Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

doi:10.1016/j.jmb.2008.12.052

J. Mol. Biol. (2009) 386, 1138–1152

Available online at www.sciencedirect.com

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein Gabriel Zoldák 1 , Linn Carstensen 1 , Christian Scholz 2 and Franz X. Schmid 1 ⁎ 1

Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany 2

Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg, Germany Received 29 October 2008; received in revised form 17 December 2008; accepted 18 December 2008 Available online 30 December 2008

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1–69 and 130–165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded. © 2008 Elsevier Ltd. All rights reserved.

Edited by K. Kuwajima

Keywords: FKBP; SlyD protein; prolyl isomerase; protein folding kinetics; folding transition state

*Corresponding author. E-mail address: [email protected]. Present address: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany. Abbreviations used: Ttrs, midpoint of a thermal unfolding transition; GdmCl, guanidinium chloride; [GdmCl]m, midpoint of a GdmCl-induced unfolding transition; [urea]m, midpoint of a urea-induced unfolding transition; m, cooperativity parameter of a denaturant (D)-induced unfolding transition, m = ∂ΔGD/∂[D]; SlyD, sensitive-to-lysis protein from E. coli; SlyD*, SlyD protein 1–165 followed by a hexa-His tag; IF, insert-in-flap domain of SlyD; SlyD*ΔIF, SlyD* without the chaperone domain (residues 1–69 and 130–165); SlyD*(D101W), SlyD*protein with the mutation D101W; FKBP, FK506 binding protein; ΔHvH, van`t Hoff enthalpy; ΔHcal, calorimetric enthalpy; ΔGD, Gibbs free energy of denaturation; ΔGD(H2O), ΔGD in the absence of denaturant; CEp, excess heat capacity; λ, apparent rate constant of a reaction; τ, time constant (τ = λ–1); ku, kf, microscopic rate constants of unfolding and refolding, respectively; mu, mf, kinetic m values for unfolding and refolding, defined as ∂ln ki/∂[D]. 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.

Domain Folding of SlyD

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Introduction Domains are the basic structural and evolutionary units of proteins.1–4 Extra domains convey proteins with additional functions, such as target binding or allosteric regulation, and new functions can be generated by the combination of existing domains. In fact, most proteins are composed of several domains, and the complexity of the domain organization increases from prokaryotic to eukaryotic organisms.5 Domains can be combined in various ways. In most multidomain proteins, they are arranged linearly along the protein chain, like beads on a string, connected by single covalent linkages. The stabilities and folding mechanisms of several oligodomain proteins have been analyzed.6–14 In rare cases, an extra domain can also be inserted into another domain,15 which leads to two covalent linkages between the domains. Usually, the inserted “guest” domain bulges out of a turn or loop at the surface of the “host” domain. To avoid mutual destabilization of the two domains upon insertion, the corresponding region of the host domain should not be critical for its stability, and, in the folded state, the guest domain should have its chain termini close together. Indeed, proteins with neighboring termini could be grafted into loops of other proteins to create artificial two-domain proteins.16–18 The introduction of guest proteins with remote chain termini destabilized both host and guest.19–21 To elucidate the principles that govern the stability and folding mechanism of a natural protein with an internally inserted domain, we use SlyD, the sensitive-to-lysis protein from Escherichia coli.22–25 SlyD is a prolyl isomerase of the FK506 binding protein (FKBP) family.26–28 It consists of a catalytic FKBP domain of 86 residues (residues 1–69 and 130– 146), an inserted domain of 60 residues (residues 70– 129), and an unstructured C-terminal region of 50 residues, which is largely absent in the protein variant used in this study. We denote this shortened version with residues 1–165 as SlyD*. The loop that hosts the inserted domain is close to the prolyl isomerase active site of SlyD* and is termed “flap” in other FKBP proteins. Accordingly, the 70–129 region of SlyD* that is inserted at this position is called the “insert-in-flap” (IF) domain. The three-dimensional structure of SlyD* (Fig. 1) shows that the guest IF domain is well-folded and protrudes from the FKBP domain, just above the prolyl isomerase active site. Analysis by NMR spectroscopy indicates that the two domains are not engaged in noncovalent interactions, and that they show a high level of flexibility relative to each other (Weininger and Balbach, personal communication). In its structure, SlyD* resembles the homologous MtFKBP17 protein from the archeon Methanococcus thermolithotrophicus.30 The IF domain functions as a chaperone module and promotes the binding of folding proteins when SlyD catalyzes proline-limited protein folding reactions. Excision of the IF domain from SlyD*

Fig. 1. Three-dimensional structure of E. coli SlyD* (1– 165) (Weininger et al., unpublished data). The FKBP domain (residues 1–69 and 130–146) is shown in red, and the IF domain (residues 70–129) is shown in green. The C-terminal part (residues 147–165) is shown in gray. Residue D101 is shown in space-filling representation. The figure was prepared using UCSF Chimera.29

abolished this folding activity. When inserted artificially into the flap of human FKBP12, it increased the folding activity of this FKBP by about 200-fold, although human FKBP12 and the FKBP domain of SlyD showed only a 14% sequence identity.31 SlyD* provides a simple and natural model system for studying how a guest domain that is inserted internally into a host domain affects the stabilities and folding mechanisms of the constituent domains. We first analyzed the stability and folding kinetics of the protein that consists of only the host domain (the FKBP domain) and then of full-length SlyD* with both domains. Domain insertion decreased the stability of the host protein, decelerated its refolding, and led to curved unfolding limbs in the chevron plots of the folding kinetics. This curvature is not caused by a transition-state movement, as generally assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentration. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded. The stability of the IF domain is strongly increased when the host domain is folded, and, in this form, it showed approximately 1000-fold higher unfolding and refolding rates than the FKBP domain. The kinetic uncoupling of the domains enabled us to determine the folding kinetics and stability of the IF domain separately by stopped-flow double-mixing experiments. The high conformational dynamics of the IF domain explains why the unfolding of the two-domain protein SlyD* is an apparently all-or-none cooperative process under almost all conditions.

