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• W1973644706 abstract "Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, IE, on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of IE but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of IE but do so differentially; the fluorescence and circular dichroism properties of IE differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of IE is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the IE ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available. Osmolytes stabilize proteins against denaturation, but little is known about how their stabilizing effect might affect a protein folding pathway. Here, we report the effects of the osmolytes, trimethylamine-N-oxide, and sarcosine on the stability of the native state of barstar as well as on the structural heterogeneity of an early intermediate ensemble, IE, on its folding pathway. Both osmolytes increase the stability of the native protein to a similar extent, with stability increasing linearly with osmolyte concentration. Both osmolytes also increase the stability of IE but to different extents. Such stabilization leads to an acceleration in the folding rate. Both osmolytes also alter the structure of IE but do so differentially; the fluorescence and circular dichroism properties of IE differ in the presence of the different osmolytes. Because these properties also differ from those of the unfolded form in refolding conditions, different burst phase changes in the optical signals are seen for folding in the presence of the different osmolytes. An analysis of the urea dependence of the burst phase changes in fluorescence and circular dichroism demonstrates that the formation of IE is itself a multistep process during folding and that the two osmolytes act by stabilizing, differentially, different structural components present in the IE ensemble. Thus, osmolytes can alter the basic nature of a protein folding pathway by discriminating, through differential stabilization, between different members of an early intermediate ensemble, and in doing so, they thereby appear to channel folding along one route when many routes are available. Osmolytes are specific amino acids, polyols, and methylamines (1Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3014) Google Scholar) that are synthesized by microorganisms, plants, and animals in response to environmental stress and that serve to protect proteins against denaturation (1Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3014) Google Scholar, 2Brown A.D. Simpson J.R. J. Gen. Microbiol. 1972; 72: 589-591Crossref PubMed Scopus (294) Google Scholar, 3Hochachka P.W. Somero G.N. Water-solute Adaptations: The Evolution and Regulation of Biological Solutions. Princeton University Press, Princeton, NJ1984: 304-354Google Scholar). The mechanism by which they stabilize proteins has been studied extensively (1Yancey P.H. Clark M.E. Hand S.C. Bowlus R.D. Somero G.N. Science. 1982; 217: 1214-1222Crossref PubMed Scopus (3014) Google Scholar, 4Arakawa T. Timasheff S.N. Biophys. J. 1985; 47: 411-414Abstract Full Text PDF PubMed Scopus (833) Google Scholar, 5Timasheff S.N. Somero G.N. Osmond C.B. Bolis C.L. Water and Life: A Comparative Analysis of Water Relationships at the Organismic, Cellular and Molecular Levels. Springer-Verlag, Berlin, Germany1992: 70-84Crossref Google Scholar, 6Timasheff S.N. Annu. Rev. Biophy. Biomol. Struct. 1993; 22: 67-97Crossref PubMed Scopus (976) Google Scholar). An osmolyte increases the chemical potential of a protein via weak interactions (7Schellman J.A. Biophys. Chem. 1990; 37: 121-140Crossref PubMed Scopus (190) Google Scholar). The unfavorable interaction of the osmolyte with the peptide backbone causes the preferential exclusion of the osmolyte from the protein-water interface, and it dominates over any favorable interaction of the osmolyte with the side chains of amino acids of the protein (8Liu Y. Bolen D.W. Biochemistry. 1995; 34: 12884-12891Crossref PubMed Scopus (426) Google Scholar, 9Wang A. Bolen D.W. Biochemistry. 