Disulfide engineering at the dimer interface of lactobacillus casei thymidylate synthase: Crystal structure of the T155C/E188C/C244T mutant

The crystal structure of a covalently cross‐linked Lactobacillus casei thymidylate synthase has been determined at 2.8 å resolution. The sites for mutation to achieve the bis‐disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.

[1]  A. Tamura,et al.  The entropy cost of protein association. , 1997, Journal of molecular biology.

[2]  Robert L. Campbell,et al.  Thermostabilization of the Bacillus circulansxylanase by the introduction of disulfide bonds , 1994 .

[3]  N Srinivasan,et al.  Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site-directed mutagenesis. , 1989, Protein engineering.

[4]  D. Powers,et al.  In vivo formation and stability of engineered disulfide bonds in subtilisin. , 1986, The Journal of biological chemistry.

[5]  M. Yaguchi,et al.  Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds. , 1994, Protein engineering.

[6]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[7]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[8]  Cavity-creating mutation at the dimer interface of Plasmodium falciparum triosephosphate isomerase: restoration of stability by disulfide cross-linking of subunits. , 1999, Biochemistry.

[9]  R M Stroud,et al.  Atomic structure of thymidylate synthase: target for rational drug design. , 1987, Science.

[10]  P. Balaram,et al.  Synthetic interface peptides as inactivators of multimeric enzymes: inhibitory and conformational properties of three fragments from Lactobacillus casei thymidylate synthase. , 1998, Biochemistry.

[11]  N. Scrutton,et al.  Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli , 1988, FEBS letters.

[12]  R. Wetzel.  Harnessing disulfide bonds using protein engineering , 1987 .

[13]  R. Sauer,et al.  An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor. , 1986, Biochemistry.

[14]  Barry C. Finzel,et al.  The use of an imaging proportional counter in macromolecular crystallography , 1987 .

[15]  D. Santi,et al.  Thermal stabilization of thymidylate synthase by engineering two disulfide bridges across the dimer interface. , 1994, Journal of molecular biology.

[16]  D. Santi,et al.  Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase , 1996, Protein science : a publication of the Protein Society.

[17]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .