Solvent‐tuning the collapse and helix formation time scales of λ6‐85*

The λ6‐85* pseudo‐wild type of lambda repressor fragment is a fast two‐state folder (kf ≈ 35 μsec−1 at 58°C). Previously, highly stable λ6‐85* mutants with kf > 30 μsec−1 have been engineered to fold nearly or fully downhill. Stabilization of the native state by solvent tuning might also tune λ6‐85* away from two‐state folding. We test this prediction by examining the folding thermodynamics and kinetics of λ6‐85* in a stabilizing solvent, 45% by weight aqueous ethylene glycol at −28°C. Detection of kinetics by circular dichroism at 222 nm (sensitive to helix content) and small angle X‐ray scattering (measuring the radius of gyration) shows that refolding from guanidine hydrochloride denatured conditions exhibits very different time scales for collapse and secondary structure formation: the two processes become decoupled. Collapse remains a low‐barrier activated process, while the fastest of several secondary structure formation time scales approaches the downhill folding limit. Two‐state folding of λ6‐85* is not a robust process.

[1]  W. M. Haynes.  CRC Handbook of Chemistry and Physics , 1990 .

[2]  V. Muñoz,et al.  Experimental Identification of Downhill Protein Folding , 2002, Science.

[3]  Lisa J Lapidus,et al.  How fast is protein hydrophobic collapse? , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Oas,et al.  Fast and faster: A designed variant of the B‐domain of protein A folds in 3 μsec , 2004 .

[5]  Masao Kakudo,et al.  Small Angle Scattering of X-Rays , 1968 .

[6]  T. Oas,et al.  Submillisecond folding of monomeric lambda repressor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  T. Irving,et al.  The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source. , 2004, Journal of synchrotron radiation.

[8]  B Honig,et al.  On the pH dependence of protein stability. , 1993, Journal of molecular biology.

[9]  J. Hofrichter,et al.  Experimental tests of villin subdomain folding simulations. , 2003, Journal of molecular biology.

[10]  Athi N. Naganathan,et al.  Robustness of downhill folding: guidelines for the analysis of equilibrium folding experiments on small proteins. , 2005, Biochemistry.

[11]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

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

[13]  Martin Gruebele,et al.  Engineering a β-sheet protein toward the folding speed limit , 2005 .

[14]  K. Kuwajima,et al.  The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy. , 1996, Journal of molecular biology.

[15]  Y. Amemiya,et al.  Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy. , 1998, Journal of molecular biology.

[16]  L Serrano,et al.  Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters. , 1998, Journal of molecular biology.

[17]  Robin S. Dothager,et al.  Random-coil behavior and the dimensions of chemically unfolded proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[18]  E. Henry,et al.  [8] Singular value decomposition: Application to analysis of experimental data , 1992 .

[19]  H. Kramers.  Brownian motion in a field of force and the diffusion model of chemical reactions , 1940 .

[20]  V. Muñoz.  Thermodynamics and kinetics of downhill protein folding investigated with a simple statistical mechanical model , 2002 .

[21]  A. Roitberg,et al.  Smaller and faster: the 20-residue Trp-cage protein folds in 4 micros. , 2002, Journal of the American Chemical Society.

[22]  R. E. Bolz,et al.  CRC Handbook of tables for Applied Engineering Science , 1970 .

[23]  M. Gruebele,et al.  Observation of strange kinetics in protein folding. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Gruebele,et al.  Binary and ternary aggregation within tethered protein constructs. , 2006, Biophysical journal.

[25]  Yan Zhang,et al.  Structure-function-folding relationship in a WW domain. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Hofrichter,et al.  The protein folding 'speed limit'. , 2004, Current opinion in structural biology.

[27]  M. Arai,et al.  Refolding of β‐lactoglobulin studied by stopped‐flow circular dichroism at subzero temperatures , 2001, FEBS letters.

[28]  D Baker,et al.  The sequences of small proteins are not extensively optimized for rapid folding by natural selection. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  D. Svergun,et al.  CRYSOL : a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates , 1995 .

[30]  H. Gray,et al.  Early events in the folding of four-helix-bundle heme proteins. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. L. Baldwin.  The nature of protein folding pathways: The classical versus the new view , 1995, Journal of biomolecular NMR.

[32]  D. Baker,et al.  Chain collapse can occur concomitantly with the rate-limiting step in protein folding , 1999, Nature Structural Biology.

[33]  M. Gruebele,et al.  Detection-dependent kinetics as a probe of folding landscape microstructure. , 2004, Journal of the American Chemical Society.

[34]  M. Gruebele,et al.  Folding λ-repressor at its speed limit , 2004 .

[35]  D Baker,et al.  Limited internal friction in the rate-limiting step of a two-state protein folding reaction. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Gruebele.  Downhill protein folding: evolution meets physics. , 2005, Comptes rendus biologies.

[37]  R. Brüschweiler,et al.  Backbone dynamics and structural characterization of the partially folded A state of ubiquitin by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy. , 1997, Biochemistry.

[38]  Valerie Daggett,et al.  Ultrafast folding of α3D: A de novo designed three-helix bundle protein , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Gruebele,et al.  Formation of a Compact Structured Ensemble without Fluorescence Signature Early during Ubiquitin Folding , 2002 .

[40]  Martin Gruebele,et al.  Rate−Temperature Relationships in λ-Repressor Fragment λ6-85 Folding† , 2004 .

[41]  M. Gruebele,et al.  Kinetic equivalence of the heat and cold structural transitions of λ6–85 , 2005, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences.

[42]  Y. Nozaki.  The preparation of guanidine hydrochloride. , 1972, Methods in enzymology.

[43]  V. Muñoz,et al.  Exploring protein-folding ensembles: a variable-barrier model for the analysis of equilibrium unfolding experiments. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  K. Schulten,et al.  Multiple probes reveal a native-like intermediate during low-temperature refolding of ubiquitin. , 2004, Journal of Molecular Biology.

[45]  S Doniach,et al.  Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering. , 1999, Journal of molecular biology.

[46]  T. Sosnick,et al.  Distinguishing between two-state and three-state models for ubiquitin folding. , 2000, Biochemistry.

[47]  C. Dobson.  Protein folding and misfolding , 2003, Nature.

[48]  Martin Gruebele,et al.  Folding at the speed limit , 2003, Nature.

[49]  S. Ghaemmaghami,et al.  Folding kinetics of a fluorescent variant of monomeric lambda repressor. , 1998, Biochemistry.

[50]  M. Gruebele,et al.  On the extended β-conformation propensity of polypeptides at high temperature , 2003 .

[51]  D. Thirumalai,et al.  Viscosity Dependence of the Folding Rates of Proteins , 1997, cond-mat/9705309.

[52]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.

[53]  A. Fersht,et al.  One-state downhill versus conventional protein folding. , 2004, Journal of molecular biology.

[54]  M. Gruebele,et al.  Correlation of early orientational ordering of engineered λ6–85 structure with kinetics and thermodynamics , 2006 .

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

[56]  M. Gruebele,et al.  Kinetics are probe-dependent during downhill folding of an engineered λ6–85 protein , 2005 .

[57]  T. Sosnick,et al.  Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil. , 1999, Biochemistry.