1997; 36: 9101-9108Crossref PubMed Scopus (414) Google Scholar). Osmolytes can also induce the folding of proteins, which are otherwise unfolded. For example, in the presence of the osmolyte TMAO, 1The abbreviation used is: TMAO, trimethylamine-N-oxide. reduced carboxyamidated RNase T1 and the destabilized T62P mutant of staphylococcal nuclease A, whose unfolded ensembles dominate in native buffers, are forced to fold into forms that are native-like in their secondary and tertiary structural contents (10Baskakov I. Bolen D.W. J. Biol. Chem. 1998; 273: 4831-4834Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). TMAO has also been shown to induce structure in α-synuclein, which is unstructured in its absence (11Uversky V.N. Li J. Fink A.L. FEBS Lett. 2001; 509: 31-35Crossref PubMed Scopus (172) Google Scholar). Thus, osmolytes can not only stabilize folded proteins, but they also appear to be capable of stabilizing the more structured members in an ensemble of disordered protein molecules. It is therefore surprising that very little is known about whether and how osmolytes can affect events on protein folding pathways on which less structured intermediates transform progressively into more structured and stabilized forms. Osmolytes can be expected to perturb not only the transitions between intermediates that differ in stability (12Russo A.T. Rosgen J. Bolen D.W. J. Mol. Biol. 2003; 330: 851-866Crossref PubMed Scopus (66) Google Scholar, 13Melo E.P. Chen L. Cabral J.M. Fojan P. Petersen S.B. Otzen D.E. Biochemistry. 2003; 42: 7611-7617Crossref PubMed Scopus (30) Google Scholar) but also to perturb the equilibria between the differently structured components that may be members of an intermediate ensemble. Such effects of osmolytes on protein folding pathways have become important to study, because recent studies of the products of the submillisecond folding reactions of several proteins, including barstar (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar), ribonuclease A (15Houry W.A. Rothwarf D.M. Scheraga H.A. Biochemistry. 1996; 35: 10125-10133Crossref PubMed Scopus (50) Google Scholar, 16Houry W.A. Scheraga H.A. Biochemistry. 1996; 35: 11734-11746Crossref PubMed Scopus (61) Google Scholar), lysozyme (17Morgan C.L. Miranker A. Dobson C.M. Biochemistry. 1998; 37: 8473-8480Crossref PubMed Scopus (31) Google Scholar), cytochrome c (18Akiyama S. Takahashi S. Ishimori K. Morishima I. Nat. Struct. Biol. 2000; 7: 514-520Crossref PubMed Scopus (159) Google Scholar), and apomyoglobin (19Nishimura C. Jane Dyson H. Wright P. J. Mol. Biol. 2002; 322: 483-489Crossref PubMed Scopus (86) Google Scholar), suggest that these early intermediates are structurally heterogeneous. So far, this heterogeneity has manifested itself in two or three co-existing forms (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar, 15Houry W.A. Rothwarf D.M. Scheraga H.A. Biochemistry. 1996; 35: 10125-10133Crossref PubMed Scopus (50) Google Scholar, 16Houry W.A. Scheraga H.A. Biochemistry. 1996; 35: 11734-11746Crossref PubMed Scopus (61) Google Scholar, 17Morgan C.L. Miranker A. Dobson C.M. Biochemistry. 1998; 37: 8473-8480Crossref PubMed Scopus (31) Google Scholar, 18Akiyama S. Takahashi S. Ishimori K. Morishima I. Nat. Struct. Biol. 2000; 7: 514-520Crossref PubMed Scopus (159) Google Scholar, 19Nishimura C. Jane Dyson H. Wright P. J. Mol. Biol. 2002; 322: 483-489Crossref PubMed Scopus (86) Google Scholar, 20Pradeep L. Udgaonkar J.B. J. Mol. Biol. 2002; 324: 331-347Crossref PubMed Scopus (42) Google Scholar, 21Georgescu R.E. Li J.H. Goldberg M.E. Tasayco M.L. Chaffotte A.F. Biochemistry. 