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Domain Folding of SlyD

Results To delete the IF domain from SlyD*, residues 70– 129 were removed and replaced by 13 amino acids from the flap region of human FKBP12 (Fig. 1). Despite the very low sequence identity between SlyD and human FKBP12, stretches A67-Y68-G69 and L130-X131-F132 (numbering of SlyD), which connect the FKBP domain with the IF domain or the flap, respectively, are conserved in the two proteins. Accordingly, they were used to align the two sequences when replacing the IF domain of SlyD* by the flap of human FKBP12.31 In this way, the conformation of the two connector peptides between the domains of SlyD* could be maintained after the excision of the IF domain. The protein without the guest domain (SlyD*ΔIF) is well-folded and stable. It shows wild-type enzymatic activity for the cis/trans peptidyl–prolyl isomerization in tetrapeptides, but is inactive as a catalyst of protein folding.31 The isolated IF domain is unfolded in aqueous buffer and at ambient temperature (data not shown). Stability of the host domain in the absence and in the presence of the guest domain The thermal unfolding of SlyD* with and without the IF domain was probed by differential scanning calorimetry (DSC) at pH 7.5 in the presence of 0, 1.0, and 2.0 M urea (Fig. 2). The transitions show high reversibility, as indicated by the coincidence of the peaks obtained during the first and second heating of the samples. The insertion of the IF domain decreased the Ttrs value of SlyD* from 53.7 °C (SlyD*ΔIF) to 46.0 °C (SlyD*). For SlyD*ΔIF, the ratio of van't Hoff enthalpy to calorimetric enthalpy is close to 1, indicating that, in the absence of the IF domain, thermal unfolding is a two-state reaction. In the presence of the IF domain, this ratio is 1.4, suggesting that at high protein concentrations, as used in the DSC measurements, SlyD* has a tendency to form dimers. The presence of partially folded intermediates would decrease the ratio between the two enthalpies to values smaller than 1.32 SlyD* does not contain Trp residues. Among its four Tyr residues, three are located in the FKBP domain, and one is located in the neck region between the host and the guest domain. The IF domain is devoid of fluorescent probes. Tyr fluorescence thus probes the unfolding of the host (FKBP) domain of SlyD*. It increases by 2- or 2.4-fold during unfolding in the absence (SlyD*ΔIF) or in the presence of the IF domain (SlyD*), respectively. Figure 3 shows the urea-induced transitions of the two proteins at 25 °C. They are reversible and apparently cooperative. The single-domain protein SlyD*ΔIF shows a higher transition midpoint (2.6 M urea) than the two-domain protein SlyD* (2.2 M urea). The cooperativities of the two transitions are also different, and the corresponding m values are 4.6 kJ mol− 1 M− 1 for SlyD*ΔIF and 6.4 kJ mol− 1 M− 1 for SlyD*. This suggests that the cooperative unit of unfolding is larger when both domains are present,

Fig. 2. Differential scanning microcalorimetry of SlyD* and SlyDΔIF. (a) Calorimetric trace for 150 μM SlyD* after subtraction of the buffer. (b and c) The excess heat capacity functions for (b) 50 μM SlyD* and (c) 60 μM SlyD*ΔIF in the presence of (from right to left) 0, 1.0, and 2.0 M urea. Reheating scans are shown as broken lines. The buffer was composed of 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5), and the scan rate was 90 K h− 1. The following thermodynamic parameters were obtained: 0 M urea for SlyD*: T trs = 46 ± 0.2 °C, ΔH vH = 330 ± 10 kJ mol − 1 , ΔH cal = 234 ± 10 kJ mol − 1 ; 0 M urea for SlyD*ΔIF: Ttrs = 53.7 ± 0.1 °C, ΔHvH = 295 ± 13 kJ mol− 1, ΔHcal = 265 ± 10 kJ mol− 1; 1 M urea for SlyD*: Ttrs = 40.6 ± 0.1 °C, ΔHvH = 265 ± 12 kJ mol− 1, ΔHcal = 190 ± 6 kJ mol− 1; 1 M urea for SlyD*ΔIF: Ttrs = 47.6 ± 0.1 °C, ΔHvH = 278 ± 20 kJ mol− 1, ΔHcal = 250 ± 10 kJ mol− 1; 2 M urea for SlyD*: Ttrs = 34.4 ± 0.1 °C, ΔHvH = 251 ± 15 kJ mol− 1, ΔHcal = 167 ± 10 kJ mol− 1; 2 M urea for SlyD*ΔIF: Ttrs = 44.5 ± 0.1 °C, ΔHvH = 253 ± 20 kJ mol− 1, ΔHcal = 220 ± 10 kJ mol− 1.

presumably because they unfold in a concerted fashion. The insertion of the IF domain thus destabilizes the FKBP domain of SlyD* in the transition region. The difference in stability between the two proteins decreases, however, when the urea concentration is lowered because the m values are different. In fact, at 0 M urea, the two-domain protein (SlyD*) shows a higher extrapolated stability. The same result is obtained when the thermal unfolding transitions in Fig. 2 are extrapolated to 25 °C. In summary, the analysis of the thermal and denaturant-induced unfolding transitions suggests that the equilibrium unfolding of SlyD* is welldescribed as an apparent two-state reaction without

Domain Folding of SlyD

Fig. 3. Urea-induced equilibrium unfolding transitions of SlyD* (squares) and SlyD* ΔIF (circles). Closed symbols represent unfolding experiments that started with folded protein, and open symbols represent refolding experiments that started with protein that was first unfolded in 8.0 M urea. The transitions of 2 μM protein in 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5) at 25 °C were followed by Tyr fluorescence at 304 nm after excitation at 280 nm. The midpoints are at 2.2 M (SlyD*) and 2.6 M (SlyD* ΔIF). Continuous lines represent leastsquares fit analyses of the experimental data based on a linear two-state model.33 For SlyD*, the calculated free energy difference ΔGD(H2O, 25 °C) = 14.2 kJ mol− 1 and m = 6.4 kJ mol− 1 M− 1. For SlyD*ΔIF, ΔGD(H2O, 25 °C) = 12.0 kJ mol− 1 and m = 4.6 kJ mol− 1 M− 1.

1141 ku = 5.0 × 10− 2 s− 1 for unfolding, and ΔGD(H2O) = 12.0 kJ mol− 1. This ΔGD(H2O) (ΔGD in the absence of denaturant) value from the kinetic analysis and the difference in the kinetic m values agree well with ΔGD (H2O) = 12.0 kJ mol− 1 and m = 4.7 kJ mol− 1 M− 1, as obtained from the equilibrium unfolding transition at 25 °C (Fig. 3). The final values of the unfolding and refolding kinetics traced the equilibrium unfolding transitions, and the observed amplitudes corresponded to the total change in fluorescence as expected from the equilibrium transitions (data not shown). All these properties indicate that the unfolding and refolding of the FKBP domain of SlyD are a twostate reaction. Next, we examined how the IF domain changes the folding kinetics. In its presence (in SlyD*), refolding is about 10-fold decelerated at all temperatures and urea concentrations (Fig. 5), but the kinetic m value of refolding remained unchanged. This remarkable finding indicates that the amount of native-like structure in the folding transition state is the same irrespective of whether or not the IF domain is present. Probably, all relevant interactions in the folding transition state are confined to the

