Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo.

An important question in protein folding is whether the natural amino and carboxyl termini and the given order of secondary structure segments are critical to the stability and to the folding pathway of proteins. Here it is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli. The variants are enzymically active and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria, despite the creation of new termini and the cleavage of a surface loop. This novel genetic approach should be useful for protein folding studies both in vitro and in vivo.

[1]  T. Jardetzky,et al.  Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Carbon,et al.  Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene. , 1980, Gene.

[3]  S. Harrison,et al.  Is there a single pathway for the folding of a polypeptide chain? , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Creighton,et al.  Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor. , 1983, Journal of molecular biology.

[5]  Cyrus Chothia,et al.  The 14th barrel rolls out , 1988, Nature.

[6]  H. Fritz,et al.  The gapped duplex DNA approach to oligonucleotide-directed mutation construction. , 1984, Nucleic acids research.

[7]  E. W. Miles,et al.  An active proteolytic derivative of the alpha subunit of tryptophan synthase. Identification of the site of cleavage and characterization of the fragments. , 1979, Biochemistry.

[8]  R. Jaenicke,et al.  Folding and association of proteins. , 1982, Biophysics of structure and mechanism.

[9]  Janet M. Thornton,et al.  Prediction of protein structure from amino acid sequence , 1978, Nature.

[10]  H. Scheraga,et al.  Experimental and theoretical aspects of protein folding. , 1975, Advances in protein chemistry.

[11]  P. Maycox,et al.  Posttranslational processing of concanavalin A precursors in jackbean cotyledons , 1986, The Journal of cell biology.

[12]  G. Braus,et al.  The role of the TRP1 gene in yeast tryptophan biosynthesis. , 1988, The Journal of biological chemistry.

[13]  J M Thornton,et al.  Amino and carboxy-terminal regions in globular proteins. , 1983, Journal of molecular biology.

[14]  C. Matthews,et al.  Folding of homologous proteins: conservation of the folding mechanism of the alpha subunit of tryptophan synthase from Escherichia coli, Salmonella typhimurium, and five interspecies hybrids. , 1988, Biochemistry.

[15]  M. Goldberg.  The second translation of the genetic message: protein folding and assembly , 1985 .

[16]  T. Jardetzky,et al.  [48] Phosphoribosylanthranilate isomerase—indoleglycerol-phosphate synthase from Escherichia coli , 1987 .

[17]  E. Padlan,et al.  Three-dimensional structure of the tryptophan synthase alpha 2 beta 2 multienzyme complex from Salmonella typhimurium. , 1988, The Journal of biological chemistry.

[18]  O. Ptitsyn,et al.  Why do globular proteins fit the limited set of folding patterns? , 1987, Progress in biophysics and molecular biology.

[19]  L. Hood,et al.  A gas-liquid solid phase peptide and protein sequenator. , 1981, The Journal of biological chemistry.

[20]  G M Edelman,et al.  Favin versus concanavalin A: Circularly permuted amino acid sequences. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[21]  K. Yutani,et al.  Guanidine hydrochloride induced unfolding of the alpha subunit of tryptophan synthase and of the two alpha proteolytic fragments: evidence for stepwise unfolding of the two alpha domains. , 1982, Biochemistry.

[22]  G. Reeke,et al.  Three-dimensional structure of favin: saccharide binding-cyclic permutation in leguminous lectins. , 1986, Science.

[23]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.