FOLDING OF BARNASE IN PARTS

Stretches of residual structure in the unfolded states of proteins cou ld possibly constitute crucial regions that initiate protein folding. We are searching for such regions in barnase by dividing it into fragm ents. By this means, we can search for regions that just form within l ocal sequences. We are also employing methods that can detect low leve ls of residual structure. In this study, we examine the fragment 1-22 and a large fragment (23-110) that contains all of the catalytic resid ues. Fragment 1-22 contains the first alpha-helix, and fragment 23-110 contains the second alpha-helix and beta-sheet structure-forming resi dues of native barnase. These fragments bind together rapidly and tigh tly upon association to form a fully native like complex. Studies by c ircular dichroism and fluorescence spectroscopy indicate that each fra gment is mainly disordered. However, we find by a procedure of titrati on with trifluoroethanol that about 3% of fragment 1-22 is helical in water at 25-degrees-C. Importantly, we have detected residual catalyti c activity in fragment 23-110 toward GpUp and RNA and the ability to b ind the polypeptide inhibitor of barnase, barstar, suggesting that thi s 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 th e activity is lost in much lower concentrations of urea than are requi red 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 bin ding of CGAC, a tight-binding substrate analogue of barnase. This impl ies that there is small amount of preexisting structure in the fragmen t 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 fo lded regions of structure.