PubMed Health⌕ Search

Biomedical subjects

D D Axe

Publications and source records attributed to D D Axe.

5 recordsLinked to original sources
PubMed2000

Extreme functional sensitivity to conservative amino acid changes on enzyme exteriors.

Mutagenesis studies and alignments of homologous sequences have demonstrated that protein function typically is compatible with a variety of amino-acid residues at most exterior non-active-site positions. These observations have led to the current view that functional constraints on sequence are minimal at these positions. Here, it is shown that this inference assumes that the set of acceptable residues at each position is independent of the overall sequence context. Two approaches are used to test this assumption. First, highly conservative replacements of exterior residues, none of which would cause significant functional disruption alone, are combined until roughly one in five have been changed. This is found to cause complete loss of function in vivo for two unrelated monomeric enzymes: barnase (a bacterial RNase) and TEM-1 beta-lactamase. Second, a set of hybrid sequences is constructed from the 50 %-identical TEM-1 and Proteus mirabilis beta-lactamases. These hybrids match the TEM-1 sequence except for a region at the C-terminal end, where they are random composites of the two parents. All of these hybrids are biologically inactive. In both experiments, complete loss of activity demonstrates the importance of sequence context in determining whether substitutions are functionally acceptable. Contrary to the prevalent view, then, enzyme function places severe constraints on residue identities at positions showing evolutionary variability, and at exterior non-active-site positions, in particular. Homologues sharing less than about two-thirds sequence identity should probably be viewed as distinct designs with their own sets of optimising features.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1996

Active barnase variants with completely random hydrophobic cores.

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution. To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives. Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function. Of the active mutants produced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes. Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.

Amino Acid Sequence↗