The study of protein folding and dynamics by determination of intramolecular distance distributions and their fluctuations using ensemble and single-molecule FRET measurements.

The folding and dynamics of globular proteins is a multidimensional problem. The structures of the heterogeneous population of refolding protein molecules are characterized by multiple distances and time constants. Deciphering the mechanism of folding depends on studies of the processes rather than the folded structures alone. Spectroscopy is indispensable for these sorts of studies. Herein, it is shown that the determination of intramolecular distance distributions by ensemble and single-molecule FRET experiments enable the exploration of partially folded states of refolding protein molecules.

[1]  A. Bax,et al.  NMR evidence for similarities between the DNA-binding regions of Drosophila melanogaster heat shock factor and the helix-turn-helix and HNF-3/forkhead families of transcription factors. , 1994, Biochemistry.

[2]  E. Haas,et al.  A general strategy for site-specific double labeling of globular proteins for kinetic FRET studies. , 2002, Bioconjugate chemistry.

[3]  J. Lakowicz,et al.  Influence of end-to-end diffusion on intramolecular energy transfer as observed by frequency-domain fluorometry. , 1990 .

[4]  P. Privalov,et al.  On the entropy of protein folding , 1996, Protein science : a publication of the Protein Society.

[5]  Lorna J. Smith,et al.  Long-Range Interactions Within a Nonnative Protein , 2002, Science.

[6]  W Bialek,et al.  Protein dynamics and reaction rates: mode-specific chemistry in large molecules? , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  T. Steitz,et al.  Space-filling models of kinase clefts and conformation changes. , 1979, Science.

[8]  A Volkmer,et al.  Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. , 2001, Journal of biotechnology.

[9]  M. Pennington,et al.  Site-specific chemical modification procedures. , 1994, Methods in molecular biology.

[10]  U. Wille.  Self-terminating, oxidative radical cyclizations: a novel reaction of acyloxyl radicals. , 2002, Journal of the American Chemical Society.

[11]  L. Brand,et al.  Orientation factor in steady-state and time-resolved resonance energy transfer measurements. , 1992, Biochemistry.

[12]  D Baker,et al.  Mechanisms of protein folding. , 2001, Current opinion in structural biology.

[13]  J. Deisenhofer,et al.  Crystallographic refinement of the structure of bovine pancreatic trypsin inhibitor at l.5 Å resolution , 1975 .

[14]  R. L. Baldwin,et al.  Making a Network of Hydrophobic Clusters , 2002, Science.

[15]  K. Teilum,et al.  Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling. , 2002, Journal of molecular biology.

[16]  C. Cepko,et al.  Electroporation and RNA interference in the rodent retina in vivo and in vitro , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[17]  A. C. Albrecht.  ``Forbidden'' Character in Allowed Electronic Transitions , 1960 .

[18]  R. Clegg.  Fluorescence resonance energy transfer and nucleic acids. , 1992, Methods in enzymology.

[19]  A. Cooper,et al.  Protein fluctuations and the thermodynamic uncertainty principle. , 1984, Progress in biophysics and molecular biology.

[20]  J. Lakowicz,et al.  Time-resolved energy transfer measurements of donor-acceptor distance distributions and intramolecular flexibility of a CCHH zinc finger peptide. , 1993, Biochemistry.

[21]  E. Rhoades,et al.  Watching proteins fold one molecule at a time , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[22]  L. Nilsson,et al.  Structural fluctuations between two conformational states of a transmembrane helical peptide are related to its channel-forming properties in planar lipid membranes. , 1993, European journal of biochemistry.

[23]  Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar. , 2003, Biochemistry.

[24]  G. Weber,et al.  Electronic energy transfer in haem proteins , 1959 .

[25]  C. Dobson,et al.  Transition states for protein folding have native topologies despite high structural variability , 2004, Nature Structural &Molecular Biology.

[26]  J. Onuchic,et al.  Understanding protein folding with energy landscape theory Part II: Quantitative aspects , 2002, Quarterly Reviews of Biophysics.

[27]  W. Bennett,et al.  Structural and functional aspects of domain motions in proteins. , 1984, CRC critical reviews in biochemistry.

[28]  Kresten Lindorff-Larsen,et al.  Determination of an ensemble of structures representing the denatured state of the bovine acyl-coenzyme a binding protein. , 2004, Journal of the American Chemical Society.

[29]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[30]  E. Haas,et al.  The natively helical chain segment 169-188 of Escherichia coli adenylate kinase is formed in the latest phase of the refolding transition. , 2002, Journal of molecular biology.

[31]  T. Főrster,et al.  10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation , 1959 .

