Folding pathways of proteins with increasing degree of sequence identities but different structure and function

Much experimental work has been devoted in comparing the folding behavior of proteins sharing the same fold but different sequence. The recent design of proteins displaying very high sequence identities but different 3D structure allows the unique opportunity to address the protein-folding problem from a complementary perspective. Here we explored by Φ-value analysis the pathways of folding of three different heteromorphic pairs, displaying increasingly high-sequence identity (namely, 30%, 77%, and 88%), but different structures called GA (a 3-α helix fold) and GB (an α/β fold). The analysis, based on 132 site-directed mutants, is fully consistent with the idea that protein topology is committed very early along the pathway of folding. Furthermore, data reveals that when folding approaches a perfect two-state scenario, as in the case of the GA domains, the structural features of the transition state appear very robust to changes in sequence composition. On the other hand, when folding is more complex and multistate, as for the GBs, there are alternative nuclei or accessible pathways that can be alternatively stabilized by altering the primary structure. The implications of our results in the light of previous work on the folding of different members belonging to the same protein family are discussed.

[1]  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.

[2]  M. Brunori,et al.  Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain , 2010, Nature Structural &Molecular Biology.

[3]  M. Brunori,et al.  Exploring the Cytochrome c Folding Mechanism , 2003, Journal of Biological Chemistry.

[4]  P. Alexander,et al.  A minimal sequence code for switching protein structure and function , 2009, Proceedings of the National Academy of Sciences.

[5]  Ellinor Haglund,et al.  Changes of Protein Folding Pathways by Circular Permutation , 2008, Journal of Biological Chemistry.

[6]  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.

[7]  E I Shakhnovich,et al.  Specific nucleus as the transition state for protein folding: evidence from the lattice model. , 1994, Biochemistry.

[8]  V Muñoz,et al.  Folding dynamics and mechanism of beta-hairpin formation. , 1997, Nature.

[9]  Valerie Daggett,et al.  The Denatured State Dictates the Topology of Two Proteins with Almost Identical Sequence but Different Native Structure and Function* , 2010, The Journal of Biological Chemistry.

[10]  E. Cota,et al.  Folding studies of immunoglobulin-like beta-sandwich proteins suggest that they share a common folding pathway. , 1999, Structure.

[11]  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.

[12]  Characterisation of transition state structures for protein folding using 'high', 'medium' and 'low' {Phi}-values. , 2008, Protein engineering, design & selection : PEDS.

[13]  T. Kiefhaber,et al.  Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate. , 1997, Journal of molecular biology.

[14]  John Orban,et al.  The design and characterization of two proteins with 88% sequence identity but different structure and function , 2007, Proceedings of the National Academy of Sciences.

[15]  M. J. Parker,et al.  An integrated kinetic analysis of intermediates and transition states in protein folding reactions. , 1995, Journal of molecular biology.

[16]  J. Onuchic,et al.  Funnels, pathways, and the energy landscape of protein folding: A synthesis , 1994, Proteins.

[17]  John Orban,et al.  NMR structures of two designed proteins with high sequence identity but different fold and function , 2008, Proceedings of the National Academy of Sciences.

[18]  Engineered symmetric connectivity of secondary structure elements highlights malleability of protein folding pathways. , 2009, Journal of the American Chemical Society.

[19]  M. Vendruscolo,et al.  Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins , 2008, Proceedings of the National Academy of Sciences.

[20]  Valerie Daggett,et al.  GB1 is not a two-state folder: identification and characterization of an on-pathway intermediate. , 2011, Biophysical journal.

[21]  M. Brunori,et al.  A common folding mechanism in the cytochrome c family. , 2004, Trends in biochemical sciences.

[22]  Alan R. Fersht,et al.  From the first protein structures to our current knowledge of protein folding: delights and scepticisms , 2008, Nature Reviews Molecular Cell Biology.

[23]  A. Fersht Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[24]  David Baker,et al.  Experiment and theory highlight role of native state topology in SH3 folding , 1999, Nature Structural Biology.

[25]  Luis Serrano,et al.  The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved , 1999, Nature Structural Biology.

[26]  D. Baker,et al.  A surprising simplicity to protein folding , 2000, Nature.

[27]  D. W. Bolen,et al.  Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. , 1988, Biochemistry.

[28]  Yale E Goldman,et al.  Lever-arm mechanics of processive myosins. , 2011, Biophysical journal.

[29]  A. Fersht,et al.  Phi-value analysis and the nature of protein-folding transition states. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Oliveberg,et al.  Malleability of protein folding pathways: a simple reason for complex behaviour. , 2007, Current opinion in structural biology.

[31]  J. Hofrichter,et al.  Sub-microsecond protein folding. , 2006, Journal of molecular biology.

[32]  M. Oliveberg,et al.  Transient aggregates in protein folding are easily mistaken for folding intermediates. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Stefan M. Larson,et al.  The family feud: do proteins with similar structures fold via the same pathway? , 2005, Current opinion in structural biology.

[34]  P. Jemth,et al.  A conserved folding mechanism for PDZ domains , 2007, FEBS letters.

[35]  V. Muñoz,et al.  Folding dynamics and mechanism of β-hairpin formation , 1997, Nature.

[36]  P. Alexander,et al.  Engineering subtilisin into a fluoride-triggered processing protease useful for one-step protein purification. , 2004, Biochemistry.

[37]  P. Jemth,et al.  Folding and stability of globular proteins and implications for function. , 2009, Current opinion in structural biology.

[38]  Sheena E Radford,et al.  Structural analysis of the rate-limiting transition states in the folding of Im7 and Im9: similarities and differences in the folding of homologous proteins. , 2003, Journal of molecular biology.