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• W2096008480 abstract "Article1 May 2008free access Biological function in a non-native partially folded state of a protein Francesco Bemporad Francesco Bemporad Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Joerg Gsponer Joerg Gsponer Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Harri I Hopearuoho Harri I Hopearuoho Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Georgia Plakoutsi Georgia Plakoutsi Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Gianmarco Stati Gianmarco Stati Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Massimo Stefani Massimo Stefani Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Niccolò Taddei Niccolò Taddei Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Michele Vendruscolo Michele Vendruscolo Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Fabrizio Chiti Corresponding Author Fabrizio Chiti Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Francesco Bemporad Francesco Bemporad Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Joerg Gsponer Joerg Gsponer Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Harri I Hopearuoho Harri I Hopearuoho Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Georgia Plakoutsi Georgia Plakoutsi Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Gianmarco Stati Gianmarco Stati Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Massimo Stefani Massimo Stefani Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Niccolò Taddei Niccolò Taddei Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Michele Vendruscolo Michele Vendruscolo Department of Chemistry, University of Cambridge, Cambridge, UK Search for more papers by this author Fabrizio Chiti Corresponding Author Fabrizio Chiti Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy Search for more papers by this author Author Information Francesco Bemporad1, Joerg Gsponer2, Harri I Hopearuoho2, Georgia Plakoutsi1, Gianmarco Stati1, Massimo Stefani1, Niccolò Taddei1, Michele Vendruscolo2 and Fabrizio Chiti 1 1Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy 2Department of Chemistry, University of Cambridge, Cambridge, UK *Corresponding author. Dipartimento di Scienze Biochimiche, Università di Firenze, viale Morgagni 50, Firenze I-50134, Italy. Tel.: +39 055 459 8319; Fax: +39 055 459 8905; E-mail: [email protected] The EMBO Journal (2008)27:1525-1535https://doi.org/10.1038/emboj.2008.82 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info As structural flexibility is known to be required for enzyme catalysis and pattern recognition and a significant fraction of eukaryotic proteins appear to be unfolded or contain unstructured regions, biological activity of conformational states distinct from fully folded structures could be more common than previously thought. By applying a procedure that allows the recovery of enzymatic activity to be monitored in real time, we show that a non-native state populated transiently during folding of the acylphosphatase from Sulfolobus solfataricus is enzymatically active. The structural characterization of this partially folded state reveals that enzymatic activity is possible even if the catalytic site is structurally heterogeneous, whereas the remainder of the structure acts as a scaffold. These results extend the spectrum of biological functions carried out in the absence of a folded state to include enzyme catalysis. Introduction Proteins are among the most abundant macromolecules in living systems and carry out a vast number of functions, including the catalysis of virtually every chemical transformation occurring in cell biology and the transduction of signals inside and between cells. Although it is well known that the attainment of well-defined, folded three-dimensional structures is crucial in determining their function, increasing evidence is accumulating about the existence of proteins or protein domains that adopt unstructured but functional states under physiological conditions (Dunker et al, 2001; Fink, 