Folding and Association of an Extremely Stable Dimeric Protein from Sulfolobus islandicus

doi:10.1016/j.jmb.2003.12.003

J. Mol. Biol. (2004) 336, 227–240

Folding and Association of an Extremely Stable Dimeric Protein from Sulfolobus islandicus Markus Zeeb1, Georg Lipps2, Hauke Lilie3 and Jochen Balbach1* 1 Laboratorium fu¨r Biochemie III Universita¨t Bayreuth D-95440 Bayreuth, Germany 2

Laboratorium fu¨r Biochemie II Universita¨t Bayreuth D-95440 Bayreuth, Germany

3

Institut fu¨r Biotechnologie Martin-Luther-Universita¨t Halle-Wittenberg D-06120 Halle, Germany

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of 2 85.1 kJ/mol at 25 8C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (tm) below the boiling point of water. Linear extrapolation of tm to 0 M GdmCl yielded a tm of 107.5 8C (5 mM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (kf, ku), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both ln kf and ln ku depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 £ 107 M21 s21) and unfolds extremely slowly (5.7 year21). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms. q 2003 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: protein dimer; protein stability; NMR; folding kinetics; Sulfolobus islandicus

Introduction The archaeon Sulfolobus islandicus is a hyperthermophilic and acidophilic member of the crenarchaeota, which grows in acidic hot springs with an optimal growth temperature of about 80 8C at pH 3. Two closely related plasmids pRN1 and pRN2 were isolated from a strain of this Abbreviations used: GdmCl, guanidinium chloride; CD, circular dichroism; HSQC, heteronuclear single quantum coherence. E-mail address of the corresponding author: [email protected]

archaeon.1 Plasmid pRN1 encodes three open reading frames (orf56, orf80, orf904) with gene products of very distinct properties. The protein ORF80 is a site-specific DNA binding protein and contains a novel type of a basic leucine zipper.2 ORF904 is a unique replicative enzyme with ATPase, primase and DNA polymerase activity.3 The small basic protein ORF56 comprises 56 amino acid residues. It has a calculated isoelectric point of 9.2 and a molecular mass of 6.5 kDa. ORF56 shares a 22% sequence identity and a high positive net charge with the CopG protein of plasmid pLS1 from Streptococcus species. The open reading frames of pRN1 and pLS1 are both upstream of a gene

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

228

encoding a protein involved in plasmid replication.4 CopG controls the copy number of the plasmid pLS1 by binding to its own promotor. Like CopG, ORF56 binds sequence-specifically as a tetramer to double-stranded DNA fragments derived from its own promotor region.4 Lipps et al. suggested from sequence homology, secondary structure prediction and far-UV circular dichroism (CD) spectroscopy that ORF56 adopts a topology similar to CopG.4 The crystal structure of CopG comprises a helix-turn-helix motif and a short N-terminal b-strand, which forms an antiparallel intersubunit b-sheet.5 An NMR structure determination of ORF56 confirms this topology (G.L. et al., unpublished results), which also resembles the NMR solution structure of Arc repressor from bacteriophage P22.6 Small globular and monomeric proteins of mesophilic organisms are often used to study protein folding and stability.7,8 To elucidate the folding mechanism of dimeric proteins, again mostly representatives of mesophilic species were chosen, e.g. Trp aporepressor from Escherichia coli,9,10 P22 Arc repressor,11,12 as well as porcine cytosolic and mitochondrial malate dehydrogenases.13,14 Only limited insights into the folding pathways of hyperthermostable dimeric proteins and their stabilization against thermal, pH or denaturantinduced unfolding has been gained so far.15 – 19 Here, we characterized the thermodynamic stability and the un- and refolding kinetics of ORF56 from S. islandicus. Native ORF56 forms a homodimer between protein concentrations of 4.6 mM and 0.8 mM as shown by analytical ultracentrifugation. The protein is extremely stable in thermal, denaturant and pH-induced unfolding experiments. Thermal unfolding can only be achieved by adding large amounts of GdmCl, which shifts the melting temperature below the boiling point of water. ORF56 remains native within a wide pH range reaching from pH 2 up to pH 12. To elucidate the folding mechanism, various equilibrium and kinetic experiments under fluorescence, circular dichroism and NMR detection were performed. From the joint analysis, we could conclude that ORF56 follows a two-state

Folding and Association of Dimeric ORF56

mechanism implying concurrent folding and association of two polypeptide chains.

Results Stability of the ORF56 protein The unfolding reaction of ORF56 containing a single tryptophan and three tyrosine residues was followed by fluorescence and far-UV circular dichroism spectroscopy. Fluorescence spectra of native and unfolded ORF56 are depicted in Figure 1(a). Unfolding the protein in 8.5 M GdmCl results in a loss of fluorescence intensity of about 80% and is accompanied by a red shift of the emission maximum from 335 nm to 348 nm. Figure 1(b) shows the far-UV CD spectra of folded and GdmCl-unfolded ORF56. The high a-helical content dominates the spectrum of the native protein with characteristic minima of the ellipticity at 220 nm and 208 nm, which vanish upon unfolding. Since S. islandicus is a hyperthermophilic organism ORF56 remains native even at very high temperatures. Changes in the far-UV CD signal at 220 nm were used to follow thermal unfolding, but only a negligible thermal denaturation could be detected up to 100 8C. In order to achieve complete thermal unfolding of ORF56 GdmCl $ 1 M was added, which shifts the melting temperature (tm) below the boiling point of water. Thermal unfolding transitions measured between 1.5 M and 3.5 M GdmCl are shown in Figure 2. In the presence of 2.5 M GdmCl the tm of 5 mM ORF56 is 85.0(^ 0.2) 8C. The melting temperature in the absence of denaturant (tm(H2O)) was extracted by linear extrapolation of the GdmCl dependence of tm to 0 M GdmCl (inset Figure 2). The tm(H2O) of ORF56 yields 107.5(^ 1.8) 8C at a concentration of 5 mM. Equilibrium unfolding of dimeric ORF56 follows a two-state model ORF56 has been described as a dimeric protein in solution with a theoretical mass of 6654.8 per

Figure 1. Optical spectra of folded (continuous line) and unfolded (broken line) ORF56 in 0 M and 8 M GdmCl, respectively, in 20 mM sodium phosphate (pH 7.5) at 25 8C. (a) Fluorescence emission spectra of 1 mM ORF56 after excitation at 280 nm in a 1 cm cell. (b) Far-UV CD spectra recorded with 20 mM samples in a 0.1 cm cell.

