Biomedical subjects

G Winter

Publications and source records attributed to G Winter.

215 records · Page 12Linked to original sources

PubMedDate not supplied

A large increase in enzyme-substrate affinity by protein engineering.

A single point mutation has been engineered in the tyrosyl-tRNA synthetase that improves its affinity (KM) for its substrate ATP by a factor of 100. In the crystal structure of the tyrosyl tRNA synthetase (of Bacillus stearothermophilus), the side-chain hydroxyl of Thr 51 appears to make a weak hydrogen bond with the AMP moiety of the substrate intermediate, tyrosyl adenylate. In the absence of substrate, however, the hydroxyl group should make a strong hydrogen bond with water which would favour dissociation of the enzyme-substrate complex. We have used oligodeoxynucleotide-directed mutagenesis to construct two point mutants at this site: one to remove the hydroxyl group (Thr 51 leads to Ala 51) and the other, in addition, to distort the local polypeptide backbone (Thr 51 leads to Pro 51). We report here that both mutants have increased activity (kcat/KM for ATP) but one mutant (Pro 51) shows a massive 25-fold increase due mainly to a lowered KM for ATP. This demonstrates dramatically the potential of in vitro mutagenesis for improving the affinity of an enzyme for its substrate.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

Hydrogen bonding and biological specificity analysed by protein engineering.

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

A model of synthetase/transfer RNA interaction as deduced by protein engineering.

The recognition of transfer-RNA by their cognate aminoacyl-tRNA synthetases is the crucial step in the translation of the genetic code. In order to construct a structural model of the complex between the tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus and tRNATyr, 40 basic residues at the surface of the TyrTS dimer have been mutated by site-directed mutagenesis and heterodimers created in vitro by recombining subunits derived from different mutants. As reported here a cluster of basic residues (Arg 207-Lys 208) in the N-terminal domain of one TyrTS subunit interacts with the acceptor stem of tRNATyr and two separated clusters of basic residues (Arg 368-Arg 371; Arg 407-Arg 408-Lys 410-Lys 411) in the C-terminal domain of the other subunit interact with the anticodon arm. The TyrTS would thus clamp the tRNA in a fixed orientation. The precise alignment of the flexible... ACCA 3' end of the tRNA for attack on the tyrosyl adenylate is made by contacts closer to the catalytic groups of the enzyme, such as with Lys 151.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

Replacing the complementarity-determining regions in a human antibody with those from a mouse.

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.

Amino Acid Sequence↗

PubMedDate not supplied