Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain.

The transition state for the folding pathway of the activation domain of human procarboxypeptidase A2 (ADA2h) has been analyzed by the protein engineering approach. Recombinant ADA2h is an 81-residue globular domain with no disulfide bridges or cis-prolyl bonds, which follows a two-state folding transition. Its native fold is arranged in two alpha-helices packing against a four-stranded beta-sheet. Application of the protein engineering analysis for 20 single-point mutants spread throughout the whole sequence indicates that the transition state for this molecule is quite compact, possessing some secondary structure and a hydrophobic core in the process of being consolidated. The core (folding nucleus) is made by the packing of alpha-helix 2 and the two central beta-strands. The other two strands, at the edges of the beta-sheet, and alpha-helix 1 seem to be completely unfolded. These results, together with previous analysis of ADA2h with either of its two alpha-helices stabilized through improved local interactions, suggest that alpha-helix 1 does not contribute to the folding nucleus, even though it is partially folded in the denatured state under native conditions. On the other hand, alpha-helix 2 folds partly in the transition state and is part of the folding nucleus. It is suggested that a good strategy to improve folding speed in proteins would be to stabilize the helices that are not folded in the denatured state but are partly present in the transition state. Comparison with other proteins shows that there is no clear relationship between fold and/or size with folding speed and level of structure in the transition state of proteins.

[1]  A. Fersht,et al.  Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[2]  A. Fersht,et al.  Mapping the transition state and pathway of protein folding by protein engineering , 1989, Nature.

[3]  G Vriend,et al.  WHAT IF: a molecular modeling and drug design program. , 1990, Journal of molecular graphics.

[4]  L. Regan,et al.  Surface point mutations that significantly alter the structure and stability of a protein's denatured state , 1996, Protein science : a publication of the Protein Society.

[5]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[6]  L Serrano,et al.  The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability. , 1992, Journal of molecular biology.

[7]  Andreas Matouschek,et al.  Transient folding intermediates characterized by protein engineering , 1990, Nature.

[8]  A. Fersht.  Nucleation mechanisms in protein folding. , 1997, Current opinion in structural biology.

[9]  A. Fersht,et al.  The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding. , 1995, Journal of molecular biology.

[10]  Luis Serrano,et al.  Different folding transition states may result in the same native structure , 1996, Nature Structural Biology.

[11]  A. Fersht,et al.  Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. , 1991, Biochemistry.

[12]  L Serrano,et al.  Development of the multiple sequence approximation within the AGADIR model of alpha-helix formation: comparison with Zimm-Bragg and Lifson-Roig formalisms. , 1997, Biopolymers.

[13]  M. Billeter,et al.  The Sequence and Conformation of Human Pancreatic Procarboxypeptidase A2 , 1995, The Journal of Biological Chemistry.

[14]  L Serrano,et al.  Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding transition. , 1994, Biochemistry.

[15]  R. Sauer,et al.  P22 Arc repressor: transition state properties inferred from mutational effects on the rates of protein unfolding and refolding. , 1995, Biochemistry.

[16]  L Serrano,et al.  The folding of an enzyme. IV. Structure of an intermediate in the refolding of barnase analysed by a protein engineering procedure. , 1992, Journal of molecular biology.

[17]  A. Fersht,et al.  Protein folding and stability: the pathway of folding of barnase , 1993 .

[18]  Serrano,et al.  Structure of the transition state for folding of the 129 aa protein CheY resembles that of a smaller protein, CI-2. , 1995, Folding & design.

[19]  A. Fersht,et al.  Movement of the position of the transition state in protein folding. , 1995, Biochemistry.

[20]  T. Sosnick,et al.  The barriers in protein folding , 1994, Nature Structural Biology.

[21]  R. L. Baldwin.  Pulsed H/D-exchange studies of folding intermediates , 1993 .

[22]  P. Alexander,et al.  Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G. , 1992, Biochemistry.

[23]  L. Serrano,et al.  Obligatory steps in protein folding and the conformational diversity of the transition state , 1998, Nature Structural &Molecular Biology.

[24]  A. Fersht,et al.  The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. , 1992, Journal of molecular biology.

[25]  F M Poulsen,et al.  Fast and one-step folding of closely and distantly related homologous proteins of a four-helix bundle family. , 1996, Journal of molecular biology.

[26]  Terrence G. Oas,et al.  The energy landscape of a fast-folding protein mapped by Ala→Gly Substitutions , 1997, Nature Structural Biology.

[27]  L Serrano,et al.  Favourable native-like helical local interactions can accelerate protein folding. , 1997, Folding & design.

[28]  Villegas,et al.  Stabilization of proteins by rational design of alpha-helix stability using helix/coil transition theory. , 1995, Folding & design.

[29]  F. Avilés,et al.  The three‐dimensional structure of human procarboxypeptidase A2. Deciphering the basis of the inhibition, activation and intrinsic activity of the zymogen , 1997, The EMBO journal.

[30]  A. Fersht,et al.  Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[32]  M. Marahiel,et al.  Extremely rapid protein folding in the absence of intermediates , 1995, Nature Structural Biology.

[33]  A. Fersht,et al.  Characterizing transition states in protein folding: an essential step in the puzzle. , 1995, Current opinion in structural biology.

[34]  L Serrano,et al.  The folding of an enzyme. III. Structure of the transition state for unfolding of barnase analysed by a protein engineering procedure. , 1992, Journal of molecular biology.

[35]  L Serrano,et al.  Evidence for a two-state transition in the folding process of the activation domain of human procarboxypeptidase A2. , 1995, Biochemistry.

[36]  L. Serrano,et al.  1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein. , 1993, European journal of biochemistry.

[37]  G. R. Taylor,et al.  PCR: a practical approach. , 1991 .

[38]  Luis Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters , 1994, Nature Structural Biology.