2005). This raises the important point that biological function can be attained by proteins under conformational states distinct from fully folded structures. The mechanisms, however, by which biological function can be carried out in the absence of a well-defined fold is not completely understood. In this study, we have focused our attention on the acylphosphatase from the archaeon Sulfolobus solfataricus (Sso AcP). We will show that this protein retains an ability to function as an enzyme when adopting a non-native state in which the catalytic site is largely unstructured and flexible. Sso AcP is a 101-residue protein belonging to the acylphosphatase-like structural family. The structure of the native state of Sso AcP was recently determined by nuclear magnetic resonance spectroscopy and X-ray crystallography (Corazza et al, 2006). This protein shares the same βαββαβ topology, typical of the ferredoxin-like fold, with the other acylphosphatases so far characterized (Thunnissen et al, 1997; Zuccotti et al, 2004; Miyazono et al, 2005; Pagano et al, 2006). By contrast to related proteins, however, Sso AcP contains an unstructured, 12-residue N-terminal tail (Corazza et al, 2006). Sso AcP is able to hydrolyse benzoylphosphate (BP), with kCAT and kM values of 198±20 s−1 and 0.36±0.04 mM, respectively, at pH 5.3 and 25°C, and to be competitively inhibited by inorganic phosphate (Corazza et al, 2006). The kCAT value of the enzyme is low at 25°C, but increases with temperature and reaches at 81°C—the living temperature for the Archaeon S. solfataricus—a value close to those previously reported for the mesophilic enzymes at 25°C (Corazza et al, 2006). The native state of Sso AcP is thermodynamically very stable with a free energy change of unfolding (ΔGU−FH2O) of 47±1 kJ mol−1 at 37°C (Corazza et al, 2006). The midpoint of thermal unfolding of the protein (Tm) is 100.8±4.1°C and at 81°C the ΔGU−FH2O is as high as 20.6±0.3 kJ mol−1, similar to that of human muscle acylphosphatase at 28°C (Corazza et al, 2006). The folding mechanism of Sso AcP was described previously at pH 5.5 and 37°C (Bemporad et al, 2004). After removal of the denaturant, the unfolded state of this protein collapses on the microsecond timescale into an ensemble of partially folded conformations. This ensemble is capable of binding the fluorescent dye 8-anilino-1-naphthalenesulfonic acid suggesting the presence of hydrophobic clusters exposed to the solvent and presents a far-UV mean residue ellipticity comparable to that of the fully native state, indicating that a native-like secondary structure is already formed in this state (Bemporad et al, 2004). The partially folded ensemble converts into the fully folded state with a rate constant of 5.4±1.0 s−1; a small fraction of molecules (∼10%) folds slower with a rate constant of ∼0.2 s−1 as their folding process is rate-determined by the cis to trans conversion of the Leu49–Pro50 peptide bond (Bemporad et al, 2004). The presence of a relatively stable partially folded state accumulating during folding of Sso AcP offers a very favourable opportunity to study the function of a protein in a conformational state different from the native and folded one, but still populated under non-denaturing conditions. In this work, the functional properties of the partially folded state of Sso AcP accumulating during folding are investigated using a procedure that allows the recovery of enzymatic activity during folding to be determined in real time (Chiti et al, 1999). The protein engineering method and Φ-value analysis (Matouschek et al, 1989) are then used to obtain information on the degree of structure formation, at the level of the mutated residues, in both the partially folded and transition states of the protein. We shall show that the partially folded state of Sso AcP accumulating during folding is enzymatically active despite structural heterogeneity at the level of the active site. In addition, molecular dynamics (MD) simulations using Φ-values as restraints