Novel folded protein domains generated by combinatorial shuffling of polypeptide segments.

It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a beta-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

[1]  M. Deem,et al.  A hierarchical approach to protein molecular evolution. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Whisstock,et al.  Serpins in the Caenorhabditis elegans genome , 1999 .

[3]  G. Winter,et al.  Proteolytic selection for protein folding using filamentous bacteriophages. , 1998, Folding & design.

[4]  J E Darnell,et al.  Speculations on the early course of evolution. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Whisstock,et al.  Serpins in the Caenorhabditis elegans genome. , 1999, Proteins.

[6]  H R Hoogenboom,et al.  Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. , 1991, Nucleic acids research.

[7]  B. Rost,et al.  Improved prediction of protein secondary structure by use of sequence profiles and neural networks. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[8]  D. Söll,et al.  Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli , 1988, Microbiological reviews.

[9]  M. Riley,et al.  Organization of the bacterial chromosome , 1990, Microbiological reviews.

[10]  A G Murzin,et al.  SCOP: a structural classification of proteins database for the investigation of sequences and structures. , 1995, Journal of molecular biology.

[11]  Alan R. Fersht,et al.  Directed evolution of new catalytic activity using the α/β-barrel scaffold , 2000, Nature.

[12]  L. Riechmann,et al.  An antibody VH domain with a lox‐Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain , 1995, FEBS letters.

[13]  L. H. Bradley,et al.  Protein design by binary patterning of polar and nonpolar amino acids. , 1993, Methods in molecular biology.

[14]  L. Riechmann,et al.  The C-Terminal Domain of TolA Is the Coreceptor for Filamentous Phage Infection of E. coli , 1997, Cell.

[15]  W. Stemmer,et al.  DNA shuffling of a family of genes from diverse species accelerates directed evolution , 1998, Nature.

[16]  C. Blake.  Molecular biology: Exons — present from the beginning? , 1983, Nature.

[17]  S J de Souza,et al.  Origin of genes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[19]  A. Fersht,et al.  Assignment of the backbone 1H and 15N NMR resonances and secondary structure characterization of barstar , 1993, FEBS letters.

[20]  R. Hartley,et al.  Directed mutagenesis and barnase-barstar recognition. , 1993, Biochemistry.

[21]  M. Inouye,et al.  Major cold shock protein of Escherichia coli. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[22]  C. Pace,et al.  Conformational stability of globular proteins. , 1990, Trends in biochemical sciences.

[23]  J. H. Shim,et al.  Combinatorial protein engineering by incremental truncation. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  V. I. Ugarov,et al.  Nonhomologous RNA Recombination in a Cell-Free System: Evidence for a Transesterification Mechanism Guided by Secondary Structure , 1997, Cell.

[25]  Phillip A. Sharp,et al.  On the origin of RNA splicing and introns , 1985, Cell.

[26]  L Regan,et al.  Proteins to order? , 1998, Structure.

[27]  M. Zuker,et al.  Testing the exon theory of genes: the evidence from protein structure. , 1994, Science.

[28]  C. Blake,et al.  Do genes-in-pieces imply proteins-in-pieces? , 1978, Nature.

[29]  B. Mcclintock,et al.  Chromosome organization and genic expression. , 1951, Cold Spring Harbor symposia on quantitative biology.

[30]  H. Edelhoch,et al.  The thermodynamic basis of the stability of proteins, nucleic acids, and membranes. , 1976, Advances in protein chemistry.

[31]  W. Gilbert.  Why genes in pieces? , 1978, Nature.

[32]  W. Gilbert.  Origin of life: The RNA world , 1986, Nature.

[33]  Mark Proctor,et al.  The Solution Structure of the S1 RNA Binding Domain: A Member of an Ancient Nucleic Acid–Binding Fold , 1997, Cell.

[34]  L Serrano,et al.  Effect of active site residues in barnase on activity and stability. , 1992, Journal of molecular biology.

[35]  L. Riechmann,et al.  Backbone assignment, secondary structure and Protein A binding of an isolated, human antibody VH domain , 1995, Journal of biomolecular NMR.

[36]  M. Hecht,et al.  De novo design of beta-sheet proteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[37]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[38]  M. Lai.  RNA recombination in animal and plant viruses. , 1992, Microbiological reviews.

[39]  U Heinemann,et al.  Crystal structure of CspA, the major cold shock protein of Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[40]  G N Murshudov,et al.  The structural basis of sequence-independent peptide binding by OppA protein. , 1994, Science.

[41]  J. Richardson,et al.  Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[42]  G. Winter,et al.  A method for the selection of catalytic activity using phage display and proximity coupling. , 1999, Angewandte Chemie.

[43]  C. Chothia,et al.  Protein folds in the all-beta and all-alpha classes. , 1997, Annual review of biophysics and biomolecular structure.

[44]  D N Woolfson,et al.  Core-directed protein design. I. An experimental method for selecting stable proteins from combinatorial libraries. , 1999, Biochemistry.

[45]  G. Fasman,et al.  Computed circular dichroism spectra for the evaluation of protein conformation. , 1969, Biochemistry.

[46]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[47]  J. Udgaonkar,et al.  Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride. , 1995, Biochemistry.

[48]  A. Plückthun,et al.  Selecting proteins with improved stability by a phage-based method , 1998, Nature Biotechnology.