1998; 37: 10286-10297Crossref PubMed Scopus (50) Google Scholar). Because structural heterogeneity is likely to be a consequence of the availability of multiple folding routes, these results suggest that only a few, and not many (22Dill K.A. Chan H.S. Nat. Struct. Biol. 1997; 4: 10-19Crossref PubMed Scopus (2014) Google Scholar, 23Dinner A.R. Sali A. Smith L.J. Dobson C.M. Karplus M. Trends Biochem. Sci. 2000; 25: 331-339Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 24Dobson C.M. Sali A. Karplus M. Angew. Chem. Int. Ed. 1998; 37: 868-893Crossref PubMed Scopus (759) Google Scholar, 25Onuchic J.N. Luthey-Schulten Z. Wolynes P.G. Annu. Rev. Phys. Chem. 1997; 48: 545-600Crossref PubMed Scopus (1701) Google Scholar), pathways may be available for folding and unfolding, as borne out by experimental studies of the folding and unfolding of several proteins (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar, 20Pradeep L. Udgaonkar J.B. J. Mol. Biol. 2002; 324: 331-347Crossref PubMed Scopus (42) Google Scholar, 26Zaidi F.N. Nath U. Udgaonkar J.B. Nat. Struct. Biol. 1997; 4: 1016-1024Crossref PubMed Scopus (102) Google Scholar, 27Goldbeck R.A. Thomas Y.G. Chen E. Esquerra R.M. Kliger D.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2782-2787Crossref PubMed Scopus (83) Google Scholar, 28Juneja J. Udgaonkar J.B. Biochemistry. 2002; 41: 2641-2654Crossref PubMed Scopus (56) Google Scholar, 29Sridevi K. Udgaonkar J.B. Biochemistry. 2002; 41: 1568-1578Crossref PubMed Scopus (49) Google Scholar, 30Wright C.F. Lindorff-Larsen K. Randles L.G. Clarke J. Nat. Struct. Biol. 2003; 10: 658-662Crossref PubMed Scopus (151) Google Scholar). It is of great interest to determine whether different protein folding pathways predominate in the presence of different osmolytes, because this will imply that the folding pathways utilized in the cell depend on the conditions present within it. The 89-amino acid residue, single domain protein barstar is the intracellular inhibitor of the extracellular ribonuclease, barnase in Bacillus amyloliquefaciens. The folding mechanism of barstar has been characterized in detail (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar, 20Pradeep L. Udgaonkar J.B. J. Mol. Biol. 2002; 324: 331-347Crossref PubMed Scopus (42) Google Scholar, 31Shastry M.C.R. Agashe V.R. Udgaonkar J.B. Protein Sci. 1994; 3: 1409-1417Crossref PubMed Scopus (42) Google Scholar, 32Schreiber G. Fersht A.R. Biochemistry. 1993; 32: 11195-11203Crossref PubMed Scopus (134) Google Scholar, 33Agashe V.R. Shastry M.C.R. Udgaonkar J.B. Nature. 1995; 377: 754-757Crossref PubMed Scopus (188) Google Scholar). Under strongly stabilizing conditions, the folding of barstar can be represented as follows: U↔KUIIE→kIL→slowNSCHEME 1 IE represents an early intermediate that is populated at a few milliseconds of folding and that equilibrates with U prior to the major structural transition to IL, a late intermediate. Earlier studies had indicated that IE is compact and possesses solvent-exposed hydrophobic patches (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar). In marginally stabilizing conditions (1–1.2 m guanidine HCl), no specific structure is found to form at a few milliseconds of folding, suggesting either that IE was devoid of specific structure (33Agashe V.R. Shastry M.C.R. Udgaonkar J.B. Nature. 1995; 377: 754-757Crossref PubMed Scopus (188) Google Scholar) or that IE does not accumulate significantly under these folding conditions. There is strong evidence for structural heterogeneity in IE, from several studies. In the first of these studies, which was the first demonstration of structural heterogeneity in the folding intermediate of any protein, IE was shown to comprise of at least two structural components that form on competing folding pathways from the same population of U molecules (14Shastry M.C.R. Udgaonkar J.B. J. Mol. Biol. 1995; 247: 1013-1027Crossref PubMed Scopus (98) Google Scholar). More recent studies indicate that IE is an ensemble of at least three different structural forms, each of which is stabilized differentially, and hence populated differentially, in the absence and presence of salts (20Pradeep L. Udgaonkar J.B. J. Mol. Biol. 2002; 324: 331-347Crossref PubMed Scopus (42) Google Scholar). IL also shows structural heterogeneity (34Bhuyan A.K. Udgaonkar J.B. Biochemistry. 