illustrate how this state is made up by an ensemble of conformations displaying native-like topology. This forces catalytic residues to be in close proximity and allows this conformational state to retain enzymatic activity. Results The partially folded state of Sso AcP populated during folding exhibits enzymatic activity We first monitored the folding process of Sso AcP using intrinsic fluorescence as a spectroscopic probe. This protein possesses one tryptophan in the N-terminal segment and seven tyrosines at various positions along the sequence, mainly positioned in the β-sheet (Supplementary Figure S1). Figure 1A shows the change of intrinsic fluorescence when one volume of Sso AcP unfolded in 5.5 M guanidinium hydrochloride (GdnHCl) is mixed with 19 volumes of refolding buffer. In this trace, which is in very good agreement with that previously reported (Bemporad et al, 2004), three phases are observed. A first rapid increase of fluorescence, occurring on a timescale shorter than 10 ms and escaping experimental detection with the stopped-flow device utilized here, was shown to correspond to the conversion of the fully unfolded state into the partially folded state (Bemporad et al, 2004). The following rapid decrease of fluorescence, with a rate constant (kI → F) of 5.3±1.0 s−1 and a relative amplitude of 90±2%, was shown to correspond to the conversion of this state into the fully native conformation, whereas the second slower decrease, with a rate constant of 0.18±0.04 s−1 and a relative amplitude of 10±2%, arises from the cis–trans isomerization of a small fraction of protein molecules with the Leu49–Pro50 peptide bond initially in a non-native cis configuration. The downward curvature observed at low GdnHCl concentrations in the plot reporting ln(kI → F) versus denaturant concentration (Figure 1B) was shown to arise from the formation of the partially folded ensemble in the dead time of the stopped-flow experiment (Bemporad et al, 2004). Figure 1.The partially folded ensemble of Sso AcP displays enzymatic activity. (A) Folding trace of Sso AcP recorded using intrinsic fluorescence as a probe in 0.275 M GdnHCl, 50 mM acetate buffer pH 5.5, 37°C. The inset shows the first second of recording. (B) Observed folding/unfolding rate constant versus denaturant concentration (readapted from Bemporad et al 2004). The continuous line represents the expected plot for a two-state model. The presence of a downward curvature at low GdnHCl concentrations, along with other experimental evidence, indicates the accumulation of a partially folded ensemble during folding. (C) Time course of BP absorbance recorded at 283 nm in the presence of native Sso AcP. (D) Time course of enzymatic activity of the native state, calculated from the trace reported in (C) (see Materials and methods). The continuous line represents the best fit to Equation (1). (E) BP absorbance recorded at 283 nm after dilution of GdnHCl-unfolded Sso AcP into a refolding buffer; final conditions are 0.275 M GdnHCl (continuous line) and 0.275 M GdnHCl, 7 M urea (dotted line). In both conditions, the native protein is thermodynamically more stable than any other states, making it possible to monitor the kinetics of folding. The inset shows the first second of recording. (F) Development of enzymatic activity, calculated from the traces reported in (E) (see Materials and Methods), during the refolding of wild-type Sso AcP in the absence (upper trace) and presence (lower trace) of 7 M urea. The continuous lines represent the best fits to Equation (1). The activities of fully folded, partially folded and unfolded states are shown. (G) A comparison between the time courses (first 2 s) of recovery of native conformation and enzymatic activity. After 3.6 ms, when the partially folded ensemble is populated more than 99%, Sso AcP exhibits 79% of the native enzymatic activity. (H) Ratio between the main folding rate constant recorded in the presence of phosphate (○) or phenyl phosphate (•) (kobs) and that recorded