intermediates in the absence and in the presence of the IF domain. Folding kinetics in the absence and in the presence of the IF domain The folding kinetics of the FKBP domain of SlyD were followed by changes in Tyr fluorescence in the absence and in the presence of the IF domain. The unfolding at 5.0 M urea is independent of the IF domain. It is monoexponential and shows identical time courses in the presence and in the absence of IF (Fig. 4a). The refolding kinetics are, however, strongly different. They are also monoexponential (Fig. 4b) and account for the entire change in fluorescence, but the time constants of refolding differ by more than 10-fold (0.3 s for SlyD* ΔIF and 3.45 s for SlyD*), showing that the insertion of the IF domain retards the refolding of the FKBP domain. The unfolding and refolding kinetics in the absence of the IF domain (of SlyD*ΔIF) were measured as a function of urea concentration at 15, 25, and 45 °C (Fig. 5). The kinetics were monoexponential under all conditions, and identical rates were observed for unfolding and refolding in the transition region. The chevrons observed for the folding of SlyD*ΔIF show linear limbs. The ratio of the kinetic m values results in a βT value of 0.7 (at 25 °C), indicating that, by this criterion, the folding transition state is about 70% native-like. Extrapolation to 0 M urea gave microscopic rate constants of kf = 6.9 s− 1 for refolding,

Fig. 4. (a) Unfolding at 5.0 M urea and (b) refolding at 0.6 M urea of 2 μM SlyD* (blue curves) and SlyD*ΔIF (red curves). The kinetics were monitored at 25 °C by fluorescence above 300 nm after excitation at 280 nm in 0.1 M potassium phosphate buffer and 1 mM EDTA (pH 7.5). Solid lines are monoexponential functions fitted to the data. They gave the following parameters: (a) for unfolding of SlyD*, τ = 1.32 s and SlyD*ΔIF τ = 1.28 s; (b) for refolding of SlyD*, τ = 3.4 s and of SlyD*ΔIF τ = 0.3 s.

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Fig. 5. Dependence of the apparent rate constant λ of folding of SlyD*(closed symbols) and SlyD*ΔIF (open symbols) on urea concentration at (a) 15 °C, (b) 25 °C, and (c) 45 °C. The kinetics were monitored by the change in fluorescence above 300 nm after excitation at 280 nm in 0.1 M potassium phosphate buffer and 1 mM EDTA (pH 7.5). The protein concentration was 2 μM. λ values smaller than 0.1 s− 1 are derived from manual-mixing experiments. Otherwise, the reactions were followed after stopped-flow mixing. Nonlinear curve fits according to Eqs. (4) and (5) are shown as solid lines. For SlyD* at 45 °C (c), only the unfolding limb was analyzed. For SlyD*, the data between 0 and 5 M urea were analyzed. From the analyses, the following parameters were obtained: (a) for SlyD*, ku = 6.4 × 10− 5 s− 1, kf = 0.2 s− 1, mu = 1.6 M− 1, mf = −1.2 M− 1; for SlyD*ΔIF, ku = 8.8 × 10− 3 s− 1, kf = 2.4 s− 1, mu = 0.65 M− 1, mf = − 1.4 M− 1; (b) for SlyD*, ku = 1.25 × 10− 3 s − 1 , kf = 0.48 s− 1 , mu = 1.3 M− 1 , m f = −1.2 M− 1; for SlyD*ΔIF, ku = 5 × 10− 2 s− 1, kf = 6.9 s− 1, mu = 0.55 M− 1, mf = −1.4 M− 1; (c) for SlyD*, ku = 3.9 s− 1, mu = 0.5 M− 1; for SlyD*ΔIF, ku = 5.1 s− 1, kf = 14.1 s− 1, mu = 0.4 M− 1, mf = −1.7 M− 1.

FKBP domain. Eventual folding in the IF domain is apparently not important for reaching the transition state. The 10-fold decrease in the refolding rate caused by the IF domain probably reflects its contribution to the entropy of the unfolded protein. The unfolding limbs of the chevrons for SlyD* show a complex nonlinear behavior. Near the end of

Domain Folding of SlyD

the equilibrium unfolding transitions (3–5 M urea), its slope is increased relative to SlyD*ΔIF (Fig. 5). When the urea concentration is further increased, however, the slopes of the unfolding limbs decrease; at high denaturant concentration, they approximate the mu (kinetic m value for unfolding, defined as ∂lnki/∂[D]) values as observed in the absence of the IF domain. This coincidence of the mu values and the general similarity of the unfolding kinetics at high denaturant concentration in the presence and in the absence of the IF domain is most clearly seen at 45 °C, where the unfolding limbs are long. This suggests that, under strongly denaturing conditions, the observed rate and cooperativity of unfolding are determined by the FKBP domain alone. In the presence of the IF domain, the minima of the kinetic chevrons are shifted to lower urea concentrations. This reflects the destabilization by the guest domain, as also observed in the equilibrium transitions. Destabilization is particularly pronounced at elevated temperature, as expected from the increased cooperativity of thermal unfolding in the presence of the IF domain (Fig. 2). Amplitude analysis shows that the final values of the unfolding and refolding kinetics trace the equilibrium unfolding transition, and that the observed kinetics virtually account for the entire change in fluorescence (Fig. S1, Supplementary Information). This suggests that no faster or slower phases were missed in the kinetic experiments. An analysis of the final fluorescence values by the twostate formalism gave a ΔGD(H2O) value of 14.2 kJ mol− 1 (at 25 °C), in excellent agreement with the ΔGD(H2O) of 14.3 kJ mol− 1, as obtained from the ratio of the microscopic rate constants of unfolding and refolding between 0 and 5 M urea. The difference in the kinetic m values (6.0 kJ mol− 1 M − 1 ) also agrees well with the m value from equilibrium unfolding (6.4 kJ mol− 1 M− 1). Folding in the presence of the IF domain is thus also well-described by a simple kinetic two-state mechanism, but refolding is about 10-fold decelerated, and the unfolding limb of the chevron is curved downward. Such a nonlinearity is usually assumed to originate from a movement of the transition state towards the native state under unfavorable folding conditions.34–41 Kinetic tests for silent reactions in unfolding and refolding Two sets of double-mixing experiments were performed to search for silent folding reactions in SlyD*: double-jump assays for isomerizations in the unfolded state42,43 and interrupted refolding assays for silent reactions during refolding.44 In the folding of human FKBP12, conformational folding is relatively slow and coupled with prolyl isomerization.45–47 To search for potential prolyl isomerizations in the unfolded form of our proteins, we performed double-jump experiments in which the protein was unfolded for variable time intervals and then transferred to refolding conditions. Thus,

