Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing

For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600–700 μs), which is followed by a slower phase with a denaturant-dependent time constant (2–30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U [rlhar2 ] I [rlhar2 ] N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.

[1]  L Mayne,et al.  Ultrafast signals in protein folding and the polypeptide contracted state. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

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

[3]  J. Hofrichter,et al.  Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Marqusee,et al.  The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions , 1997, Nature Structural Biology.

[5]  Mixing liquids in microseconds , 1985 .

[6]  T. Creighton.  How important is the molten globule for correct protein folding? , 1997, Trends in biochemical sciences.

[7]  H. Roder,et al.  Evidence for barrier-limited protein folding kinetics on the microsecond time scale , 1998, Nature Structural Biology.

[8]  D. Baker,et al.  Contact order, transition state placement and the refolding rates of single domain proteins. , 1998, Journal of molecular biology.

[9]  J. Hofrichter,et al.  Submillisecond protein folding kinetics studied by ultrarapid mixing. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Fersht,et al.  Submillisecond events in protein folding. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  A. Gronenborn,et al.  A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. , 1993, Science.

[12]  H. Roder,et al.  A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale. , 1998, Biophysical journal.

[13]  Valerie Daggett,et al.  Molecular dynamics simulations of hydrophobic collapse of ubiquitin , 1998, Protein science : a publication of the Protein Society.

[14]  G. Wider,et al.  NMR determination of residual structure in a urea-denatured protein, the 434-repressor. , 1992, Science.

[15]  J. Hofrichter,et al.  Laser temperature jump study of the helix<==>coil kinetics of an alanine peptide interpreted with a 'kinetic zipper' model. , 1997, Biochemistry.

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

[17]  C. M. Jones,et al.  Fast events in protein folding initiated by nanosecond laser photolysis. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  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.

[19]  H. Roder,et al.  Kinetic role of early intermediates in protein folding. , 1997, Current opinion in structural biology.

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

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

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

[23]  M. Gruebele,et al.  Direct observation of fast protein folding: the initial collapse of apomyoglobin. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Jackson,et al.  How do small single-domain proteins fold? , 1998, Folding & design.

[25]  L Serrano,et al.  Role of a nonnative interaction in the folding of the protein G B1 domain as inferred from the conformational analysis of the alpha-helix fragment. , 1997, Folding & design.

[26]  R. L. Baldwin,et al.  On-pathway versus off-pathway folding intermediates. , 1996, Folding & design.

[27]  A M Gronenborn,et al.  1.67-A X-ray structure of the B2 immunoglobulin-binding domain of streptococcal protein G and comparison to the NMR structure of the B1 domain. , 1992, Biochemistry.

[28]  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.

[29]  H. Scheraga,et al.  The nature of the initial step in the conformational folding of disulphide-intact ribonuclease A , 1995, Nature Structural Biology.

[30]  M. J. Parker,et al.  Topology, sequence evolution and folding dynamics of an immunoglobulin domain , 1998, Nature Structural Biology.

[31]  Patricia A. Jennings,et al.  Evidence for an obligatory intermediate in the folding of lnterleukin-1β , 1997, Nature Structural Biology.

[32]  R. Dyer,et al.  Fast events in protein folding: the time evolution of primary processes. , 1998, Annual review of physical chemistry.

[33]  V. Muñoz,et al.  Kinetics and Dynamics of Loops, α-Helices, β-Hairpins, and Fast-Folding Proteins , 1998 .

[34]  C L Brooks,et al.  Calculations on folding of segment B1 of streptococcal protein G. , 1998, Journal of molecular biology.

[35]  Soon-Ho Park,et al.  An early intermediate in the folding reaction of the B1 domain of protein G contains a native-like core. , 1997 .

[36]  A. Fersht,et al.  Folding intermediates of wild-type and mutants of barnase. I. Use of phi-value analysis and m-values to probe the cooperative nature of the folding pre-equilibrium. , 1998, Journal of molecular biology.

[37]  Tobin R. Sosnick,et al.  The burst phase in ribonuclease A folding and solvent dependence of the unfolded state , 1998, Nature Structural Biology.

[38]  R. Dyer,et al.  Fast events in protein folding: relaxation dynamics of secondary and tertiary structure in native apomyoglobin. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Dyer,et al.  Fast events in protein folding: helix melting and formation in a small peptide. , 1996, Biochemistry.

[40]  A. Gronenborn,et al.  Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G , 1994, Protein science : a publication of the Protein Society.

[41]  T. Kiefhaber,et al.  Kinetic traps in lysozyme folding. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[42]  M. J. Parker,et al.  Thermodynamic properties of transient intermediates and transition states in the folding of two contrasting protein structures. , 1998, Biochemistry.

[43]  D. Yee,et al.  Principles of protein folding — A perspective from simple exact models , 1995, Protein science : a publication of the Protein Society.

[44]  T. Oas,et al.  Microsecond protein folding through a compact transition state. , 1996, Journal of molecular biology.

[45]  S. Radford,et al.  The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate. , 1999, Journal of molecular biology.

[46]  J. Udgaonkar,et al.  Folding of tryptophan mutants of barstar: evidence for an initial hydrophobic collapse on the folding pathway. , 1997, Biochemistry.

[47]  H. Roder,et al.  Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease. , 1997, Biochemistry.

[48]  G. Schwarz,et al.  Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements. , 1979, Biophysical chemistry.

[49]  J. Onuchic,et al.  Fast-folding experiments and the topography of protein folding energy landscapes. , 1996, Chemistry & biology.

[50]  S. Khorasanizadeh,et al.  Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues , 1996, Nature Structural Biology.

[51]  H. Scheraga,et al.  A very fast phase in the refolding of disulfide-intact ribonuclease A: implications for the refolding and unfolding pathways. , 1994, Biochemistry.