without substrate-analogue in the sample (kI → F) plotted versus substrate-analogue concentration. Download figure Download PowerPoint The time course of recovery of enzymatic activity has then been studied in real time during Sso AcP folding. The substrate BP, unlike its hydrolysis products benzoate and phosphate, has a significant optical absorption at 283 nm. By measuring the decay of absorbance at 283 nm in small and sequential time intervals Δt, in the presence of Sso AcP undergoing the folding process, the time course of enzymatic activity during folding can be reconstructed (see Materials and methods for details). In a first control experiment, a solution containing native Sso AcP has been mixed with the refolding buffer containing BP and a non-denaturing concentration of GdnHCl. Final conditions were 0.02 mg ml−1 protein, 5 mM BP, 0.275 M GdnHCl, 50 mM acetate buffer, pH 5.5, 37°C. The change of absorbance at 283 nm has been monitored in real time (Figure 1C) and analysed to yield the time course of enzymatic activity (Figure 1D). In this control experiment, the protein is expected to remain native all the way through. Indeed, the absorbance at 283 nm arising from BP decays linearly with time (Figure 1C) and the enzymatic activity remains high and constant during the 20 s of recording (Figure 1D), as a result of ongoing catalysis. To monitor the time course of the enzymatic activity during Sso AcP refolding, a sample of GdnHCl-unfolded Sso AcP has been mixed with the refolding buffer containing BP. Final conditions were the same as in the first experiment. Figure 1E and F show the time-dependent changes of absorbance at 283 nm and of the corresponding enzymatic activity, respectively. Immediately after mixing, when the partially folded state is maximally populated, enzymatic activity is already present, corresponding to 79±10% of that of the native protein under the same conditions (Figure 1F). The activity then shows a small increase with a rate constant of 3.5±1.5 s−1, in good agreement with the kI → F value determined with intrinsic fluorescence for the conversion of the partially folded state into the folded state (Figure 1F). The time-dependent changes of intrinsic fluorescence and enzymatic activity in the first two seconds of recording are summarized in Figure 1G. This figure emphasizes that at the beginning of the folding trace, when the fraction of native protein is equal to 0 and the protein populates the partially folded ensemble, the activity is equal to 79% of the native protein. The early enzymatic activity of Sso AcP is not contributed by the unfolded or native states The GdnHCl-unfolded protein has been diluted into a solution containing urea to a final concentration of 7 M. Owing to the high conformational stability of Sso AcP, these conditions are not yet denaturing and refolding is possible. However, the plot reporting ln(k) versus urea concentration shows a downward curvature in the range of 0–5 M, indicating that in 7 M urea, the partially folded state is destabilized and the protein folds according to a two-state model (Supplementary Figure S2). The results show that enzymatic activity is absent immediately after mixing, when only the unfolded state is populated (Figure 1F). Upon refolding, the activity then increases with a rate constant of 0.08±0.02 s−1 (Figure 1F) corresponding to the folding rate constant under these conditions (Supplementary Figure S2). To rule out that a substantial fraction of the native protein is present after the dead time of the stopped-flow experiment, when enzymatic activity is observed already, a double jump experiment has been carried out (Supplementary Figure S3). In this experiment, the GdnHCl-unfolded protein has been diluted into the refolding buffer (first jump) and then, after 10 ms, transferred again to solutions containing GdnHCl at final concentrations ranging from 4.2 to 7.0 M (second jump). Although such final conditions promote the unfolding of native Sso AcP and produce a single exponential