Domain Folding of SlyD

transient species that differ from the fully unfolded form can be detected in the rate of refolding. This assay is generally useful for detecting spectroscopically silent reactions during or after unfolding, and the formation of off-pathway intermediates. Briefly, SlyD* was first unfolded in 5.5 M urea at 15 °C for either 10 s, 300 s, or 72 h, and then refolding was initiated by dilution to 1.0 M urea. The observed refolding kinetics were identical (Fig S2, Supplementary Information), and thus no evidence could be obtained for silent reactions (such as prolyl isomerizations) in unfolded SlyD*. Hidden intermediates during refolding were searched for by interrupted refolding assays. These assays detect folding intermediates that show nativelike spectroscopic properties, but unfold more rapidly than the fully native molecules because they have not yet crossed the major energy barrier of folding. The refolding reaction of SlyD* was initiated by diluting the unfolded protein from 6.0 to 0.5 M urea (at 15 °C). Then, after variable intervals of refolding, samples were transferred to 5.0 M urea, and the kinetics of unfolding were followed. All unfolding reactions were monophasic and showed the same rate of unfolding as native SlyD*. The increase in the amplitude of unfolding paralleled the time course of the fluorescence-detected refolding reaction (Fig. S3). Thus, evidence for hidden intermediates in refolding could not be obtained. The folding kinetics of the IF domain The IF domain of SlyD* does not contain a spectroscopic probe; therefore, its contribution to the overall folding of the two-domain protein could not be measured. To monitor its folding and how it is coupled with the folding of the FKBP domain, we introduced a tryptophan at position 101 into the IF domain. This position is located in a solventexposed loop (Fig. 1) and is not conserved in SlyD homologs. In the corresponding D101W variant, the fluorescence of Trp101 monitors selectively the folding of the IF domain, whereas the Tyr fluorescence follows the folding of the FKBP domain, as in the wild-type protein. The D101W substitution led to a small increase in the stability of SlyD* (Fig. 6). Unfolding of the FKBP domain, as followed by Tyr fluorescence at 285 nm, and unfolding of the IF domain, as followed by Trp fluorescence at 330 nm (after excitation at 280 or 295 nm) (Fig. 6a), led, after normalization, to similar transitions with midpoints at 2.7 and 2.6 M and m values of 6.8 and 7.6 kJ mol− 1 M− 1 for Tyr and Trp fluorescence, respectively (Fig. 6b). This confirmed that, at equilibrium, the two domains of SlyD* in fact unfold as a single cooperative unit. The transitions are also similar to those obtained for the wild-type protein ([urea]1/2 = 2.2 M and m = 6.4 kJ mol− 1 M− 1), indicating that the introduction of Trp101 did not change the folding mechanism of SlyD*. The unfolding of SlyD*(D101W) (SlyD* protein with the mutation D101W) by guanidinium chloride (GdmCl) was characterized as well because this

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Fig. 6. (a) Urea-induced unfolding transitions of 2 μM SlyD*(D101W) in 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5) at 25 °C. Fluorescence was measured at 285 nm after excitation at 275 nm (open squares), at 330 nm after excitation at 280 nm (open circles), and at 330 nm after excitation at 295 nm (filled circles). Continuous lines represent least-squares fit analyses of the experimental data based on a linear two-state model.33 They gave values of ΔGD(H2O) = 20.0 kJ mol− 1 and m = 7.4 kJ mol− 1 M− 1 (open squares); ΔGD(H2O) = 16 kJ mol− 1 and m = 6.2 kJ mol− 1 M− 1 (open circles); and ΔGD (H2O) = 17.4 kJ mol− 1 and m = 6.7 kJ mol− 1 M− 1 (filled circles). (b) Normalized transitions, as obtained after the two-state analyses of the data. The broken line represents the unfolding transition of SlyD* (taken from Fig. 3).

strong denaturant was required for analyzing the folding kinetics of the IF domain by double-mixing experiments (see below). The GdmCl-induced transitions at 15 and at 25 °C showed midpoints of 1.3 and 1.1 M and m values of 14.0 and 13.0 kJ mol− 1 M− 1, respectively (Fig. S4, Supplementary Information). The subsequent kinetic analysis was performed at 15 °C because the time resolution of stopped-flow mixing was not sufficient to resolve the fast folding kinetics of the IF domain at 25 °C. For refolding, identical kinetic traces were observed for wild-type SlyD*, when followed by the Tyr fluorescence of the FKBP domain, and for the refolding of SlyD*(D101W), when followed by the fluorescence of Trp101 in the IF domain (Fig. 7a). Identical kinetics were also observed when the probes in the two domains were employed to follow unfolding at 4.0 M urea (Fig. 7b), which is near the end of the transition region (Fig. 6). The measured amplitudes accounted for the entire change in fluorescence, as expected from the equilibrium transitions. This suggests that, under these conditions, the unfolding and refolding reactions of the two domains are governed by the

1144

Domain Folding of SlyD

constant τ = 380 ms (τ = λ− 1)] that accounted for the entire change in Tyr fluorescence. In contrast, the unfolding, monitored by the Trp fluorescence of the IF domain of SlyD* D101W, was a biphasic reaction. In addition to the slow phase (τ = 340 ms), a major 60fold-faster reaction with τ = 7 ms was detected, which accounted for about 80% of the total change in Trp fluorescence. No such fast reaction was observed by Tyr fluorescence for SlyD* and SlyD*ΔIF.

Fig. 7. Unfolding and refolding kinetics of SlyD* (blue) and SlyD*(D101W) (green) at 15 °C. Black lines represent monoexponential or biexponential functions fitted to the data. (a) Refolding after dilution of the unfolded protein from 6.0 to 0.8 M urea. For SlyD*, τ = 12 s; for SlyD*(D101W), τ = 10 s. (b) Unfolding kinetics in the presence of 4.0 M urea. For SlyD*, τ = 19 s; for SlyD*(D101W), τ = 22 s. (c) Unfolding kinetics in the presence of 8.1 M urea. For SlyD*, τ = 0.38 s; for SlyD*(D101W), two phases were observed with τ1 = 0.34 s and τ2 = 0.007 s. The kinetics of 3 μM SlyD* were monitored by fluorescence at 304 nm after excitation at 280 nm in 0.1 M potassium phosphate buffer, 1 mM EDTA (pH 7.5), and the indicated urea concentrations. For comparison, the kinetic curves were normalized such that they coincide at 0 s and at infinity. The kinetics of 1 μM SlyD*(D101W) were monitored by fluorescence at 330 nm after excitation at 295 nm in the presence of urea, 0.1 M potassium phosphate buffer, and 1 mM EDTA (pH 7.5).

same rate-limiting process, which probably is the unfolding of the FKBP domain. Different unfolding kinetics were observed, however, by the two probes under strongly denaturing conditions (8.1 M urea; Fig. 7c). Unfolding, followed by the Tyr fluorescence of the FKBP domain, remained to be a monoexponential process [time

