FOLDING OF BARNASE IN PARTS

Stretches of residual structure in the unfolded states of proteins could possibly constitute crucial regions that initiate protein folding. We are searching for such regions in barnase by dividing it into fragments. By this means, we can search for regions that just form within local sequences. We are also employing methods that can detect low levels of residual structure. In this study, we examine the fragment 1-22 and a large fragment (23-110) that contains all of the catalytic residues. Fragment 1-22 contains the first alpha-helix, and fragment 23-110 contains the second alpha-helix and beta-sheet structure-forming residues of native barnase. These fragments bind together rapidly and tightly upon association to form a fully native like complex. Studies by circular dichroism and fluorescence spectroscopy indicate that each fragment is mainly disordered. However, we find by a procedure of titration with trifluoroethanol that about 3% of fragment 1-22 is helical in water at 25-degrees-C. Importantly, we have detected residual catalytic activity in fragment 23-110 toward GpUp and RNA and the ability to bind the polypeptide inhibitor of barnase, barstar, suggesting that this fragment can form a nativelike conformation in water. The catalytic activity does not result from a small amount of contaminating impurity of parent enzyme or other ribonuclease, since the activity requires a 1:1 mole ratio of fragment to barstar for complete inhibition, and the activity is lost in much lower concentrations of urea than are required to denature the parent enzyme. There is a very weak signal in the near-UV CD spectrum of the large fragment. This is enhanced on the binding of CGAC, a tight-binding substrate analogue of barnase. This implies that there is small amount of preexisting structure in the fragment that is enhanced upon the binding of GpUp or barstar. Thus, evidence from this study on fragments, and from earlier studies on the intact enzyme, shows that barnase can fold by association of independently folded regions of structure.