change of intrinsic fluorescence (Bemporad et al, 2004), no significant fluorescence changes have been observed in any of these experiments, suggesting that the native protein is not present 10 ms after the folding process has been initiated (Supplementary Figure S3). This result indicates that the enzymatic activity observed at 10 ms does not arise from Sso AcP adopting a native conformation. Sso AcP refolding has also been followed using intrinsic fluorescence in the presence of phosphate and phenyl phosphate, two competitive inhibitors of Sso AcP that are stable analogues of BP. Experimental conditions were the same as for the enzymatic-activity experiments. Neither the first nor the second folding rate constants are affected by either compound (Figure 1H). These findings rule out the possibility that folding of Sso AcP is accelerated by BP and that the enzymatic activity observed at the beginning of the folding process is due to an early substrate-induced folding of the protein. Taken together, these data suggest the idea that the partially folded ensemble accumulating during folding of Sso AcP possesses significant enzymatic activity. The enzymatic activity observed in the Sso AcP partially folded state is highly sensitive to mutations The recovery of enzymatic activity during folding has also been recorded for a set of Sso AcP mutants. Several mutants, such as the K92A variant, show a behaviour similar to that of the wild-type protein with enzymatic activity detected for both the partially folded and native states (Figure 2A and B, Table I). The traces recorded for the N48A and R30A variants show full inactivation of their partially folded and native states (Figure 2C and D, Table I). This result confirms the key role of these two residues in the catalysis of the native state of acylphosphatases and suggests their major role in the catalytic mechanism of the partially folded state as well (Stefani et al, 1997). The partially structured ensembles of the V24A and V27A mutants do not show residual enzymatic activity (Figure 2E and F, Table I). In the case of V24A, the enzymatic activity increases with a rate constant of 3.2±1.0 s−1, a value that is, within experimental error, similar to that measured by following folding of the mutant using intrinsic fluorescence (Table II). This observation indicates that the observed recovery of enzymatic activity in the V24A variant is due to the conversion of the partially folded ensemble into the native state during folding. Finally, two variants with mutations within the 49–52 loop have been investigated. Similarly to V24A, both P50A and G52A variants display fully inactive, partially folded states, but native structures with significant activity that are recovered with rate constants highly consistent with those measured for folding using intrinsic fluorescence (Figure 2G and H, Tables I and II). These data show that the ability to hydrolyse the substrate of the partially folded ensemble is more sensitive to mutations than the native state and provide insight into regions of the structure that are more important for enabling enzymatic activity. Figure 2.The enzymatic activity of the partially folded state of Sso AcP is sensitive to mutations. (A, C, E, G) BP absorbance at 283 nm after dilution of the indicated GdnHCl-unfolded mutants into a refolding buffer. (B, D, F, H) Development of enzymatic activity, calculated from the traces reported in the corresponding panels on the left (see Materials and methods), during the refolding of the indicated Sso AcP mutants. The continuous lines represent the best fits to Equation (1). All traces are labelled to indicate the mutants to which they refer. Download figure Download PowerPoint Table 1. Catalytic parameters for a set of Sso AcP variants measured in 50 mM acetate buffer at pH 5.5, 37°C using BP as a substrate Variant Native state kCAT (s−1) Partially folded state kCAT (s−1)a Activity in the partially folded state (% of that from the native state)b WT 222±20 175±30 79±10 