Fig. 8. (a) Dependence on urea concentration of the macroscopic rate constants λ1 (squares) and λ2 (circles, triangles) for the folding reactions of 1 μM SlyD*(D101W). Circles represent stopped-flow experiments, and triangles represent manual-mixing experiments. The kinetics were monitored by the change in fluorescence above 320 nm (or at 330 nm after manual mixing) after excitation at 295 nm in 0.1 M potassium phosphate buffer and 1 mM EDTA (pH 7.5) at 15 °C. Filled circles denote refolding (initiated by a dilution of the unfolded protein from 8.1 M urea to the indicated urea concentration); open circles and triangles denote unfolding experiments. Filled squares denote refolding kinetics that were initiated after a 60-ms unfolding pulse at 6.0 M urea. Continuous lines represent fits on the basis of a linear two-state model. For the slow phase, the following parameters were obtained: ku = 1.1 × 10− 4 s− 1, kf = 0.5 s− 1, mu = 1.5 M− 1, mf = −1.6 M− 1. For the fast folding phase, no parameters are given because the refolding limb of the chevron is illdefined (dashed line). (b) Amplitudes of the unfolding reaction for the fast (λ1; open squares) and slow (λ2; open circles) kinetic phases and the sum of the amplitudes (filled triangles). The unfolding amplitudes obtained after manual mixing (open triangles, right ordinate) are aligned such that they coincide with those from the stopped-flow experiments in the overlap region between 4 and 5 M urea. The lines are guides for the eye only.

Domain Folding of SlyD

The unfolding and refolding of SlyD*( D101W), as monitored by Trp fluorescence, were subsequently measured as a function of the urea concentration. Figure 8 shows how the rates and amplitudes vary between 0 and 8 M urea. Below 4 M urea, only a single slow reaction is seen in unfolding and refolding, and its rate is identical with the rate measured by Tyr fluorescence for the wild-type protein. The amplitudes reflect the entire change in fluorescence. The amplitude of the slow unfolding reaction decreases in a sigmoid fashion when the urea concentration is further increased (Fig. 8b). In turn, a very fast unfolding reaction is monitored. It gains in amplitude; near 8 M urea, it accounts for about 80% of the entire resolved amplitude (Fig. 8b). Its rate is almost independent of the urea concentration and shows a value near 240 s− 1 (Fig. 8a). This fast unfolding reaction is detected only by the fluorescence of Trp101, which resides in the IF domain and thus monitors the unfolding of this domain. We

1145 suggest that the increase in the amplitude of the fast phase and the corresponding decrease in the amplitude of the slow phase in Fig. 8b reflect the progressive unfolding of the IF domain when it is linked with the folded form of the FKBP domain. The IF domain reaches its folding equilibrium in this rapid reaction because it unfolds at least 80 times faster than the FKBP domain (Fig. 8a). The high midpoint for this transition (near 5.3 M urea; Fig. 8b) suggests that the IF domain is strongly stabilized when its chain termini are immobilized by the linkage with the still-folded FKBP domain. In contrast, the isolated IF domain is unfolded in aqueous buffer. The m value for the urea-induced unfolding of a 60-residue protein such as the IF domain is estimated to be b 3.7 kJ mol− 1 M− 1;48 consequently, the end point of its transition (as defined by the amplitude of its unfolding reaction) could not be reached at 8 M urea (Fig. 8b). Therefore, we analyzed the unfolding and refolding kinetics of

Fig. 9. (a) Dependence on GdmCl concentration of the macroscopic rate constants λ1 (squares) and λ2 (circles), of the folding of SlyD*(D101W) at 15 °C. Filled circles denote the λ2 value for the monophasic refolding of the protein that was unfolded for 2 h in 3.0 M GdmCl. Fast refolding (λ1; filled squares) was measured after a short 60-ms unfolding pulse at 3.0 M GdmCl in double-mixing experiments. Unfolding (open symbols) of the native protein shows two kinetic phases. Continuous lines represent fits on the basis of a linear two-state model. The analysis of the data for the slow phase between 0 and 3.0 M GdmCl gave values of ku = 2.64 × 10− 3 s− 1, kf = 2.0 s− 1, mu = 2.2 M− 1, and mf = −3.6 M− 1. For the fast phase, ku = 19 s− 1, kf = 458 s− 1, mu = 0.64 M− 1, and mf = −1.6 M− 1 were obtained. (b) Relative amplitudes of the fast (squares) and slow (circles) unfolding reactions. Broken lines are guides for the eye only. (c) Amplitudes of refolding as obtained after a 60-ms unfolding pulse (filled squares). The continuous line represents an analysis in terms of an unfolding transition, based on a linear two-state model.33 It gave a midpoint at 1.6 M GdmCl, ΔGD(H2O) = 14.4 kJ mol− 1, and m = 9.2 kJ mol− 1 M− 1. (d) Amplitudes of the refolding reaction (filled circles) measured after complete unfolding of the protein as a function of GdmCl concentration. The continuous line represents an analysis in terms of an unfolding transition. The two-state analysis gave a midpoint at 1.0 M GdmCl, ΔGD(H2O) = 12.8 kJ mol− 1, and m = 12.8 kJ mol− 1 M− 1. All kinetics were monitored by the change in fluorescence above 320 nm after excitation at 295 nm in 0.1 M potassium phosphate buffer and 1 mM EDTA (pH 7.5) at 15 °C. The protein concentration was 1–3 μM.

1146 SlyD*(D101W) also in the presence of the stronger denaturant GdmCl. The corresponding chevron plot is shown in Fig. 9a, and the amplitudes of unfolding are shown in Fig. 9b. The results were similar to those obtained in the presence of urea. Again, unfolding becomes biphasic beyond the transition midpoint (1.0 M GdmCl) because the IF domain starts to unfold rapidly. Unlike in the case of ureainduced unfolding, the GdmCl-induced unfolding of IF domain becomes slightly faster when the denaturant concentration is increased (Fig. 9a). Above 4 M GdmCl, the IF domain is apparently fully destabilized, and the amplitude of its unfolding accounts for almost the entire change in Trp fluorescence (Fig. 9b). Interrupted unfolding assays for characterizing the stability and refolding kinetics of the IF domain At 3.0 M GdmCl, the IF domain unfolds very fast with τ = 5 ms, even at 15 °C. The FKBP domain unfolds slowly with τ = 450 ms (Fig. 9a). We made use of this 100-fold difference in the rate of unfolding to selectively produce, by a short 60-ms unfolding pulse, SlyD* molecules in which the FKBP domain is folded and the IF domain is unfolded. These molecules were then used to study selectively the refolding kinetics of the IF domain. Figure 10 shows the fast refolding of the IF domain at 0.8 M GdmCl after such a 60-ms unfolding pulse. The apparent rate constants of refolding of the IF domain, as measured by this technique, are shown in Fig. 9a as a function of the denaturant concentration. They yield the refolding limb of the chevron for the IF domain and connect smoothly with the unfolding limb, which had been obtained directly from the single-mixing unfolding experiments. The chevron was analyzed using a linear two-state mechanism. This analysis reveals that the IF domain is moderately stable and is in a highly dynamic conformational equilibrium when it is linked with the folded FKBP domain. Under most conditions, it refolds and unfolds more than 100-fold faster than the FKBP domain. The microscopic rate constants extrapolated to 0 M GdmCl show values of 460 s− 1 for refolding and 20 s − 1 for unfolding. Near the transition midpoint, the apparent rate is 100 s− 1, which implies that the unfolded and folded forms of IF equilibrate within 50 ms. The m values for refolding and unfolding are −3.8 and 1.4 kJ mol− 1 M− 1, suggesting that the folding transition state of IF is about 70% native-like. The analysis results in a transition midpoint of 1.44 M GdmCl and a ΔGD (Gibbs free energy of denaturation) of 7.5 kJ mol− 1 at 0 M GdmCl. The amplitude of the rapid refolding reaction traces the unfolding transition of the IF domain (Fig. 9c). It shows a midpoint of 1.56 M GdmCl and an apparent m value of 9.2 kJ mol− 1 M− 1, which is in fair agreement with the data derived from the kinetic chevron for the IF domain in Fig. 9a. In control experiments, unfolding at 3.0 M GdmCl was allowed to proceed to completion for both

