A SEARCH FOR SINGLE SUBSTITUTIONS THAT ELIMINATE ENZYMATIC FUNCTION IN A BACTERIAL RIBONUCLEASE

Exhaustive-substitution studies, where many amino acid replacements are individually tested at all positions in a natural protein, have proven to be very valuable in probing the relationship between sequence and function. The broad picture that has emerged from studies of this sort is one of functional tolerance of substitution. We have applied this approach to barnase, a 110-residue bacterial ribonuclease. Because the selection system used to score barnase mutants as active or inactive detects activity down to a level that can be approached by nonenzyme catalysts, mutants that test inactive are essentially devoid of enzymatic function. Of the 109 barnase positions subjected to substitution, only 15 (14%) are vulnerable to this extreme level of inactivation, and only 2 could not be substituted without such inactivation. A total of 33 substitutions (amounting to 5% of the explored substitutions) were found to render barnase wholly inactive. The profoundly disruptive effects of all of these inactivating substitutions appear to result from either (1) replacement of a side chain that is directly involved in substrate binding or catalysis, (2) replacement of a substantially buried side chain, (3) introduction of a proline residue, or (4) replacement of a glycine residue. Although substitutions of these types are functionally tolerated more often than not, the system used here indicates that only these sorts of substitution are capable of single-handedly reducing catalytic function to, or nearly to, levels that can be achieved by nonenzyme catalysts. It is hoped that investigations of protein folding will ultimately yield a comprehensive understanding of the relationship between protein sequence and structure. Al- though this is an ambitious undertaking, it is only half of the larger effort aimed at elucidating the relationship between sequence and function. The other half, aimed at understand- ing the structure-function relationship, is no less ambitious. While a general solution to the grand problem of relating protein sequence to function will clearly be some time in the making, we do have at our disposal, in the form of natural proteins, thousands of specific solutions to this problem. It therefore makes good sense for us to glean as much raw data as we possibly can from these natural solutions. Given the complexities and subtleties of the sequence-function relationship, it also makes sense for us to collect these data in a manner that is entirely unbiased by any a priori expectations we may hold regarding the nature of that relationship. The simplest and most direct way to obtain such an unbiased data set is to produce and test a large collection of mutant proteins where all possible single-residue substitutions are represented. This might be termed the exhaustive- substitution approach. Since all single-replacement pos- sibilities are examined by this approach, the data lack any imprint of the experimenter's expectations, and they are complete in the sense that the entire molecule is examined