229

Folding and Association of Dimeric ORF56

Figure 2. Thermal unfolding of ORF56 at various GdmCl concentrations. The 5 mM protein solutions in 20 mM sodium cacodylate/HCl (pH 7.5) were used in the presence of 1.5 M (S), 2 M (L), 2.5 M (A), 3 M (K) and 3.5 M (W) GdmCl (from right to left curve). The inset shows the GdmCl dependence of the melting temperature tm. The continuous line represents a linear regression resulting in a slope of 2 9.6 deg. C/M and an extrapolated tm(H2O) in the absence of denaturant of 107.5(^ 1.8) 8C.

peptide chain. However, it is able to form tetramers upon binding to its cognate DNA.4 In order to ensure that tetramerization does not take place at high protein concentrations even in the absence of DNA the association state of ORF56 was analyzed by analytical ultracentrifugation. Independent of protein concentration in the range of 4.6 mM – 0.8 mM the apparent molecular mass was determined to be Mr ¼ 14,060(^ 600), indicating that ORF56 is still dimeric even at millimolar concentrations (Figure 3). This result was confirmed by sedimentation velocity experiments. An apparent s value of 1.85 £ 10213 S, typical for a globular protein of a mass of 13 kDa, could be determined at a protein concentration of 30 mM and 0.8 mM. Thus, ORF56 is dimeric in solution even at the highest concentrations used in this study. Consequently, the folding reaction starts with unfolded monomers (U) and ends with native dimers (N2). The simplest possible folding mechanism for dimeric proteins is described by the two-state model in equation (1): kf

2U O N2

ð1Þ

ku

At this equilibrium only folded dimers and unfolded monomers exist and the midpoint of the unfolding transition depends on the protein concentration. The dimeric state is favored at higher protein concentrations, which results in a higher midpoint of the transition. The extracted thermodynamic parameters DGU and DGU(H2O) refer to a protein concentration of 1 M and are concentration-independent.20 The thermodynamic stability of ORF56 was elucidated by thermal and denaturant-induced unfolding transitions using various protein concentrations. For thermal unfolding in the presence of 4 M GdmCl the melting temperature increases from 52 8C to 75 8C when the protein concentration is increased from 0.2 mM

Figure 3. The dimeric state of ORF56 as determined by analytical ultracentrifugation. ORF56 in 50 mM potassium phosphate (pH 7.5) was analyzed by sedimentation equilibrium at 20,000 rpm, 25 8C. The protein was used at monomer concentrations of 4.6 mM (W), 30 mM (X), and 0.8 mM (L). Data were collected at 230 nm (4.6 mM), 280 nm (30 mM), and 300 nm (0.8 mM), respectively. A global fit yielded a molecular mass of Mr ¼ 14,060(^ 600), independent of the protein concentration.

to 28 mM, respectively (data not shown). Besides the extremely high thermal stability of ORF56 the stability against chemical denaturation is also remarkable. The protein is fully native in 20 mM sodium cacodylate/HCl (pH 7.5) in the presence of 10 M urea. Therefore, strong denaturants such as GdmCl or GdmSCN have to be used to accomplish complete unfolding. GdmCl-induced unfolding transitions of 0.5 mM and 5 mM ORF56 at 25 8C monitored by fluorescence as well as far-UV CD are depicted in Figure 4(a). The different spectroscopic probes show superimposable transitions, indicating a simultaneous loss of secondary and tertiary structure. Unfolding free energies (DGU) were calculated for different protein concentrations according to equations (3) and (4), revealing equivalent results. Figure 4(b) shows DGU determined in the transition region as a function of the GdmCl or GdmSCN concentration at various protein concentrations. The linear extrapolation of

230

Folding and Association of Dimeric ORF56

Figure 4. Concentration dependence of ORF56 denaturation. (a) GdmCl-induced unfolding of 0.5 mM (W) and 5 mM (S,X) ORF56 in 20 mM sodium cacodylate/HCl (pH 7.5) at 25 8C. Transitions indicated with (W,X) were detected by fluorescence at 335 nm after excitation at 280 nm and (S) was probed by far-UV CD at 225 nm. (b) DGU derived from fluorescence data as a function of the denaturant concentration. GdmCl-induced unfolding of 0.5 mM (W) or 5 mM (X) ORF56 and GdmSCN-induced unfolding of 1 mM (A) or 5 mM (B) ORF56.

DGU to 0 M denaturant (equation (5)) yields the unfolding free energy in the absence of denaturant (DGU(H2O)). The intercept provides a DGU(H2O) of 85.1 kJ/mol for both GdmCl and GdmSCNinduced unfolding. Slopes of 2 9.5 kJ/(mol M) and 2 31.3 kJ/(mol M) determine the m-values for GdmCl and GdmSCN, respectively, indicating that GdmSCN is a threefold more efficient denaturant than GdmCl. It should be noted that all denaturation curves were monophasic and showed very similar cooperativities. From these results we can conclude that equilibrium unfolding of ORF56 is a two-state process between folded dimers and unfolded monomers. Temperature dependence of the unfolding free energy DGU Thermodynamic parameters such as DGU, DHU and DSU vary as a function of temperature depending upon the change in heat capacity DCp.21 To obtain these parameters a stability profile was determined by combining GdmCl-induced unfolding transitions between 10 8C and 75 8C with heatinduced transitions at 1.5– 3.5 M GdmCl. The former were probed by fluorescence spectroscopy and DGU was derived by equations (3) and (4) (open symbols in Figure 5) and the latter free unfolding energies were calculated at the melting temperature of heat-induced unfolding transitions ( fU ¼ 0.5), which were monitored by far-UV CD. Since thermal unfolding was performed in the presence of various GdmCl concentrations between 1.5 M and 3.5 M (Figure 2) free energies at tm in the absence of denaturant DGU(H2O) were estimated according to the linear extrapolation by equation (5) (filled symbols in Figure 5). The m-value determined in Figure 4(b) was used as the slope for this calculation and assumed to be temperature-independent. Open and filled symbols in Figure 5 coincide at 75 8C, indicating the feasibility of a joint fit of the two datasets and all assumptions for the construction of the stability

profile from the two-state model. The profile displays a maximum around 30 8C. Fitting of equations (4) and (6) to the stability profile yields DHm ¼ 525(^ 10) kJ/mol, tm ¼ 105.2(^ 0.5) 8C and DCp ¼ 5.8(^ 0.2) kJ/(mol K) for 5 mM monomer concentration and 131.1(^ 0.5) 8C for 1 M protein. NMR spectroscopy confirms the dimeric twostate model for ORF56 The cooperativity of the folding reaction of ORF56 was further analyzed in great detail by heteronuclear 2D NMR spectroscopy, which provides many individual probes distributed over the entire protein. A protein concentration of 1 mM was used in these NMR experiments, and therefore the midpoint of the denaturant-induced unfolding transition of dimeric ORF56 is shifted to a higher denaturant concentration compared to the fluorescence and CD data, which were measured at

Figure 5. Temperature dependence of the equilibrium stability of ORF56. (W) DGU(H2O) derived from GdmClinduced unfolding transitions at the respective temperature. (X) DGU(H2O) calculated from the midpoints of heat-induced unfolding transitions (KU ¼ Pt in equation (4)) at various GdmCl concentrations using equation (5) and m ¼ 29.5 kJ/(mol K). The continuous line represents a fit of equations (4) and (6) to the free energies giving DHm ¼ 525(^ 10) kJ/mol, Tm ¼ 378.3(^ 0.5) K, and DCp ¼ 5.8(^ 0.2) kJ/(mol K).

231

Folding and Association of Dimeric ORF56

causing significant spectral overlap. At the midpoint of the unfolding transition (Figure 6(b)) the two conformations give rise to cross-peaks caused by the slow chemical exchange between the native and unfolded state with respect to the NMR chemical shift timescale. HSQC spectra over the entire GdmSCN concentration range show only crosspeaks of the native or the unfolded state, indicating that no additional species are present. Assignment of the cross-peaks at pH 7.5 was straightforward due to the gradual shift of the resonance frequencies with pH. Figure 7 depicts a selection of the GdmSCN-induced transitions for signals of both the native (Figure 7 upper row) and the unfolded state (Figure 7 lower row). The respective crosssignals of these selected residues are highlighted in Figure 6. Cross-peaks of the unfolded state are not assigned to individual residues except for W20 H11 but numbered consecutively. Individual unfolding transitions could thus be obtained for 31 cross-peaks of the native and 18 cross-peaks of the unfolded state. All transitions of both states exhibit the same cooperativity within experimental error. In the middle of the top row in Figure 7 the fit of the cross-peak intensities of the unfolded state is plotted as a broken line. The transition midpoints of the native and unfolded protein fraction are identical. Therefore, the normalized populations fN and fU of all individual probes distributed over the entire protein intersect at a fractional change of 0.5, strongly emphasizing the equilibrium two-state folding mechanism of ORF56. pH dependence of the thermodynamic stability of ORF56

Figure 6. 2D 1H/15N NMR correlation spectra (HSQC) of (a) native ORF56, (b) at the midpoint of the unfolding transition (2.8 M GdmSCN) and (c) the fully unfolded protein (4.5 M GdmSCN) at 25 8C. Labeled cross-peaks indicate residues for which the entire unfolding transition is given in Figure 7.

5 mM ORF56 (see Figure 4). Consequently, GdmSCN was used as a very strong denaturant because GdmCl could not completely unfold 1 mM ORF56 at pH 7.5 and 25 8C. Throughout the unfolding transition, 28 2D 1H/15N heteronuclear single quantum coherence (HSQC) spectra at various GdmSCN concentrations between 0 M and 4.5 M were recorded. In Figure 6 the spectra of (a) native and (c) fully unfolded ORF56 are shown as well as (b) the spectrum measured at the midpoint of the unfolding transition at 2.8 M GdmSCN. The well-dispersed cross-peaks of the folded protein vanish with increasing GdmSCN concentration and cross-peaks of the unfolded state emerge. Most of the latter depict 1 H chemical shifts between 8.0 ppm and 8.5 ppm

The thermal stability of ORF56 depends on the pH. Nevertheless, at 10 8C there are no changes between pH 2 and pH 12 related to the far-UV CD spectrum of folded ORF56, indicating that even at extreme pH values no detectable changes of the structure occur (Figure 8(a)). Thermal unfolding transitions over this pH range were performed at a protein concentration of 5 mM (Table 1 and Figure 8(b)). Since, in the absence of GdmCl the melting temperature between pH 5 and pH 9 exceeds the boiling point of water, thermal unfolding was also carried out in the presence of 4 M GdmCl. The obtained pH profile of tm displays a maximum around pH 7, which is close to the intra-cellular pH of Sulfolobus cells.22 At 25 8C and in the presence of 4 M GdmCl ORF56 is folded between pH 2 and pH 11. Refolding of ORF56 depends on the protein concentration The equilibrium data showed that ORF56 folding is a two-state process in which two unfolded monomers associate to form the native dimers (equation (1)). The kinetics of refolding should thus depend on the protein concentration. Therefore, refolding kinetics were measured as a

232

Folding and Association of Dimeric ORF56

Figure 7. GdmSCN-induced unfolding transitions of ORF56 monitored by backbone amide cross-peaks of individual residues. Assigned cross-peaks of the native state are given in the respective graph. Cross-peaks of the unfolded state are not assigned but numbered consecutively. Normalized populations of the native (upper row) and the unfolded protein (lower row) were determined from peak intensities in 28 2D 1H/15N HSQC spectra recorded at various GdmSCN concentrations at 25 8C. Continuous lines represent fits of a two-state model for dimeric proteins (equations (3)– (5)). The broken line in the middle of the top row represents the fit of the cross-peak intensities of the unfolded protein. The two lines intersect at 0.5, clearly indicating a two-state behavior of ORF56. Table 1. pH dependence of the thermal stability of ORF56 pH

tm (8C)a

tm (8C)a in 4 M GdmCl

2 3 4 5 6 7 8 9 10 11 12

61.7 82.1 97.0 n.ab n.ab n.ab n.ab 99.6 96.0 81.6 37.1

43.0 55.5 63.9 67.9 72.1 78.6 68.6 64.8 57.0 41.6 n.ac

All experiments were performed with 5 mM ORF56. a 20 mM sodium phosphate, 20 mM sodium citrate was used as a buffer at the respective pH. b No complete denaturation in the absence of GdmCl. c ORF56 is fully unfolded at the starting conditions (10 8C).

function of the ORF56 concentration at 20 8C and pH 7.5. Refolding was initiated by an 11-fold dilution of unfolded ORF56 (in 8.5 M GdmCl) in a stopped-flow apparatus. All kinetic traces were monophasic and apparent rate constants (kapp) were calculated by equation (8), which is the appropriate function to analyze the kinetics (the top inset in Figure 9(a) shows the residual after fitting). Adjusting a single exponential function to the data results in large residuals (bottom inset in Figure 9(a)). Figure 9(b) depicts kapp as a function of the protein concentration. As expected for the two-state model (equation (7)), refolding depends linearly on the protein concentration up to about 5 mM (equation (9)). At higher

Figure 8. Secondary structure and thermodynamic stability of 20 mM ORF56 in 20 mM sodium phosphate, 20 mM sodium citrate at various pH values between 2 and 11. (a) Far-UV CD spectra of 10 mM ORF56 at pH 2 (black), pH 3 (dark blue), pH 4 (marine blue), pH 5 (light blue), pH 7 (red), pH 9 (magenta), pH 10 (pink) and pH 11 (green) depict an invariant degree of secondary structure throughout the entire pH range. (b) Thermal unfolding curves of 5 mM ORF56 in 20 mM sodium phosphate, 20 mM sodium citrate as a function of pH followed by the change of the CD signal at 225 nm. (A) pH 2, (O) pH 3, (X) pH 4, (W) pH 10 and (K) pH 11. The continuous lines represent the non-linear least-squares fits of equation (6) to the data according to the two-state model for dimeric proteins (equation (1)). The midpoints of the thermal unfolding transitions are given in Table 1.

Folding and Association of Dimeric ORF56

233

Figure 9. Concentration dependence of the apparent rate constant kapp of refolding in 20 mM sodium cacodylate/ HCl (pH 7.5) and 0.8 M GdmCl at 20 8C. (a) Representative kinetic trace with a final concentration of 1 mM ORF56 and the best fit with equation (8) (continuous line). The upper inset depicts the residuals revealed for the fit of equation (8) to the data and the lower inset shows the residuals of the fit of a single-exponential equation to the data. This indicates that refolding can be adequately described by a single second-order kinetic phase. (b) Apparent rate constant kapp of refolding as a function of the ORF56 concentration. The continuous line indicates the best fit using equation (9) with kf ¼ 4.9 £ 106 M21 s21 as the slope. The symbol size exceeds the error bars for kapp below 9 mM ORF56.

concentrations kapp becomes smaller than predicted, indicating a deviation from this simple model. The slope of the straight line in Figure 9 was calculated by using equation (9) and revealed a bimolecular rate constant kf of 4.9 £ 106 M21 s21 for ORF56 concentrations below 5 mM. To measure the unfolding kinetics, native dimeric ORF56 was

diluted 40-fold by manual mixing with a buffer containing 8.2 M GdmCl. The unfolding rate constant is independent of the protein concentration between 0.25 mM and 5 mM (data not shown), thus supporting the two-state model for this range of ORF56 concentrations (equations (11) and (12)).

Figure 10. Folding kinetics of 5 mM ORF56 at 10 8C in 20 mM sodium cacodylate/HCl (pH 7.5) monitored by fluorescence at 335 nm (K) or CD at 225 nm (W). (a) Kinetic traces of refolding to final conditions of 2 M GdmCl. Continuous lines are the best fits to equation (8) with a kapp of 1.36(^ 0.01) s21 and 0.78(^ 0.12) s21 for fluorescence and CD data, respectively, which reveal a kf of 6.8 £ 105 M21 s21 and 3.9 £ 105 M21 s21, respectively. (b) GdmCl dependence of the refolding rates. The refolding rate in the absence of denaturant kf(H2O) is 7.0(^0.7) £ 107 M21 s21 and mf is 22.44(^0.02) M21. (X) Refolding rates derived from unfolding experiments using equation (10). (c) Kinetic traces of unfolding to final conditions of 7.6 M GdmCl. Continuous lines are the best fits to a single exponential equation with a ku of 0.0039(^0.0001) s21 and 0.0041(^0.0001) s21 for fluorescence and CD detection, respectively. (d) GdmCl dependence of the unfolding rates reveals a 0 M GdmCl intercept of ku(H2O) ¼ 1.8(^ 0.6) £ 1027 s21 and mu ¼ 1.33(^0.06) M21. (X) Unfolding rates calculated from refolding experiments using equation (10).

234

Folding and Association of Dimeric ORF56

GdmCl concentration dependence of the folding kinetics of ORF56 Refolding and unfolding of ORF56 was followed by fluorescence and far-UV CD spectroscopy. Fast folding events at concentrations between 0.7 M and 3.7 M GdmCl were analyzed in a stoppedflow apparatus, whereas refolding above 3.7 M GdmCl was measured after manual mixing. A representative refolding experiment with both fluorescence and circular dichroism detection is shown in Figure 10(a). Continuous lines represent best fits of monophasic curves to the data by using equations (8) and (9). Refolding rates (kf) at denaturant concentrations up to 5 M GdmCl were determined with equations (8) and (9). In the transition zone (5.2 – 6.4 M GdmCl) values of kf were derived according to equation (10), which takes into account both the unfolding and refolding reaction as described by Milla & Sauer.12 Fluorescence as well as CD-detected kinetics reveal identical refolding rates. Figure 10(b) depicts the GdmCl dependence of the refolding rate constant. Linear regression (equation (13)) results in a refolding rate constant of 7.0 £ 107 M21 s21 in the absence of denaturant and a slope (mf-value) of 2 2.44 M21. Kinetic traces of ORF56 unfolding in 7.6 M GdmCl monitored by fluorescence and circular dichroism are presented in Figure 10(c). The reaction is monophasic and both probes exhibit the same time-course. Because unfolding is independent of the protein concentration, the rate constant ku between 6.6 M and 8 M GdmCl can be calculated with the single-exponential equation (equation (12)). As mentioned above for refolding in the transition zone, unfolding rates between 5.2 M and 6.4 M GdmCl were calculated from the experimental data by equation (10).12 The GdmCl dependence of ln ku is shown in Figure 10(d). It reveals an unfolding rate of 1.8 £ 1027 s21 (i.e. 5.7 year21) at 0 M GdmCl and an mu-value of 1.33 M21 by using equation (14). Correlation between the folding kinetics and the equilibrium stability Relating parameters from kinetic data with results from equilibrium experiments provides simple criteria to test whether a particular folding reaction is well described by the proposed model. Table 2. Thermodynamic and kinetic data of the folding transition of ORF56 Method Equilibrium Kinetics

KU (M)

DGU(H2O) (kJ/mol)

m (kJ/(mol M))

5.3(^0.1) £ 10216 5.1(^1.3) £ 10215

82.8 ^ 0.3 77.5 ^ 0.6

9.5 ^ 0.5 8.9 ^ 0.2a

In 25 mM sodium cacodylate/HCl (pH 7.5) at 10 8C. a The kinetic m-value was calculated by m ¼ mu 2 mf and multiplied by RT using mf ¼ 22:44ð^0:02Þ M21 and mu ¼ 1:33ð^0:06Þ M21 :

The calculation of the equilibrium constant of unfolding from folding kinetics with Kkin U ¼ 2ku(H2O)/kf(H2O) reveals values that are in agreement with those derived from equilibrium transitions (Table 2). The tenfold deviation of KU between the equilibrium and kinetic analysis can be tolerated considering the long extrapolations shown in Figures 4 and 10(d). The virtually identical kinetic and equilibrium m-value also supports the validity of the applied two-state model for dimeric proteins. This coincidence corroborates well the assumed linearity for ln ku down to 0 M GdmCl (Figure 10(d)) as well as the determination of tm(H2O) and DGU(H2O) at various temperatures, where we had to extrapolate the GdmCl data to 0 M GdmCl (e.g. Figure 4(b)).

Discussion The homodimeric protein ORF56 from hyperthermophilic and acidophilic S. islandicus exhibits an extremely high stability towards heat, denaturant, and acid/base-induced unfolding. Such exceptional stabilities have been observed for other proteins from hyperthermophilic organisms and explained by both the thermodynamic and kinetic properties of their polypeptide chains. Oligomerization is one of several mechanisms of thermal stabilization of a protein but no general adaptive rules have been established.16,23 – 25 Two-state folding of ORF56 Self-organization of the native state of oligomeric and multimeric proteins includes both folding and association steps.26 The folding mechanism and stability of only a few homodimeric proteins have been studied in detail so far. Prominent examples are Arc repressor,11,12 mitochondrial and cytosolic malate dehydrogenase,13,14 and Trp aporepressor,9,10 which all fold in the absence of detectable equilibrium intermediates. ORF56 belongs therefore together with dihydrofolate reductase from Thermotoga maritima,16 human papillomavirus strain-16 E2 DNA-binding domain,27 a model coiled coil leucine zipper,28 and spherulin 3a29 to this class of proteins, which is supported by the present findings. The midpoint of denaturation depends on the protein concentration for heat as well as denaturant-induced unfolding. Equilibrium unfolding transitions monitored by far-UV CD and fluorescence coincide, indicating a simultaneous loss of secondary and tertiary structure. But this is not always a reliable proof for a two-state behavior. Folding studies of rat intestinal fatty acid binding protein,30 ferricytochrome c,31 and human CDK inhibitor p19INK4d,32 revealed superimposable unfolding transitions when measured by various optical probes and therefore an apparent two-state behavior. A detailed characterization by NMR, however, uncovered for these proteins the presence

235

Folding and Association of Dimeric ORF56

of equilibrium intermediates. This can be ruled out in the case of ORF56 because all 2D NMR spectra recorded during the entire GdmSCN-induced unfolding transition contained only cross-peaks of the native and unfolded conformation. In all, 31 and 18 analyzed transitions of resonances of the native and denatured state, respectively, exhibit equal midpoints and the fraction of native and unfolded protein intersect at 0.5, which leaves no room for the population of intermediates. For K55 at the C terminus the cross-peaks of the native and unfolded state superimpose. Further evidence for the two-state characteristics of ORF56 unfolding was provided by the kinetic folding investigations. The monophasic unfolding and refolding kinetics reveal corresponding folding rates (kf, ku) when followed by fluorescence and far-UV CD. Only refolding rates depend on the protein concentration. Furthermore, ln kf and ln ku depend linearly on the GdmCl concentration, indicating that the rate-limiting step does not change throughout the entire GdmCl concentration range. In the transition region (5.2 – 6.4 M GdmCl) identical folding rates were obtained from both unfolding and refolding experiments (filled symbols in Figure 10). Coinciding results for DGU and the m-value derived from equilibrium and kinetic analyses also support the two-state mechanism. In several cases such a simple kinetic two-state behavior has been reported for Arc repressor12 and particular GCN4 peptides.33,34 In contrast, kinetic intermediates and more complex refolding mechanisms of dimeric proteins have been detected for dihydrofolate reductase from T. maritima,16 human papillomavirus strain-16 E2 DNA-binding domain,35 Trp aporepressor,9,10 stabilized leucine zipper peptides,28,36,37 and phage 434 Cro protein.38 Stability profile The thermodynamic characterization of ORF56 was carried out according to equations (3) – (6), which are based on a two-state model for dimeric proteins. The latter state was present under all conditions used in this study. Analytical ultracentrifugation confirmed earlier reported results about the dimeric character of ORF56 in the absence of its cognate DNA.4 For a comprehensive definition of the temperature profile of the free energy of unfolding (Figure 5) we combined data from denaturant and heat-induced unfolding transitions under fluorescence and far-UV CD detection, respectively, to sample a range of 85 deg. C. Thermal transitions were acquired at different GdmCl concentrations with subsequent extrapolation of DGU to the absence of denaturant. Consequently, all data points of the stability profile were derived by a linear extrapolation to 0 M GdmCl of experimental data measured in solutions containing at least 1 M GdmCl. Therefore, the extrapolated Gibbs free energy at pH 7.5 represents the stability of ORF56 under high-salt conditions. The same argument holds for the comparison of

thermodynamic parameters derived from equilibrium and time-resolved experiments in Table 2. We found the maximum stability of ORF56 around 30 8C, which is about 50 deg. C below the optimal growth temperature of S. islandicus. Such a behavior is generally observed for thermophilic proteins.24,39 This indicates that many proteins from thermophilic and hyperthermophilic organisms are stabilized by an increased Gibbs free energy rather than by a shift of the maximum stability towards higher temperatures. The difference in heat capacity between unfolded and native state of 5.8 kJ/(mol K) is in good agreement with the expected value of about 6 kJ/(mol K) for a protein of the size of ORF56.40 At 80 8C, ORF56 has lost only about 30% of its maximum stability and more than 99% of all molecules are folded even at a concentration as low as 10 pM, indicating that ORF56 is always fully functional at the optimal growth temperature of S. islandicus. The maximal affinity of ORF56 to a double-stranded DNA fragment from its own promotor was found around 40 8C with a dissociation constant of 4 nM.4 The extrapolated dissociation constant at 80 8C can be estimated to be below 1 mM. An examination of whether ORF56 follows the general concept of corresponding states24,25 between mesophilic and thermophilic organisms as found for example for the maltose-binding protein from the hyperthermophilic bacterium T. maritima41 is not possible because no mesophilic archaeon corresponding to S. islandicus is known. In addition to its remarkable thermal stability, ORF56 is also highly stable against acid and baseinduced unfolding. The native structure does not change significantly between pH 2 and pH 11 and thermal unfolding transitions at pH 2 and pH 11 reveal melting temperatures that still depend on the protein concentration. This implies that ORF56 remains natively folded as a dimer even under extreme pH conditions and net charges as expected for a protein from a hyperthermophilic and acidophilic organism. Folding kinetics Up to an ORF56 concentration of 5 mM, the apparent refolding rate kapp increases linearly with the protein concentration, whereas the unfolding reaction is concentration-independent. This is expected for a unibimolecular mechanism without populated intermediate states under strongly native or strongly denaturing conditions (equations (7) and (11)). Above 5 mM, kapp deviates from linearity towards smaller rates than predicted from the protein concentration. This deviation suggests that a unimolecular step either before or after the bimolecular step becomes co-rate-limiting for the refolding reaction. Such a deviation from linearity was also observed for the Arc repressor.12 The bimolecular refolding rate constant kf of 1 £ 107 M21 s21 at 0.8 M GdmCl is approximately two orders of magnitude below the diffusion limit

236 of about 1 £ 109 M21 s21, suggesting that the association of two polypeptide chains is not diffusionlimited. The unimolecular unfolding rate constant ku of about 1026 s21 under the conditions of these refolding experiments (0.8 M GdmCl) is far too low to contribute to the measured kinetics. It is unlikely that ORF56 forms monomeric kinetic intermediates in a rate-limiting unimolecular folding reaction at protein concentrations above 5 mM. Monomeric intermediates should populate prior to the association of two chains at decreasing rather than at increasing protein concentrations if the association reaction is not diffusion-limited. We can also exclude that transient species are populated only under strong native conditions, because the same deviation from linearity of kapp above 5 mM ORF56 was observed for various refolding conditions up to 3.75 M GdmCl (data not shown). A change in the folding mechanism because of the formation of higher oligomeric states under higher protein concentrations can also be excluded from the analytical ultracentrifugation data. These considerations follow closely the lines for understanding the refolding kinetics of the structurally and functionally homologous Arc repressor,12 indicating that both proteins follow a common mechanism. Bimolecular refolding rates derived between 1 M and 6 M GdmCl cover a remarkable range of five orders of magnitude between 40 M21 s21 and 5 £ 106 M21 s21. ln kf and ln ku depend linearly on the denaturant concentration, which strongly emphasizes that the rate-limiting step of refolding and unfolding does not change in this GdmCl concentration range. The coincidence of the folding rates under far-UV CD and fluorescence detection and the equivalence of kf and ku in the transition zone (5.2 –6.4 M GdmCl) determined from both unfolding and refolding reactions also support the two-state model (filled black symbols in Figure 10(b) and (d)). The kinetic m-values derived from these data coincide with the m-value from the equilibrium experiments (Table 2) validating the assumed simple folding models. The fractional change of the m-value during refolding a (a ¼ mf/(mf 2 mu)) yields 0.65, indicating that the dimeric activated state of folding resembles more the native state in its solvent-accessible surface area as the unfolded state. In this property ORF56 also resembles the Arc repressor, which showed an a-value of 0.73.12 Unfolding of ORF56 becomes extremely slow at 0 M GdmCl (1.8 £ 1027 s21). Such a retardation of unfolding by several orders of magnitude has been observed for other hyperthermophilic and dimeric proteins with respect to their mesophilic homologs. In the case of dihydrofolate reductase from T. maritima, the unfolding reaction is 108 times slower compared to the representative from E. coli.16 A very detailed comparison of the folding of homologous cold shock proteins from mesophilic, thermophilic, and hyperthermophilic organisms was possible for

Folding and Association of Dimeric ORF56

CspB from Bacillus subtilis, Bc-Csp from Bacillus caldolyticus, and Tm-Csp from T. maritima.23 All monomeric homologs follow a two-state folding mechanism with close refolding rates between 500 s21 and 1500 s21. On the contrary, the unfolding rates are inversely proportional to the thermal stability: 10 s21 for CspB, 0.64 s21 for Bc-Csp, and 0.018 s21 for Tm-Csp. Further examples are rubredoxin from Pyrococcus furiosus, which unfolds 2000 times slower compared to mesophilic rubredoxin from Clostridium pasteuriamus,42 as well as pyrrolidone carboxy peptidase from P. furiosus, which unfolds at room temperature 107 times slower compared to the protein from Bacillus amyloliquefaciens.43 The respective refolding rates are almost invariant for the homologous proteins.

Conclusions To extend our knowledge about folding of proteins from extremophilic organisms from monomeric to dimeric proteins, we studied the stability, folding, and association of ORF56 from S. islandicus. This small protein is composed of two-times 56 amino acid residues and revealed a remarkable stability against denaturants of DGU(H2O) ¼ 85.1 kJ/mol. Both the equilibrium and kinetic folding reaction are two-state processes, indicating that folding and association of ORF56 are concurrent events. Together with structural similarities, this protein resembles closely the folding mechanism of Arc repressor from bacteriophage P22.11,12 The bimolecular refolding rates of both proteins are of the order of 107 M21 s21. The major differences between the mesophilic Arc repressor and the hyperthermophilic ORF56 is the thermal stability (tm ¼ 54 8C versus tm ¼ 107 8C) and DGU(H2O) from denaturant-induced unfolding transitions (26 kJ/mol versus 85 kJ/mol). The main reason for these differences is the extremely low unfolding rate of ORF56, which is 1.8 £ 1027 s21 compared to 0.1 s21 for Arc repressor. Such a correlation of high protein stability and slow unfolding rates has been often observed from comparisons of mesophilic and (hyper)thermophilic homologs. It seems that this correlation is one important and realized way during the evolution of (hyper)thermophilic proteins25 independent from their oligomeric states. With the Arc repressor and ORF56 we have a very rare case, where the mesophilic and hyperthermophilic protein follow under equilibrium and non-equilibrium conditions the same folding model. This opens new insights into the elementary folding processes of homodimers and their stability.