1999; 38: 9158-9168Crossref PubMed Scopus (44) Google Scholar). Most recently, the use of a multisite time-resolved fluorescence resonance energy transfer approach has shown that the extent of structural heterogeneity depends on how stable the folding conditions are and that different structural components predominate in IL under different folding conditions (35Sridevi K. Lakshmikanth G. Krishnamoorthy G. Udgaonkar J.B. J. Mol. Biol. 2004; 337: 699-711Crossref PubMed Scopus (51) Google Scholar). Obviously, barstar is a good model system for exploring the effects of osmolytes on the structural heterogeneity that now appears characteristic of protein folding reactions. In this study, fluorescence and circular dichroism have been used to study the effects of osmolytes on the stability of the native state and on the heterogeneity of the protein folding reactions. It is seen that the free energy of unfolding of the N state of barstar to the U form has a linear dependence on TMAO as well as on sarcosine concentration, suggesting that the mechanism of stabilization by both osmolytes primarily involves their preferential exclusion from the protein surface. Next, it is shown that the structure of the early intermediate ensemble, IE, is altered, significantly and differentially, in the presence of 1 m TMAO and sarcosine. The heterogeneity in IE has been characterized, and it is shown that the two osmolytes, TMAO and sarcosine, differentially stabilize structure in IE by shifting the equilibrium between different structural components to favor the more structured components. Finally the use of osmolytes has led to a better understanding of the folding pathway of barstar; the transition from the unfolded protein in refolding conditions to IE, is shown to occur through more than one step in the presence of the osmolytes. The Escherichia coli strain MM294 was used for protein expression. The expression plasmid for wild-type barstar was pMT316. The method used to purify barstar has been described in detail (36Khurana R. Udgaonkar J.B. Biochemistry. 1994; 33: 106-115Crossref PubMed Scopus (130) Google Scholar). Protein concentrations were calculated using an extinction coefficient of 23,000 m–1 cm–1 (36Khurana R. Udgaonkar J.B. Biochemistry. 1994; 33: 106-115Crossref PubMed Scopus (130) Google Scholar). Mass spectroscopy using a Micromass Q-TOF Ultima showed that the protein was pure and had a mass of 10342, which indicated that the N-terminal methionine residue had remained uncleaved during synthesis. 30 mm Tris-HCl (pH 8) (ultrapure, 99.9% from Invitrogen), 250 μm EDTA (disodium salt, dihydrate, 99+% from Sigma), and 250 μm dithiothreitol (ultrapure from Invitrogen) constituted the native buffer used for all equilibrium and kinetic experiments. Unfolding buffer was native buffer containing 9 to 10 m urea (ultrapure, 99.9% from United States Biochemical). The concentrations of stock solutions of urea were determined by measuring the refractive index using an Abbe 3L refractometer from Milton Roy. For folding studies in the presence of osmolyte (TMAO or sarcosine), the osmolyte was present in the refolding as well as in the unfolding buffer. The unfolding buffers containing 1 m TMAO (dihydrate, ultrapure from Sigma) or 1 m sarcosine (ultrapure from Sigma) had a maximum urea concentration of 9 m, because of solubility limitations. All of the buffers and solutions were filtered through 0.22-μm filters before use and were degassed prior to the kinetic experiments. CD spectra were collected on a Jasco J720 spectropolarimeter, using a bandwidth of 1 nm, a response time of 1 s, and a scan speed of 50 nm/min. Each spectrum was an average of three scans monitored between 210 and 250 nm. The protein concentration used was typically10–15 μm for the far-UV CD experiments, and the path length of the cuvette was 0.2 cm. Fluorescence spectra were collected on a SPEX DM 3000 spectrofluorimeter. The protein was excited at 295 nm, and the emission was monitored between 300 and 400 nm with a bandwidth of 0.37 nm for excitation and 10 nm for emission. Each spectrum was an average of three scans. The protein concentration was typically 2–4 μm, and the path length of the cuvette used was 1 cm. Protein stability at equilibrium was determined by urea-induced denaturation studies using two probes. The CD at 222 nm and fluorescence at 320 nm were monitored as described above. Prior to the CD and fluorescence measurements, the samples were equilibrated for at least 4 h. Identical results were obtained if the time of incubation was 24 h. Kinetic experiments were performed on a Biologic SFM-4 stopped-flow mixing module. Folding was monitored using either intrinsic tryptophan fluorescence at 320 nm or far-UV CD at 222 nm as the probe. Intrinsic Tryptophan Fluorescence Measurements—For intrinsic tryptophan fluorescence measurements, the excitation wavelength was set at 295 nm, and emission was monitored at 320 nm using an Oriel bandpass filter with a bandwidth of 10 nm. The protein concentration during refolding was between 15 and 30 μm. In all experiments, an FC-08 cuvette with a path length of 0.8 mm was used, the total flow rate was 6.0 ml/s, and the dead time of the instrument was 1.5 ms. For refolding experiments, barstar was unfolded in 9 m urea (unfolding buffer) for at least 4 h (the unfolding buffer contained TMAO or sarcosine when refolding was studied in the presence of osmolyte). In refolding experiments, the final concentration of urea was between 0.9 and 2.7 m in the absence of osmolyte and between 0.9 and 4.5 m in the presence of 1 m TMAO or 1 m sarcosine. Far-UV CD Measurements—For far-UV CD measurements, a polarizer/modulator assembly was installed on the Biologic SFM-4 stopped-flow mixing module. A photomultiplier and its controller (model PMS 400) was used to collect data in the CD mode. A test experiment was performed to check the performance of the CD recording system for proper alignment of the optics and the polarizer/modulator assembly, in which the alkaline hydrolysis of glucuronolactone at 225 nm was studied. For all of the kinetic studies on the folding of barstar, the wave-length was set to 222 nm, and the retardation was set to ¼ wavelength. The protein concentration during refolding was between 15 and 25 μm; the upper limit was decided based on a pilot experiment, wherein linearity in the CD signal was found to be lost at concentrations above 25 μm. In all experiments, an FC-20 cuvette with a path length of 2.0 mm was used, the total flow rate was 6.0 ml/s, and the dead time was 9.0 ms. For refolding experiments, barstar was unfolded in 9 m urea (unfolding buffer) for at least 4 h (the unfolding buffer contained TMAO or sarcosine when refolding was studied in the presence of osmolyte). In refolding experiments, the final concentration of urea was between 0.9 and 2.7 m in the absence of osmolyte and between 0.9 and 4.5 m in the presence of 1 m TMAO or 1 m sarcosine. The kinetics on this module could be collected only for 20 s because of bleaching. Far-UV CD kinetics for longer time periods were collected manually on a Jasco J720 spectropolarimeter, using a bandwidth of 1 nm, a response time of 1 s, and a sensitivity of 50 millidegrees. Each kinetic trace was an average of three kinetic runs at 222 nm. The protein concentration used was typically 15–25 μm for the far-UV CD experiments, the path length of the cuvette was 2.0 mm, and the dead time for manual kinetics was 20 s. Equilibrium Studies—According to the weak interaction (linear free energy) model for describing the interaction of urea (D) with a protein (7Schellman J.A. Biophys. Chem. 1990; 37: 121-140Crossref PubMed Scopus (190) Google Scholar, 37Schellman J.A. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 115-137Crossref PubMed Scopus (297) Google Scholar), the change in free energy, ΔG/, that occurs upon unfolding of any form of a protein, j, in the presence of D, is linearly dependent on denaturant concentration, [D]. ΔGUj′=ΔGUj+mUj[D](Eq. 1) mUj is the change in free energy associated with the preferential interaction of the denaturant with the unfolded form, U, relative to the folded (partially or fully) form, j. When the form j is a partially folded intermediate I, ΔGUI/, represents the free energy of unfolding of I, and when j is the native state, N, ΔGUN/ represents the free