Domain Folding of SlyD

Fig. 10. Refolding kinetics of SlyD* D101W at 0.8 M GdmCl after unfolding at 2.8 M GdmCl for 60 ms. The kinetics was followed by tryptophan fluorescence above 320 nm after excitation at 295 nm. The final concentration of the protein was 1.0 μM in 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5) at 15 °C. The continuous line represents a fitted monoexponential function, giving τ = 6.7 ms.

domains by extending the duration of the unfolding pulse to 7 s. The refolding of these fully unfolded molecules is slow because it is limited in rate by the folding of the FKBP domain. Its refolding amplitude (Fig. 9d) follows the equilibrium unfolding transition of SlyD*(D101W), with a midpoint at 1.1 M GdmCl and an m value of 12.8 kJ mol− 1 M− 1. Double-mixing experiments were also used to follow how, during unfolding, the form with a still folded FKBP and an unfolded IF domain is converted into the fully unfolded form. In these experiments, the duration of unfolding in 3.0 M GdmCl was varied between 30 ms and 24 s before, in the second step, refolding was initiated and monitored by the fluorescence of Trp101 in the IF domain. Figure 11a shows three refolding traces observed after 60 ms, 240 ms, and 24 s of unfolding. After 60 ms, unfolding of the IF domain is already complete, but the FKBP domain is still folded. Refolding is therefore dominated by the fast refolding of the IF domain. With increasing time of unfolding, the FKBP domain unfolds as well, and molecules with both domains unfolded accumulate. They refold much more slowly because the refolding of the IF domain is linked with the slow refolding of the FKBP domain. Consequently, the amplitude of fast refolding decreases, and the amplitude of slow refolding increases with prolonged time of unfolding in the first step. Figure 11b shows the kinetics of both the decrease in the fast-refolding molecules, in which only the IF domain is unfolded, and the increase in the slow-refolding molecules, in which both domains are unfolded. The two reactions occur in a reciprocal fashion with a common rate constant of 2.2 s− 1, which is identical with the rate constant of the slow unfolding reaction, as measured directly by fluorescence (Fig. 9a). A minor fast refolding reaction (5–10%) seems to persist even after prolonged unfolding. It cannot be excluded, however,

Domain Folding of SlyD

Fig. 11. Interrupted unfolding experiments to follow the sequential unfolding reaction of SlyD*(D101W). (a) Kinetics of refolding after unfolding for 60 ms (red), 240 ms (blue), or 24 s (black). Black lines represent biexponential functions that were fitted to the data. The time constants for the fast refolding reaction after 60 or 240 ms of unfolding were 4 and 5 ms, respectively. After 24 s of unfolding, the fast phase had a very small amplitude and could not be analyzed by standard curve fitting. Therefore, its time constant was fixed at 4.0 ms for the fit. This fast phase might arise also from a minor mixing artifact in the double-jump experiment. (b) Dependence on the time of unfolding of the relative amplitudes for the fast (squares) and slow (circles) refolding reactions. Black lines represent monoexponential functions that were fitted to the data. They are governed by the same time constant of 450 ms. The protein was unfolded in 3.0 M GdmCl at 15 °C in 0.1 M potassium phosphate (pH 7.5) and 1 mM EDTA for the indicated time. Refolding was measured after a 6-fold dilution to 0.45 M GdmCl in the same buffer.

that it might result from a small artifact in the double-mixing stopped-flow experiments; therefore, we refrain from interpreting it. The results in Fig. 11b show that, in its refolding, the IF domain depends on the FKBP domain. The IF domain can refold only when the folded FKBP domain is present as a scaffold that fixes its chain termini in their native arrangement.

Discussion Domain insertion and protein stability SlyD* is a peculiar two-domain protein in which the domains are in a host–guest relationship. The

1147 larger FKBP domain serves as the host and provides a loop that is used to accommodate the guest domain IF. Unlike in many other cases, the guest domain protrudes from the host domain, and, in addition to the two covalent chain connections, there are no noncovalent interactions between the domains. Two factors are important for two-domain proteins such as SlyD*: (i) the local chain connectivity of the host domain is interrupted by the guest domain, interfering with the refolding of the host and changing its stability; and (ii) the guest domain profits from linkage with the host because the folded host domain keeps the chain termini of the guest domain in close proximity and thus stabilizes the folded conformation of the guest and facilitates its folding. These general principles explain why the thermal and denaturant-induced equilibrium unfolding transitions of SlyD* are apparently simple two-state reactions and why the transition midpoints of the FKBP domain are decreased in the presence of the IF domain. The guest domain can profit from the conformational stability of the host only as long as the host is folded; therefore, its unfolding is coupled with the unfolding of the host domain. In other words, unfolding of the two-domain protein is cooperative, as observed. The stabilization of the guest by the folded host is large. The IF domain of SlyD* is unfolded in isolation, but shows a stability of 7.5 kJ mol− 1 when linked to the folded FKBP domain, as measured for a kinetically produced transient SlyD* species. The kinetic results support this model. In the absence of the guest domain (SlyD* ΔIF), the host domain unfolds and refolds with monoexponential kinetics that account for the entire change in fluorescence, and both limbs of the chevron plot are linear, which are the hallmarks of simple twostate folding reactions without populated intermediates. In addition, the presence of silent intermediates could be excluded by double-mixing experiments. In the presence of the IF domain, refolding is about 10-fold slower, but the kinetic m value of refolding is unchanged. Apparently, the IF domain is still unfolded when the transition state of refolding is reached, and, therefore, the extent of folding (as reflected by the kinetic m value) is the same with and without the IF domain. Still, the IF domain retards refolding because the loop insertion decreases the activation entropy. In SlyD*ΔIF, the 60-residue IF domain is replaced by the 13-residue flap sequence of FKBP12. The difference in the refolding rate between the two forms originates presumably from the difference in the entropy of the unfolded protein. The configurational contribution of the IF sequence (ΔΔGconfig) can be estimated as 5.7 kJ mol− 1 from the relation ΔΔGconfig = − 1.5RTln (n1/n2), where n1 and n2 are the loop lengths before and after domain deletion, respectively.49 Assuming that the FKBP domain is largely folded and the IF domain is still unfolded in the transition state of folding, this difference in the entropy of the unfolded protein would lead to a 10-fold difference