Materials and Methods Materials GdmCl

(ultrapure)

was

purchased

from

ICN

237

Folding and Association of Dimeric ORF56

Biomedicals (Eschwege, Germany), GdmSCN from Acros (Germany) and polyoxyethylene-8-decylether (POE-8-DE) from Sigma; all other chemicals were obtained from Merck (Darmstadt, Germany). Denaturant concentrations were measured by the refraction of the solution.44 Cloning, expression and purification of ORF56 from S. islandicus was described recently.4 For 15 N labeling, bacteria were grown under the same conditions using M9 medium45 supplemented with 1 g/l of 15NH4Cl. Determination of protein concentrations made use of the calculated molar absorption coefficient of 9650 M21 s21 at 280 nm for the monomeric protein46 and the unity of ORF56 was proven by SDSPAGE. All experiments were carried out in 25 mM sodium cacodylate/HCl buffer (pH 7.5) unless indicated otherwise. Although every ORF56 monomer contains a single cysteine residue there was no need for using reducing agents or EDTA. For denaturant-induced equilibrium unfolding, the addition of 0.1 mM POE-8DE was required to protect the protein from adhesion to the reaction tubes during incubation of the samples. Spectroscopic methods Fluorescence emission measurements were carried out in 1 cm cells with a Hitachi F4010 spectrofluorimeter and protein concentrations of 1 mM or 5 mM at an excitation wavelength of 280 nm. CD spectra were performed in 1 mm cuvettes on a Jasco J600A spectropolarimeter at a scan speed of 20 nm/minute and a protein concentration of 20 mM. The spectra were measured six times, averaged and corrected for contributions of the respective buffer. Cuvettes were thermostated using a circulating waterbath. The temperature in the cell was measured with a calibrated precision thermometer (Brand, Wertheim, Germany). Denaturant-induced unfolding transitions For denaturation experiments, ORF56 samples (1 mM and 5 mM) were incubated for at least four hours at 25 8C in the presence of various denaturant (GdmCl, GdmSCN) concentrations. For GdmCl-induced transitions at different temperatures the samples were incubated in a waterbath at the appropriate temperature for at least four hours (10 – 45 8C) or one hour (75 8C) prior to denaturation. Equilibrium between folding and unfolding is reached in this time-period, since there is no spectral change observable at longer incubation times. The unfolding experiments were monitored in 1 cm cuvettes by the intrinsic tryptophan fluorescence emission at 335 nm after excitation at 280 nm or CD at 225 nm. The far-UV CD or fluorescence signal was recorded for 60 seconds, averaged and corrected by subtraction of the buffer value. The folding reaction is fully reversible because the renaturation curve, starting with unfolded ORF56 in 8 M GdmCl, coincides with the denaturation curve starting with fully native protein. The reversibility of thermal denaturation was shown by superimposable heat-induced unfolding transitions of native and previously thermal unfolded ORF56 (five minutes at 100 8C in the presence of 4 M GdmCl). For comparing changes of different optical properties of the protein during the transition range, each denaturation curve was normalized to the ratio of unfolded and total protein, fU: fU ¼ ðyN 2 yobs Þ=ðyN 2 yU Þ

ð2Þ

where yobs is the observed fluorescence intensity or molar residue ellipticity, respectively, at a given denaturant concentration [D]. Linear extrapolation of the base lines of native ðyN ¼ SN þ mN ½DÞ and unfolded ðyU ¼ SU þ mU ½DÞ protein yield yN and yU at the respective denaturant concentration. Thermodynamic parameters were determined by analyzing the unfolding curves on the basis of a simple two-state model for dimeric proteins.11,20 In the equilibrium only folded dimeric protein N2 and unfolded monomers U exist (equation (1)). At any point of the denaturation reaction the equilibrium constant KU was calculated according to the two-state model for dimeric proteins: KU ¼ ½U2 =½N2  ¼ 2Pt ðfU2 =ð1 2 fU ÞÞ

ð3Þ

in which Pt is the total molar concentration of protein monomers. Unfolding free energies were calculated using: DGU ¼ 2RT ln KU

ð4Þ

where R is the gas constant and T the absolute temperature. The linear dependence of the free energy of unfolding from the denaturant concentration is given by: DGU ¼ DGU ðH2 OÞ þ m½D

ð5Þ

where DGU is the Gibbs free energy of unfolding at a 1 M concentration of all reactants, DGU(H2O) is the extrapolated free energy of unfolding in the absence of denaturant44,47 and m is the cooperativity parameter.48 Values of DGU(H2O) were obtained directly by fitting the unfolding curves to equations (3) – (5).20,49 Heat-induced unfolding transitions Thermal denaturation curves were recorded under the same buffer conditions as used for the denaturantinduced unfolding transitions (25 mM sodium cacodylate/HCl, pH 7.5) unless indicated otherwise. The monomer concentration ranged between 0.2 mM and 28 mM in 1 cm cells. Far-UV CD data were collected at 225 nm, 227.5 nm, 230 nm or 232 nm due to different absorbance of the samples with a Jasco J600A spectropolarimeter equipped with a Peltier element. However, the transition midpoint and cooperativity of the unfolding curves are independent of the detected wavelength. The temperature was measured with a sensor, which was directly inserted into the cell. The accuracy of the sensor was checked with a calibrated precision thermometer. Samples were heated at 1 deg. C per minute from 10 8C to 105 8C in a sealed cell with a Teflon stopper and Parafilm to prevent evaporation. The recorded molar ellipticity per residue was normalized with equation (2). In this case yN and yU were assumed to depend linearly on the temperature and were calculated from yN ¼ y0N þ nN T and yU ¼ y0U þ nU T; respectively, where y0N and y0U are the values at 0 K and nN and nU define the temperature dependence, respectively. The transition curves were analyzed by evaluating equilibrium unfolding constants with equation (3) at any given temperature and fitting them to the following equation:28 KU ¼ Pt exp{ðDHm =RÞð1=tm 2 1=TÞ 2 ðDCp =RTÞðT 2 tm 2 T lnðT=tm ÞÞ}