energy of unfolding of N in the presence of denaturant. ΔGUj represents the free energy of unfolding of the folded (partially or fully) form j to U in the absence of any denaturant or added co-solute. An osmolyte, O, acting as a chemical perturbant, also interacts with a protein according to the weak interaction model (38Timasheff S.N. Adv. Protein Chem. 1998; 51: 355-432Crossref PubMed Google Scholar), and the free energy of unfolding of a folded form j to U, in the presence of O, has a linear dependence on osmolyte concentration, [O]. ΔGUj//=ΔGUj+mOj[O](Eq. 2) mOj is the change in free energy associated with the preferential interaction of the osmolyte with the unfolded form, U, relative to the folded (partially or fully) form, j. According to Equation 2, mOj has a positive value when the folded form is stabilized in the presence of osmolyte. When the form j is a partially folded intermediate I, ΔGUI//, represents the free energy of stabilization of I, and when j is the native state, N, ΔGUN// represents the free energy of stabilization of N, in the presence of osmolyte. Thus, in the presence of both denaturant and osmolyte, the free energy of unfolding of a folded form, j to the unfolded form U, ΔGUj/// is given by the following. ΔGUj///=ΔGUj+mOj[O]+mUj[D](Eq. 3) Equation 3 assumes that mUj is independent of [O] and that mOj is independent of [D]. The equilibrium data for the unfolding of N as a function of [D], obtained in the presence of a fixed concentration of osmolyte, were fit to a two-state U [rlhar2] N model according to the following equation, YO=YN+mN[D]+(YU+mU[D])e−(ΔGUN//+mUN[D])RT1+e−(ΔGUN//+mUN[D])RT(Eq. 4) where YO is the value of the spectroscopic property being measured as a function of [D] at fixed [O], YN and YU represents the intercepts, and mN and mU represent the slopes of the native protein and unfolded protein base lines, respectively. Thus, fits of denaturant-induced equilibrium unfolding data at different fixed values of [O] to Equation 4 yield values for ΔGUN// and mUN at each fixed [O], and a subsequent fit of the osmolyte dependence of ΔGUN// to Equation 2 yields values for ΔGUN and mON. Raw equilibrium unfolding data of N as a function of [D] were also analyzed in an alternative way (39Agashe V.R. Udgaonkar J.B. Biochemistry. 1995; 34: 3286-3299Crossref PubMed Scopus (208) Google Scholar). They were first converted to plots of fraction unfolded (fU) versus [D], using Equation 5. fU=YO−(YN+mN[D])(YU+mU[D])−(YN+mN[D])(Eq. 5) The fU values were then fit to Equation 6. fU=e−(ΔGUN//+mUN[D])RT1+e−(ΔGUN//+mUN[D])RT(Eq. 6) In Equation 6, ƒU is related to ΔGUN// by a transformation of the Gibbs-Helmholtz equation in which the equilibrium constant for unfolding in the transition zone, KUN///, is given by KUN///=fU/(1−fU), for a two-state transition. The concentration of the denaturant at which the protein is half unfolded (when ΔGUj/=0), is given by Cm and from Equation 1, Cm = ΔGUj/mUj. Kinetic Studies—The observable kinetics of folding of barstar in the pretransition zone are described by a three-exponential process when monitored by fluorescence at 320 nm: A(t)=A(∞)−A1e−λ1t−A1e−λ2t−A1e−λ3t(Eq. 7) where A(t) and A(∞) are the observed reduced amplitudes at times t and at infinity; λ1, λ2 and λ3 are the apparent rate constants of the slow, fast and intermediate phases, and A1, A2 and A3 are the respective amplitudes. The relative amplitude of each phase was determined by dividing the observed amplitude of that phase by the equilibrium amplitude of the reaction at that urea concentration. In the transition zone, the folding process is two-exponential and is described by Equation 7, with A3 = 0. The observable kinetics of folding of barstar in the pretransition zone and transition zone are described by a two-exponential process when monitored by CD at 222 nm, A(t)=A(∞)−A1e−λ1t−A1e−λ2t(Eq. 8) or by a single exponential process, by setting A1 equal to zero in Equation 8, at lower urea concentrations in the pretransition zone. In the absence of any osmolyte, for refolding at the lowest urea concentration, i.e. 