An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase.


Single amino acid residue substitutions rarely destroy the structural integrity of proteins. Substitution of glycine residues, however, is among the few sorts of alterations that can have such an effect. Here, we seek to understand what accounts for the extreme functional impairment of the bacterial ribonuclease barnase upon substitution of Gly52 or Gly53. We find that inactivation is caused by overall disruption of the folded state that manifests itself in three ways: (1) dramatically reduced stability (by 5.2 to 8.4 kcal mol-1 for mutants showing inactivation in vivo); (2) progressive loss of folded-state activity with increasing temperature, indicating a less well formed fold; and (3) substantial proteolytic degradation of mutant enzymes in vivo. Examination of two deletion mutants, missing either Gly53 or Asp54, shows that the irregular beta-bulge formed by these two residues is of vital importance to the structural integrity of barnase. The parallel behaviour of mutants carrying replacements of either of the two glycine residues therefore appears to arise from a common mechanism: disruption of local structure at the beta-bulge. The importance of this structural element to the function of barnase raises the question of whether it may be present in other RNases. The Streptomyces enzymes RNase Sa and RNase St differ considerably from barnase in both sequence and structure, yet both show significant sequence similarity to barnase over a region beginning at Gly53. Structural comparison indicates that the Streptomyces enzymes do have the barnase-like irregular beta-bulge, making this an important characteristic feature of a group of bacterial ribonucleases. The sensitivity of this feature demonstrates that detailed aspects of local structure may have a major role in determining the overall structural and functional properties of an enzyme, even where no explanation for this role is readily apparent. If this is a general characteristic of the structure-function relationship, it may pose a formidable obstacle to the de novo design of new enzymes. Study holds ProTherm entries: 6057, 6058, 6059, 6060, 6061, 14292, 14293, 14294, 14295 Extra Details: beta-bulge; microbial ribonuclease; molecular evolution;,inactivating substitutions; destabilising substitutions

Submission Details

ID: h3zaRYyM

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Axe DD;Foster NW;Fersht AR,J. Mol. Biol. (1999) An irregular beta-bulge common to a group of bacterial RNases is an important determinant of stability and function in barnase. PMID:10064710
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease P00648 RNBR_BACAM
97.3 Ribonuclease P35078 RN_BACCI