ð6Þ

DHm is the enthalpy of unfolding at the transition midpoint temperature tm ( fU ¼ 0.5) and DCp is the difference

238

Folding and Association of Dimeric ORF56

in heat capacity between the unfolded monomer and the native dimer. The fit with equation (6) yields DHm and tm. DCp was kept constant at 5.8 kJ/(mol K) for the analysis of the thermal transitions. DCp was derived by fitting equations (4) and (6) to the temperature dependence of DGU (Figure 5). Analytical ultracentrifugation ORF56 was analyzed at initial (monomer) protein concentrations of 4.6 mM – 0.8 mM in 50 mM potassium phosphate (pH 7.5) with a Beckman Optima XL-A centrifuge and an An50Ti rotor. Sedimentation equilibrium measurements (absorption at 230 nm, 280 nm, and 300 nm) were carried out in double-sector cells at 12,000 rpm and 20,000 rpm at 25 8C. Sedimentation velocity was measured at 40,000 rpm and scans were taken over six hours every ten minutes. Data were analyzed with the software provided by Beckman Instruments (Palo Alto, CA).

respectively: ku

N2 O 2U kf

d½U=dt ¼ 2kf ½U2 þ 2ku

ð10Þ

The explicit solutions of equation (10) for the unfolding and refolding reaction in the transition region are given by Milla & Sauer.12 Refolding between 3.7 M and 5.8 M GdmCl as well as unfolding was accomplished by manual mixing of 50 ml of 100 mM ORF56 solution either in 8.5 M or 0 M GdmCl, respectively, with 950 ml of buffer of the desired GdmCl concentration. The kinetics were measured once with fluorescence detection on a Hitachi F4010 spectrofluorimeter and once with CD detection on a Jasco J600A spectropolarimeter. Unfolding is independent of the protein concentration and can therefore be described between 6.6 M and 8 M GdmCl by the model in equation (11) or by the single-exponential equation (equation (12)): ku

N2 ! 2U

d½U=dt ¼ 2ku ½N2 

ð11Þ

Stopped-flow kinetics S ¼ S0 þ S1 ð1 2 expð2ku tÞÞ A DX.17MV sequential mixing stopped-flow spectrometer from Applied Photophysics (Leatherhead, UK) was used for refolding kinetics at various ORF56 concentrations. To absorb scattered light from the excitation beam, acetone was inserted in a 5 mm cell between the observation chamber and the emission photomultiplier. Refolding was started by tenfold dilution of variously concentrated ORF56 solutions in 25 mM sodium cacodylate/HCl (pH 7.5) with 8.5 M GdmCl and was monitored by the change of fluorescence above 300 nm after excitation at 280 nm. Typically, six to eight kinetic traces per protein concentration were averaged. Due to the proposed two-state model, refolding under strongly native conditions can be described by: kf

2U ! N2

d½N2 =dt ¼ kf ½U2

ð12Þ

where ku is the unfolding rate constant and the other variables are defined as above. For GdmCl concentrations between 5.8 M and 6.4 M the solution of equation (10) was used to determine the unfolding rate constant due to a significant contribution of the refolding reaction to the overall signal change. The dependence of ln kf and ln ku on the GdmCl concentration is assumed to be linear (equations (13)–(14)): ln kf ¼ ln kf ðH2 OÞ þ mf ½GdmCl

ð13Þ

ln ku ¼ ln ku ðH2 OÞ þ mu ½GdmCl

ð14Þ

NMR spectroscopy ð7Þ

where kf is the bimolecular refolding rate. Solving this differential equation yields a hyperbolic equation for the description of the refolding reaction: S ¼ S0 þ S1 ðkapp tÞ=ð1 þ kapp tÞ

ð8Þ

kapp ¼ Pt kf

ð9Þ

where S and S0 are the signal amplitude at time t and time t ¼ 0, respectively; S1 is the signal amplitude change during the refolding; kapp corresponds to the apparent rate constant and Pt is the total monomer concentration.12 The determination of the GdmCl dependence of the folding rates was carried out by stopped-flow as well as manual mixing. Refolding of ORF56 between 0.7 M and 3.7 M GdmCl was achieved with a Bio-Logic (Claix, France) PMS 400 detection system connected to a BioLogic SFM3 stopped-flow mixer with a cell path-length of 1.5 mm under simultaneous detection of fluorescence (335 nm) and far-UV CD (225 nm). It was initiated by a tenfold dilution of a 50 mM protein solution in 8.5 M GdmCl with buffers containing various amounts of GdmCl. Refolding between 0.7 M and 5.0 M was analyzed according to equations (8) and (9). In the transition zone (5.2– 6.4 M GdmCl), unfolding and refolding rates were determined using the model in equation (10), which accounts for substantial contributions of the refolding and unfolding reaction to the signal change,

All spectra were recorded at a Bruker DRX 500 spectrometer equipped with a pulsed field z-gradient unit. Sequential resonance assignments of ORF56 were achieved by standard procedures including homonuclear 2D 1H NOESY and TOCSY NMR spectra, 2D 1 H/15N HSQC spectra, as well as 15N edited 3D 1H/15N NOESY and TOCSY HSQC experiments with mixing times between 80 ms and 120 ms. Experiments were performed at 15 8C with a sample containing 2 mM unlabeled or 15N uniformly labeled ORF56 in 50 mM sodium phosphate (pH 5.0) (93%/7% H2O/2H2O). Analytical ultracentrifugation under these solvent conditions confirmed the dimeric state of ORF56 (data not shown). A GdmSCN-induced unfolding transition was monitored by 2D 1H – 15N HSQC50 with a sample of 1 mM uniformly 15N labeled ORF56 dissolved in 50 mM sodium cacodylate (pH 7.5) (93%/7% H2O/2H2O). All NMR data were processed on a Silicon Graphics O2 workstation using Felix 97 (MSI) and NDEE (Software Symbiose).

Acknowledgements We thank C. M. Dobson for experimental time at the stopped-flow CD and F. X. Schmid for helpful discussions. This research was supported by

239

Folding and Association of Dimeric ORF56

grants from the Deutsche Forschungsgemeinschaft (Ba 1821/1-1; Ba 1821/2-1) and INTAS-2001-2347. 17.

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Edited by C. R. Matthews (Received 6 August 2003; received in revised form 26 November 2003; accepted 1 December 2003)