0.9 m urea, no slow phase was observed; the signal corresponding to that of the N state was achieved in the dead time (20 s) of a manual mixing experiment, and no slow phase is observable in a stopped-flow experiment. For slightly higher urea concentrations (1.5 and 1.8 m), the t = ∞ of the stopped-flow kinetic data coincides with the t = 0 of the manual mixing kinetic data. But for the urea concentrations 2.1, 2.4, and 2.7 m, the t = ∞ of the stopped-flow kinetic data did not coincide with the t = 0 of the manual mixing kinetic data. That is, at higher concentrations of urea, there was a 5–14% discrepancy between the equilibrium folding amplitude and amplitude of the observable kinetic phases and the burst phase. Likewise, in the presence of either osmolyte, only the fast phase of refolding was observed at urea concentrations below 2.7 m. At higher urea concentrations, the t = ∞ from the stopped-flow measurements again did not coincide with the t = 0ofthe manual mixing experiments, with there being a maximum discrepancy of up to 10% in amplitudes. The discrepancy was possibly due to the contribution of linear dichroism (arising from pressure on the cuvette) to the observed protein signal, which probably is also responsible for the 2–3-millidegree discrepancy observed between the signal of unfolded protein (9 m urea) measured on the Jasco J720 spectropolarimeter and on the Biologic SFM 4. Thus, there is about a 10–15% error in the data of the amplitudes at the higher concentrations of urea used. To analyze kinetic data according to Scheme 1, it was assumed that the conformational transitions between U, UC, and IE are rapid compared with the subsequent slow conversion of IE to IL and N, so that a transient pre-equilibrium, characterized by the equilibrium constant, KUI, is established between UC and IE. To determine whether the transition between two UC and IE is two-state, the pre-equilibrium data for the denaturant-induced unfolding of IE, obtained from kinetic experiments in the presence of a fixed concentration of osmolyte, O, as well as the pre-equilibrium data for the osmolyte-induced stabilization of IE at a fixed concentration of denaturant, were fit to a two-state model according to the following equation, Y=YI+mI[X]+(YU+mU[X])e−(ΔGUI+mUI[D]+mOj[O])RT1+e−(ΔGUI+mUI[D]+moj[O]))RT(Eq. 9) where [X] is the variable [D] for experiments in which refolding is carried out at a fixed value of [O], and [X] is the variable [O] for experiments in which refolding is carried out at a fixed [D]. Y is the value of the spectroscopic property being measured as a function of the variable [X], YI and YU represent the intercepts, and mI and mU represent the slopes of the IE and UC base lines, respectively. The dependence of the rate constant, k, for the conversion of IE to IL or N (Scheme 1) is expected to decrease exponentially with an increase in [D], because the free energy of activation is expected to increase linearly with an increase in [D]. Also, k is expected to increase exponentially with an increase in [O], because the free energy of activation is assumed to decrease linearly with an increase in [O]. This is given by the following equation, k=k°e−mkD[D]emko[O](Eq. 10) where ko is the rate constant in the absence of denaturant and osmolyte, RTmkD is the free energy associated with the preferential interaction of denaturant with the transition state relative to IE, and RTmkO is the free energy associated with the preferential interaction of osmolye with the transition state relative to IE. Then the observed rate of folding, λ///, in the presence of both urea at concentration [D] and osmolyte at concentration [O], is given by the" @default.
• W1973644706 created "2016-06-24" @default.
• W1973644706 creator A5047773816 @default.
• W1973644706 creator A5057549416 @default.
• W1973644706 date "2004-09-01" @default.
• W1973644706 modified "2023-10-02" @default.
• W1973644706 title "Osmolytes Induce Structure in an Early Intermediate on the Folding Pathway of Barstar" @default.
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