1148 in the folding rate, as observed. This suggests that the deceleration of refolding in the presence of the IF domain is caused by the difficulty of closing an extended loop during refolding. Folding was also retarded when loops in proteins were artificially enlarged by inserting additional residues.49–52 Domain insertion and the folding mechanism The unfolding limb of the kinetic chevron is curved downward when the guest domain (IF) is present. At moderate denaturant concentrations, IF remains folded as long as its termini are held together by the folded FKBP domain, and, as a consequence, unfolding is a concerted reaction. It is limited in rate by the slow unfolding of the FKBP domain, and its m value reflects the unfolding of both domains. At high denaturant concentration, the IF domain is unstable, even when its ends are immobilized by the folded FKBP domain. This unfolding reaction of IF is fast and complete before the FKBP domain starts to unfold (in the ratelimiting step). This explains why, under strongly denaturing conditions, the measured m value decreases to the value that is observed for the isolated FKBP domain alone. After introducing a Trp residue as a selective reporter group into the IF domain, its fast unfolding and refolding could be measured for the molecules that contain the FKBP domain still in the folded state. This was possible because the folding dynamics of the IF domain is extremely high. These results confirmed that the curvature in the chevron plot for the slow unfolding reaction of SlyD* indeed reflects the transition from a coupled unfolding of both domains to the unfolding of the FKBP domain only. This transition is determined by the conformational stability of the IF domain, which is unfolded in isolation but shows transition midpoints of about 5 M urea or 1.5 M GdmCl when linked with the folded form of the FKBP domain. These midpoints are higher than those of the equilibrium transitions of the intact two-domain protein. This strong dependence of the stability of the IF domain on the folded state of the FKBP domain explains why the equilibrium unfolding of the two-domain protein is indistinguishable from a cooperative two-state reaction. Curved unfolding limbs in chevrons caused by ground-state effects Downward curvatures in the unfolding limbs of chevron plots are frequently observed, and this is usually explained by the movement of the transition state towards the native state in a Hammond-type behavior.34,35 Under conditions that strongly disfavor the folded conformation, fewer interactions must be broken to reach the transition state of unfolding (i.e., this state becomes more native-like). Such a shift could occur as a discrete change from a less native transition state to a more native transition state via a high-energy intermediate,36–38 or as a gradual shift when the transition state is composed

Domain Folding of SlyD

of a broad ensemble of species.34,39–41 In either case, the native state and the transition state become more similar in their interactions with the denaturant and, therefore, the kinetic m value of unfolding decreases when the denaturant concentration is increased. The curvature of the unfolding limb in the chevron of SlyD has a different origin. It is caused by an effect not on the transition state but on the native ground state. At high denaturant concentration, a part of the protein (the IF domain) unfolds prior to the ratelimiting unfolding reaction in a very fast reaction; therefore, the size of the cooperative unit that unfolds in the rate-limiting step has decreased. This fast partial unfolding is neither detectable in wild-type SlyD* because there is no spectral probe in the fast unfolding part of the molecule, nor does it change the two-state character of unfolding because it is kinetically uncoupled from the slow unfolding of the FKBP domain. Such ground-state effects are not considered in the current kinetic mechanisms for protein folding. They might be widespread, however. In the case of SlyD*, it is an entire domain that unfolds rapidly in a silent reaction. In other proteins, exposed loops might similarly lose their structure very rapidly during unfolding under strongly denaturing conditions. Such regions could be similar to those that exchange rapidly with the solvent in the local unfolding reactions that are detected by nativestate hydrogen exchange.53–55 High folding dynamics of the IF domain The high folding dynamics of the IF domain explains why the folding kinetics of the two-domain protein is so well-described by a two-state mechanism without intermediates. The IF domain unfolds and refolds about 1000 times during the average lifetime of the folded FKBP domain. Once the FKBP domain happens to unfold, this high dynamics is broken, and the IF domain remains unfolded as well. Thus, the guest IF domain behaves as a “servant” that obeys the “master” FKBP domain. It folds rapidly when the FKBP domain is folded and is maintained unfolded when the host is unfolded. This mechanistic coupling holds throughout the transition region. Only under strongly destabilizing conditions, such as at high denaturant concentration, does the IF domain unfold rapidly and independently of the FKBP domain. All proteins with an inserted domain that have been analyzed to date had been constructed by engineering an artificial guest into a surface loop of another protein. In these cases, the stabilities were usually lowered for both host and guest. 16–18 Apparently, the strong stabilization that originates from the reduction of the chain entropy of the guest is easily offset by structural deformations that result from conformational mismatches in the region where the two proteins are fused. In extreme cases, when the chain ends of the guest are too remote in the folded state, the host or guest can be forced to unfold.19–21

1149

Domain Folding of SlyD

Functional importance of the conformational dynamics of the IF domain The conformational dynamics of the IF domain is remarkably high. Even in the middle of its unfolding transition, the folded and unfolded forms equilibrate with a time constant near 10 ms (at 15 °C). This could be of importance for its chaperone function during the SlyD-mediated catalysis of protein folding. A high structural plasticity could enable the IF domain to rapidly adapt to different incompletely folded protein chains upon binding. A high conformational dynamics would ensure very rapid binding and release of protein substrates. In combination, these two properties might be important to allow an efficient transfer of folding protein chains between the chaperone domain and the prolyl isomerase active site of SlyD and to avoid trapping of SlyD in abortive long-lived complexes with misfolded proteins.

