring the intrinsic tryptophan fluorescence or the circular dichroism signal at 221 nm, showing that the transition is due to the global denaturation of the protein. Barstar contains two proline residues, one of which (Pro48) has a cis N-aminoacyl bond conformation in the folded state. A transiently generated form of the unfolded protein, which contains the proline residues in their native conformations, has a rate constant for refolding (31 s-1) similar to that for refolding of the equilibrium-unfolded protein, which results in a ''misfolded'' form of the protein (32 s-1). The two refolded states are different: the free energies of unfolding measured from kinetic constants for the native and misfolded variants are 5.4 +/- 0.3 and 2.85 +/- 0.1 kcal/mol, respectively. The rate constant for the unfolding in water of the misfolded protein is 0.87 s-1, compared with 0.068 s-1 for the unfolding of the native protein. This difference can be explained by a nonnative trans peptidyl-proline bond at position 48 in the misfolded protein. The transiently misfolded form inhibits barnase, showing that the structure is native-like. There is a trans to cis isomerization in the misfolded protein with a rate constant of 0.01 s-1, after which the protein has all the thermodynamic properties of native barstar. There is at least one further intermediate (I) on the folding pathway. DELTAG(U-I) is 0.74 kcal/mol for the folding of equilibrated unfolded protein and 1.9 kcal/mol when folding from the transiently generated unfolded protein. Thus, the folding of equilibrated unfolded barstar gives a native-like active structure, with a trans proline at position 48, which then isomerizes on a much slower time basis. The unfolding reaction goes rapidly from the folded to the unfolded state, after which the cis peptidyl-prolyl bond in the unfolded protein isomerizes to equilibrium. The last step accounts for the discrepancy between the 4.84 kcal/mol measured at equilibrium and the 5.4 kcal/mol measured from the kinetics for the free energy of unfolding, the cis nature of the peptidyl-proline bond in the native protein being a source of thermodynamic instability.