V24A 124±10 7±5 5.6±10 V27A 13±1 0±10 0.0±10 R30A 7±1 ND ND N48A 2±2 ND ND P50A 137±14 0±10 0.0±10 G52A 178±18 6±5 3.3±10 K92A 169±20 111±30 66±10 a Values determined from the best fits of the traces reported in Figure 2 to Equation (1). b Values obtained by combining data of columns 1 and 2. Table 2. Thermodynamic and kinetic parameters of (un)folding for a set of Sso AcP mutants Variant Cm (M) m (kJ mol−1 M−1) ΔΔGU−FH2O (kJ mol−1)a kI→FH2O (s−1) kF→UH2O) (s−1) ΦI Φ‡ WT 4.23±0.07 11.3±1.1 — 5.436±0.272 (6.10±0.30)·10−6 — — Hydrophobic core residues M16A 3.10±0.07 11.0±1.1 12.59±1.13 7.179±0.359 (2.04±0.10)·10−4 0.34±0.06 0.28±0.07 A18G 3.21±0.07 10.3±1.0 11.36±1.12 4.249±0.212 (3.21±0.16)·10−5 0.57±0.05 0.62±0.04 V20A 3.78±0.07 11.7±1.2 5.01±1.11 1.477±0.074 (3.3±0.17)·10−6 0.64±0.09 1.31±0.08 F29L 3.58±0.07 10.9±1.1 7.24±1.11 6.421±0.321 (1.47±0.07)·10−4 −0.07±0.17 −0.13±0.18 I42V 4.11±0.07 11.5±1.1 1.34±1.10 ND ND ND ND A46G 3.07±0.07 9.9±1.0 12.92±1.13 0.855±0.043 (1.66±0.083)·10−4 −0.03±0.09 0.34±0.06 V54A 3.11±0.07 10.6±1.1 12.47±1.13 0.754±0.038 (1.81±0.09)·10−4 −0.11±0.10 0.30±0.06 A58G 3.89±0.07 10.6±1.1 3.79±1.10 1.567±0.078 (6.14±0.31)·10−6 0.15±0.26 1.00±0.05 Y61A 4.18±0.07 14.5±1.4 0.56±1.10 ND ND ND ND Y61L 4.18±0.07 14.0±1.4 0.56±1.10 ND ND ND ND L65A 2.59±0.07 10.3±1.0 18.27±1.15 2.354±0.118 (2.78±0.14)·10−4 0.34±0.04 0.46±0.04 L68A 2.54±0.07 10.0±1.0 18.82±1.15 1.064±0.053 (3.35±0.17)·10−4 0.23±0.05 0.45±0.04 I72V 3.78±0.07 11.8±1.2 5.01±1.11 4.364±0.218 (2.22±0.11)·10−5 0.22±0.18 0.34±0.15 P76A 3.73±0.07 12.1±1.2 5.57±1.11 5.003±0.250 (3.83±0.19)·10−4 −0.96±0.39 −0.92±0.38 V81A 3.13±0.07 10.0±1.0 12.25±1.12 4.761±0.238 (7.72±0.38)·10−4 −0.05±0.10 −0.02±0.09 V84A 3.26±0.07 10.6±1.1 10.80±1.12 4.729±0.236 (2.98±1.49)·10−4 0.04±0.10 0.07±0.10 F88A 2.92±0.07 12.7±1.3 14.59±1.13 5.176±0.259 (2.01±0.10)·10−3 −0.03±0.08 −0.02±0.08 G93A 4.05±0.07 10.8±1.1 2.00±1.10 ND ND ND ND Residues important in catalysis V24Ab 3.28±0.07 9.7±1.0 10.58±1.12 4.290±0.214 (1.94±0.10)·10−4 0.10±0.10 0.16±0.09 V27Ab 3.78±0.07 11.1±1.1 5.01±1.11 6.473±0.324 (4.43±0.22)·10−5 0.07±0.21 −0.02±0.23 R30Ac 4.41±0.07 12.2±1.2 −2.00±1.10 ND ND ND ND N48A 4.34±0.07 11.2±1.1 −1.23±1.10 ND ND ND ND Salt bridge residues R15A 3.88±0.07 11.9±1.2 3.90±1.10 3.012±0.151 (2.07±0.10)·10−5 −0.20±0.35 0.19±0.23 R19A 3.56±0.07 10.4±1.0 7.46±1.11 0.652±0.033 (2.25±0.11)·10−5 −0.18±0.18 0.55±0.07 E59A 3.52±0.07 12.0±1.2 7.91±1.11 2.343±0.117 (5.49±0.27)·10−5 0.01±0.14 0.28±0.10 R71A 4.52±0.07 12.6±1.3 −3.23±1.10 10.090±0.504 (7.01±0.35)·10−6 0.62±0.15 1.11±0.07 Other residues A37G 3.36±0.07 10.1±1.0 9.69±1.12 4.996±0.250 (1.43±0.07)·10−4 0.14±0.10 0.16±0.10 L49A 3.73±0.07 11.2±1.1 5.57±1.11 1.422±0.071 (1.49±0.07)·10−5 −0.03±0.21 0.59±0.09 P50A 4.20±0.07 11.8±1.2 0.33±1.10 ND ND ND ND G52A 3.34±0.07 11.3±1.1 9.91±1.12 0.181±0.009 (1.21±0.06)·10−5 −0.06±0.12 0.82±0.03 P77A 4.19±0.07 10.1±1.0 0.45±1.10 ND ND ND ND S89A 4.17±0.07 11.2±1.1 0.67±1.10 ND ND ND ND K92A 4.07±0.07 10.1±1.0 1.78±1.10 ND ND ND ND F98L 3.01±0.07 8.2±0.8 13.59±1.13 4.211±0.211 (7.61±3.80)·10−4 0.04±0.08 0.08±0.08 a The ΔΔGU−FH2O have been calculated according to Equation (2) using the average of the m values for all mutants. b These residues also belong to the group of ‘hydrophobic core residues’. c These residues also belong to the group of ‘Salt bridge residues’. Investigation of the partially folded and transition states of Sso AcP using Φ-value analysis To characterize the partially folded and transition states of Sso AcP, we have carried out a Φ-value analysis using 34 single mutants (Matouschek et al, 1989, 1992). This method introduces the Φ-value for a certain mutation in the partially folded state Z (ΦZ) according to the equation: ΦZ=ΔΔGZ−U/ΔΔGF−U, where ΔΔGZ−U and ΔΔGF−U represent the free energy changes upon mutation of the partially folded and native states, respectively. The rationale behind this approach is that the elimination of targeted atoms from the side chain of a residue already structured in the partially