Materials and Methods Materials Urea (ultrapure) and GdmCl (ultrapure) were obtained from ICN Biomedicals (Aurora, OH, USA). The concentrations of urea and GdmCl were determined by the refractive indices of the solutions.56 The D101W variant of SlyD* was produced by site-directed mutagenesis using blunt-end PCR followed by ligation, DpnI digestion, and transformation. The mutation was verified by sequencing of the gene. SlyD* and SlyD*(D101W) were overexpressed in E. coli strain BL21(DE3)pLysS (Stratagene, La Jolla, CA, USA) using the expression plasmid pET24 (T7 promoter, terminator, His-tag, and Km +). Expression, purification, and refolding of SlyD variants were performed as described previously.31,57 SDS-PAGE showed single bands upon staining with Coomassie brilliant blue. Proteins were dialyzed against 0.1 M potassium phosphate (pH 7.5) and 1 mM ethylenediaminetetraacetic acid (EDTA; pH 7.5). Protein concentrations were determined by absorbance, using ɛ280 values of 6070 M− 1 cm− 1 for SlyD*, 4800 M− 1 cm− 1 for SlyD*ΔIF, and 11,570 M− 1 cm− 1. for SlyD*(D101W), as calculated from the protein sequences.58 Differential scanning microcalorimetry DSC experiments were performed using a VP-DSC instrument (MicroCal, Northampton, MA, USA). Protein samples (60–150 μM) were dialyzed overnight in 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5). The pH values were measured before and after the DSC experiments. Degassed and filtered buffer used for dialysis was taken as reference. Buffer baselines were recorded before and after protein measurements. The heating rate was 90 K h− 1. Measurements were performed under constant pressure. Thermograms were analyzed based on a non-two-state model according to Eq. (1):    vH exp
DH 1
TTtrs RT DHvH DHcal ð1Þ CEp =    2  RT 2 vH 1
TTtrs 1 + exp
DH RT

where CEp is the baseline- and buffer-corrected molar excess heat capacity, R is the gas constant, ΔHvH is the van't Hoff enthalpy, ΔHcal is the calorimetric enthalpy, and Ttrs is the transition temperature. Stability and fit convergence were examined by introducing small variations in the parameters. Denaturant-induced unfolding of the proteins Samples of the proteins (1–3 μM) were incubated for at least 2 h at 15 °C in the presence of 0.1 M potassium phosphate (pH 7.5), 1 mM EDTA, and varying concentrations of GdmCl or urea. The Tyr fluorescence of the samples was measured in 10-mm cells at 304 nm (SlyD*, SlyD*ΔIF) after excitation at 280 nm, or at 285 nm (SlyD*D101W) after excitation at 275 nm. For SlyD*D101W, Trp fluorescence was excited at 295 nm and measured at 330 nm. The bandwidths were 3 nm for excitation and 5 nm for emission. The experimental unfolding transitions were analyzed according to a two-state model by assuming a linear dependence on the GdmCl concentration of the fluorescence emissions of the folded and unfolded forms. A nonlinear leastsquares fit with proportional weighting of the experimental data was used to obtain ΔGD as a function of the GdmCl or urea concentration.33 For denaturation by GdmCl, linear extrapolation might not always be valid.59 Folding kinetics Folding kinetics were measured after manual or stopped-flow mixing by employing a DX.17MV stoppedflow spectrometer from Applied Photophysics (Leatherhead, UK). The path length of the observation chamber was 2 mm. The folding kinetics of 3 μM SlyD* or SlyD*ΔIF were followed by fluorescence above 300 nm after excitation at 280 nm using a 10-nm bandwidth. A filter containing a 10 mM aqueous solution of cytidine-2′phosphate in a 0.2-cm cell was inserted in front of the emission photomultiplier to remove stray light. The folding kinetics of 3 μM SlyD*(D101W) were followed by fluorescence above 320 nm after excitation at 295 nm using acetone as filter. The stability of the signal was verified by control mixing experiment with 1 μM N-acetyltryptophanamide. All experiments were performed in 0.1 M potassium phosphate and 1 mM EDTA (pH 7.5). Unfolding was initiated by dilution of the native protein, and refolding was initiated by dilution of the unfolded protein (in 8.0 M urea) with buffers containing various concentrations of urea. Kinetics were measured three to eight times under identical conditions, averaged, and analyzed as single-exponential functions. Stopped-flow double mixing was used to obtain the refolding kinetics of the IF domain of SlyD* D101W in the presence of the folded FKBP domain. In the first mixing, the protein (66–122 μM) was unfolded for 60 ms by 11-fold dilution with 3.0 M GdmCl to allow fast unfolding of the chaperone IF domain. In the second mixing, refolding was induced by a further 6-fold dilution with buffer containing varying concentrations of GdmCl. In a further set of experiments, the duration of unfolding at 2.7 M GdmCl was varied between 30 ms and 3 h, followed by refolding at a final concentration of 0.45 M GdmCl. Refolding was monitored by fluorescence above 320 nm after excitation at 295 nm (bandwidth, 10 nm). Kinetics were measured four times under identical conditions, averaged, and

1150

Domain Folding of SlyD

analyzed as monoexponential or biexponential functions. The kinetics were independent of protein concentration. The mixing and dead time of the instrument were estimated by using the reaction between N-acetyl-tryptophanamide and N-bromo-succinimide.60 The stability of the fluorescence signal was ascertained by mixing 1 μM Nacetyl-tryptophanamide with folding buffer. Analysis of the folding kinetics Monoexponential (Eq. (2)) or biexponential (Eq. (3)) functions were fitted to the kinetic data: FðtÞ = Ad expð
kd tÞ + Fl FðtÞ = A1 d expð
k1 d tÞ + A2 d expð
k2 d tÞ + Fl

ð2Þ ð3Þ

where F is the measured fluorescence at a given time t, Ai is the amplitude of the reaction, λi is the apparent rate constant, and F∞ is the final fluorescence. The monoexponential folding kinetics observed for wild-type SlyD* and its FKBP domain were analyzed as single N ↔ U two-state reactions. The two phases in the folding of SlyD*(D101W) differed by more than 100-fold in rate under all conditions; therefore, the two chevrons could also be analyzed as two separate two-state reactions. The apparent rate constant λ was decomposed into the microscopic rate constants of unfolding (ku) and refolding (kf; Eq. (4)), and the logarithms of the microscopic rate constants were assumed to depend linearly on denaturant concentration (Eq. (5)): k = ku + kf

k = ku expðmu ½DÞ + kf exp mf ½DÞ

ð4Þ

ð5Þ

where mu = ∂lnku/∂([D]) and mf = ∂lnkf/∂([D]).

Acknowledgements We thank our group members for suggestions and comments on the manuscript. This research was supported by grants from the Deutsche Forschungsgemeinschaft. G.Z. was supported by a DAAD postdoctoral fellowship under the program “Modern Applications of Biotechnology.” The threedimensional NMR structure of E. coli SlyD* in Fig. 1 was kindly provided by Ulrich Weininger and Jochen Balbach (Martin-Luther-Universität HalleWittenberg, Germany). The technical assistance of Nicole Amtmann, Franz Wagner, Laurence Thirault, and Sima Hassanzadeh-Makooi (Roche Diagnostics GmbH, Penzberg, Germany) in protein production is gratefully acknowledged.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2008.12.052

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