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(delta delta G/delta [urea]) = 1.25 +/- 0.04 kcal/mol2. The free energy of unfolding, delta GU-FH2O, 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.(ABSTRACT TRUNCATED AT 250 WORDS)