folded state Z will affect the conformational stabilities of both native and partially folded states to the same extent, resulting in a ΦZ value equal to 1. Conversely, any side-chain change of a residue unstructured in the partially folded ensemble does not affect its conformational stability, resulting in a ΦZ value equal to 0. Mutations have been chosen to probe (1) the hydrophobic core, (2) the active site (four of the investigated mutations, namely R30A, N48A, V24A and V27A, involve the catalytic site and have been found here to abolish or decrease dramatically the enzymatic activity of Sso AcP), (3) the salt bridges that are present on the protein surface and contribute to the enhanced structural stability of the protein (Corazza et al, 2006). An equilibrium GdnHCl-induced unfolding curve has been acquired for each mutant to yield the change in conformational stability upon mutation (ΔΔGU−FH2O). Conditions were 50 mM acetate buffer, pH 5.5, 37°C. Figure 3A shows representative equilibrium unfolding curves for the wild-type protein, the only mutant found to be stabilized relative to the wild type (R71A), and three destabilized mutants (V20A, A46G, L65A). All plots have been analysed with Equation (2). The results show that several mutants are destabilized (Table II). Figure 3.Thermodynamics and kinetics of (un)folding for Sso AcP variants. (A) Equilibrium unfolding curves for a set of Sso AcP variants in 50 mM acetate buffer, pH 5.5, 37°C. The continuous lines represent the best fits to the equation reported by Santoro and Bolen (Santoro and Bolen, 1988). The obtained parameters of conformational stability are reported in Table II. (B) Folding traces recorded in 0.275 M GdnHCl, 50 mM acetate buffer at pH 5.5, 37°C. (C) Unfolding traces recorded in 6 M GdnHCl, 50 mM acetate buffer at pH 5.5, 37°C. The inset shows the first 5 s of recording. In all plots, the traces refer to wild-type (black), R71A (blue), A46G (orange), L65A (red) and V20A (green) Sso AcP. Download figure Download PowerPoint The destabilized (or stabilized) mutants with ΔΔGU−FH2O values higher than 3.2 kJ mol−1 or lower than −3.2 kJ mol−1 have been analysed to obtain the Φ-values of the corresponding mutations for the partially folded and transition states. For each of these mutants, kinetic traces for folding and unfolding have been acquired at various denaturant concentrations, using intrinsic fluorescence and far-UV ellipticity as probes for folding and unfolding, respectively. Figure 3B and C show representative traces for folding and unfolding, respectively. All mutants showed, at low denaturant concentrations, a downward curvature in the folding limb of the plot reporting the folding/unfolding rate constant versus denaturant concentration. This deviation from the two-state model is similar to that observed for the wild-type protein (Figure 1B) and suggests that the partially folded ensemble forms in all mutants. For each mutant, the kinetic traces have been analysed as described in the Materials and methods, to yield the folding (kI→FH2O) and unfolding (kF→UH2O) rate constants in the absence of denaturant (kI→FH2O) refers to the rate of formation of the native state regardless of the on- or off-pathway nature of the partially folded state). A list of the kI→FH2O and kF→UH2O values obtained for all mutants is reported in Table II. The thermodynamic and kinetic data have been combined to obtain the Φ-values for the partially folded and transition states. The Φ-values for the partially folded ensemble (ΦI) are generally lower than the corresponding ones for the transition state (Φ‡), showing a gain of structure along the folding coordinate (Table II). In the partially folded state, the catalytic site does not appear to be fully structured. The R30A and N48A variants have not been analysed, due to their low ΔΔGU−FH2O Nevertheless, the ΦI values obtained for the V24A and V27A variants are close to 0, suggesting that the cata" @default.
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