[32]  E. Blout,et al.  ENERGY TRANSFER. A SYSTEM WITH RELATIVELY FIXED DONOR-ACCEPTOR SEPARATION. , 1965, Journal of the American Chemical Society.

[33]  E. Katchalski‐Katzir,et al.  Long-Range Nonradiative Transfer of Electronic Excitation Energy , 1983 .

[34]  T. Creighton,et al.  Experimental studies of protein folding and unfolding. , 1978, Progress in biophysics and molecular biology.

[35]  Jayant B Udgaonkar,et al.  Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble. , 2004, Journal of molecular biology.

[36]  R. S. Goody,et al.  Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  E. Haas,et al.  Nonlocal interactions stabilize long range loops in the initial folding intermediates of reduced bovine pancreatic trypsin inhibitor. , 1995, Biochemistry.

[38]  J M Beechem,et al.  Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements. , 1989, Biophysical journal.

[39]  J. Eisinger,et al.  The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer. , 1979, Biophysical journal.

[40]  Everett A Lipman,et al.  Single-Molecule Measurement of Protein Folding Kinetics , 2003, Science.

[41]  Linlin Qiu,et al.  Fast chain contraction during protein folding: "foldability" and collapse dynamics. , 2003, Physical review letters.

[42]  H. Roder,et al.  Early kinetic intermediate in the folding of acyl-CoA binding protein detected by fluorescence labeling and ultrarapid mixing , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Matthias Rief,et al.  Single-molecule folding. , 2003, Current opinion in structural biology.

[44]  I. Z. Steinberg,et al.  Intramolecular dynamics of chain molecules monitored by fluctuations in efficiency of excitation energy transfer. A theoretical study. , 1984, Biophysical journal.

[45]  E. Haas,et al.  Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor. , 1992, Biochemistry.

[46]  A. Johnson.  Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome. , 2004, Biochemical Society transactions.

[47]  R. E. Dale,et al.  Interpretation of intramolecular energy transfer experiments , 1974 .

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

[49]  B. Meer,et al.  Resonance Energy Transfer: Theory and Data , 1994 .

[50]  J. Kuśba,et al.  Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry. , 1991, Biophysical chemistry.

[51]  J. Lakowicz,et al.  Distance distributions and anisotropy decays of troponin C and its complex with troponin I. , 1991, Biochemistry.

[52]  E. Haas,et al.  Design consideration and probes for fluorescence resonance energy transfer studies. , 2000, Bioconjugate chemistry.

[53]  L. Stryer,et al.  Energy transfer: a spectroscopic ruler. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[54]  B. R. Thomas,et al.  Affinity purification of functional receptors for Escherichia coli heat-stable enterotoxin from rat intestine. , 1992, Biochemistry.

[55]  E. Katchalski‐Katzir,et al.  Effect of the orientation of donor and acceptor on the probability of energy transfer involving electronic transitions of mixed polarization. , 1978, Biochemistry.

[56]  P. Privalov,et al.  Energetics of protein structure. , 1995, Advances in protein chemistry.

[57]  J M Sturtevant,et al.  Cold denaturation of staphylococcal nuclease. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[58]  W. Jencks,et al.  Binding energy, specificity, and enzymic catalysis: the circe effect. , 2006, Advances in enzymology and related areas of molecular biology.

[59]  W. Eaton,et al.  Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy , 2002, Nature.

[60]  T. G. Mammadov,et al.  Deformation effects in electronic spectra of the layered semiconductors TlGaS2, TlGaSe2 and TlInS2 , 2003 .

[61]  J. Eisinger,et al.  Intramolecular energy transfer and molecular conformation. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[62]  C. Wu,et al.  Statistical interpretation of fluorescence energy transfer measurements in macromolecular systems. , 1976, Biochemistry.

[63]  L. Brand,et al.  Resonance energy transfer: methods and applications. , 1994, Analytical biochemistry.

[64]  E. Katchalski‐Katzir,et al.  Brownian motion of the ends of oligopeptide chains in solution as estimated by energy transfer between the chain ends , 1978 .

[65]  A. Fersht,et al.  Early events in protein folding. , 2003, Current opinion in structural biology.

[66]  G. Krishnamoorthy,et al.  Structure is lost incrementally during the unfolding of barstar , 2001, Nature Structural Biology.

[67]  L. Brand,et al.  Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements. , 1993, Biophysical chemistry.

[68]  L. Stryer,et al.  Diffusion-enhanced fluorescence energy transfer. , 1982, Annual review of biophysics and bioengineering.

[69]  H. Dyson,et al.  Unfolded proteins and protein folding studied by NMR. , 2004, Chemical reviews.

[70]  Julia G. Lyubovitsky,et al.  Mapping the cytochrome C folding landscape. , 2002, Journal of the American Chemical Society.

[71]  P. Privalov,et al.  Intermediate states in protein folding. , 1996, Journal of molecular biology.

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

[73]  F. Schmid,et al.  Rapid collapse precedes the fast two-state folding of the cold shock protein. , 2004, Journal of molecular biology.

[74]  L. Nilsson,et al.  Structural fluctuations of a helical polypeptide traversing a lipid bilayer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[75]  J. Beechem,et al.  Local and global dynamics during the folding of Escherichia coli dihydrofolate reductase by time-resolved fluorescence spectroscopy. , 1995, Biochemistry.

[76]  Michael Börsch,et al.  Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase , 2004, Nature Structural &Molecular Biology.

[77]  A. Fink,et al.  Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding. , 2000, Journal of molecular biology.

[78]  J. Brochon.  Maximum entropy method of data analysis in time-resolved spectroscopy. , 1994, Methods in enzymology.

[79]  A. Warshel,et al.  Electrostatic energy and macromolecular function. , 1991, Annual review of biophysics and biophysical chemistry.

[80]  G. Rose,et al.  Protein folding--what's the question? , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[81]  W. Eaton,et al.  Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[82]  R. Hochstrasser,et al.  Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[83]  E. Trifonov,et al.  Protein folding: Looping from hydrophobic nuclei , 2001, Proteins.

[84]  Helmut Grubmüller,et al.  Maximum likelihood trajectories from single molecule fluorescence resonance energy transfer experiments , 2003 .

[85]  E. Gussakovsky,et al.  The compact state of reduced bovine pancreatic trypsin inhibitor is not the compact molten globule , 1992, FEBS letters.

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

[87]  J. Kuśba,et al.  Resolution of end‐to‐end diffusion coefficients and distance distributions of flexible molecules using fluorescent donor‐acceptor and donor‐quencher pairs , 1991, Biopolymers.

[88]  R. Huber,et al.  Functional significance of flexibility in proteins , 1982, Biopolymers.

[89]  M Dahan,et al.  Ratiometric single-molecule studies of freely diffusing biomolecules. , 2001, Annual review of physical chemistry.

[90]  Charles Tanford,et al.  Physical Chemistry of Macromolecules , 1961 .

[91]  H. Grubmüller,et al.  Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1 , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[92]  Helmut Grubmüller,et al.  FRETsg: Biomolecular structure model building from multiple FRET experiments , 2004 .

[93]  I. Z. Steinberg.  Long-range nonradiative transfer of electronic excitation energy in proteins and polypeptides. , 1971, Annual review of biochemistry.

[94]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[95]  Elisha Haas,et al.  Alpha-synuclein: its biological function and role in neurodegenerative diseases. , 2003, Journal of molecular neuroscience : MN.

[96]  J. Lakowicz,et al.  Conformational flexibility of the Cys 697-Cys 707 segment of myosin subfragment-1. Distance distributions by frequency-domain fluorometry. , 1991, Biophysical chemistry.

[97]  E. Haas,et al.  Determination of intramolecular distance distribution during protein folding on the millisecond timescale. , 2000, Journal of molecular biology.

[98]  E. Haas,et al.  Estimation of intramolecular distance distributions in bovine pancreatic trypsin inhibitor by site-specific labeling and nonradiative excitation energy-transfer measurements. , 1987, Biochemistry.

[99]  Valerie Daggett,et al.  The present view of the mechanism of protein folding , 2003, Nature Reviews Molecular Cell Biology.

[100]  G. Haran.  Single-molecule fluorescence spectroscopy of biomolecular folding , 2003 .

[101]  A. Fersht,et al.  Is there a unifying mechanism for protein folding? , 2003, Trends in biochemical sciences.

[102]  E. Trifonov,et al.  Closed loops of nearly standard size: common basic element of protein structure , 2000, FEBS letters.

[103]  An instrument for time resolved monitoring of fast chemical transitions: Application to the kinetics of refolding of a globular protein , 1998 .

[104]  B. Vallee,et al.  Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[105]  Andrew B. Martin,et al.  Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[106]  L. Stryer,et al.  Fluorescence energy transfer measurements of distances in rhodopsin and the purple membrane protein. , 1982, Methods in enzymology.

[107]  O. Ptitsyn,et al.  A study of apo‐ and holo‐forms of horse liver alcohol dehydrogenase in solution by diffuse X‐ray scattering , 1986, Biopolymers.