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A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The ...
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Anal. Chem. 2006, 78, 4517-4523

Isothermal Calorimetry as a Tool To Investigate Slow Conformational Changes in Proteins and Peptides Jo 1 rg Fangha 1 nel,*,† Stephan Wawra, Christian Lu 1 cke, Dirk Wildemann, and Gunter Fischer

Max-Planck-Forschungsstelle fu¨r Enzymologie der Proteinfaltung, Weinbergweg 22, D-06120 Halle (Saale), Germany

A new calorimetric method has been developed to follow the time course of slow conformational changes during the refolding of denatured proteins. The method is based on the ability of isothermal titration calorimeters (ITC) to detect small amounts of heat continuously over a minute to an hour time range without being disturbed by baseline drift. We benchmarked the method on the basis of the slow kinetic phases resulting from prolyl cis/trans isomerization of oligopeptides. Using this method, the simultaneous investigation of the kinetics and thermodynamics of slow phases in the refolding of GdmCl-denatured RNase A by single jump techniques was performed. Time traces of heat production in the presence of a peptidyl prolyl cis/ trans isomerase support the classical model of ratelimiting prolyl trans to cis isomerizations in the folding reactions of RNase A. However, we also observed that, unlike prolyl cis/trans isomerizations in oligopeptides, those found in RNase A refolding are highly exothermic. It appears that coupling between slow prolyl trans to cis isomerization and relocation of remote backbone segments increases the number of contacting sites during formation of the native protein. The results demonstrate that calorimetrically monitored folding kinetics will be of relevance in the detection of otherwise silent folding events. Over the past decade, it has become increasingly obvious that conformational changes observed in native proteins are an important feature to regulate enzyme functions. The fast growing interest in the conformational state of native and denatured proteins motivated us to develop a calorimetric method to observe slow conformational changes during the refolding of proteins. In contrast to spectroscopic methods widely applied to investigate unfolding and refolding pathways of proteins, calorimetric methods are more advantageous due to the fact that they do not require intrinsic chromophors to report on the conformational state of the protein under investigation. Differential scanning calorimetry (DSC) has been found to be a valuable tool to characterize protein unfolding, but in only a few cases has it been demonstrated that * Corresponding author. (Germany) Phone: +49-345-5522801. Fax: +49-3455511972. E-mail: [email protected]. † Temporary address: Centre for Interdisciplinary Research Tohoku University, Aramaki, Aoba-ku, 980-8578 Sendai, Japan. Phone: +81-22-795-5759. Fax: +49-1212-5-152-38-470. 10.1021/ac052040x CCC: $33.50 Published on Web 05/25/2006

© 2006 American Chemical Society

isothermal titration calorimetry (ITC) can be employed to observe changes in protein structure.1-3 Slow refolding phases are especially difficult to detect by spectroscopic means, since the accompanying conformational change is considered to be small and locally restricted, leading in many cases to none or only minor spectroscopic signal changes. Further complications arise from the fact that different spectroscopic probes monitor different folding steps; the measured time constants of protein refolding kinetics, therefore, depend on the employed detection method.4-6 The possibility of applying a new method to the refolding of proteins, which is independent from spectroscopic means, offers the possibility of obtaining new insights into the mechanism of protein folding. In the late 1970s, Miller and Bolen detected slow conformational changes in native ribonuclease A (RNase A) at low denaturant concentrations by time-dependent ITC measurements, although they did not thoroughly follow the time course of the reaction.7 In addition, the use of ITC to follow the kinetics of enzyme reactions has been described earlier.8-10 Here, we present a method which allows us to follow slow conformational changes in proteins and peptides by exploiting the ability of modern titration calorimeters (i) to record kinetics and (ii) to be sufficiently sensitive to detect the heat associated with folding processes. To demonstrate its general use, we applied this method to bovine pancreatic RNase A, a well-studied case of a slow folding protein. Under conditions that the native form of RNase A is strongly stabilized, its refolding kinetics consist of multiple phases which arise from more than three different unfolded species.11 The major fast folding species, called UF, refolds to the native protein on a millisecond time scale, and the two different major (1) Hamada, D.; Kidokoro, S.; Fukada, H.; Takahashi, K.; Goto, Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10325-10329. (2) Liang, Y.; Du, F.; Sanglier, S.; Zhou, B. R.; Xia, Y.; Van Dorsselaer, A.; Maechling, C.; Kilhoffer, M. C.; Haiech, J. J. Biol. Chem. 2003, 278, 3009830105. (3) Wimley, W. C.; White, S. H. J. Mol. Biol. 2004, 342, 703-711. (4) Lin, L. N.; Brandts, J. F. Biochemistry 1983, 22, 564-573. (5) Schmid, F. X. Eur. J. Biochem. 1981, 114, 105-109. (6) Kim, P. S.; Baldwin, R. L. Biochemistry 1980, 19, 6124-6129. (7) Miller, J. F.; Bolen, D. W. Biochem. Biophys. Res. Commun. 1978, 81, 610615. (8) Cai, L.; Cao, A.; Lai, L. Anal. Biochem. 2001, 299, 19-23. (9) Lonhienne, T.; Baise, E.; Feller, G.; Bouriotis, V.; Gerday, C. Biochim. Biophys. Acta 2001, 1545, 349-356. (10) Todd, M. J.; Gomez, J. Anal. Biochem. 2001, 296, 179-187. (11) Wedemeyer, W. J.; Welker, E.; Scheraga, H. A. Biochemistry 2002, 41, 14637-14644.

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slow folding species, USI and USII, refold with time constants of seconds to minutes. Both the UF and the US species are completely unfolded proteins without specific long-range interactions.12 Each US species contains at least one peptidyl prolyl bond in its nonnative conformation. It is known that 20% of the unfolded protein belongs to the fast folding type, leaving a fraction of 80% to the slow folding species.4,13 The 20% fast folding species thereby consists of a 5% portion that shows very fast folding in the absence of prolyl isomerization and 13% of molecules that have to undergo a cis-to-trans prolyl isomerization.14 To help understand the slow folding kinetics of proteins, a wide variety of proline-containing oligopeptides have been used as simplified molecular models of proline-limited slow folding processes. In such peptides, the imidic peptidyl prolyl bond is characterized by a high rotational barrier of ∼20 kcal/mol. It results in slow cis/trans isomerization kinetics exhibiting relaxation times on the order of seconds to minutes, depending on the local sequence context at proline. Peptidyl prolyl cis/trans isomerases (PPIases) are able to lower the activation barrier considerably and can accelerate the cis/trans isomerization of prolyl bonds in unfolded polypeptide chains as well as native-state isomers of proteins.15 In the absence of strong intramolecular forces, the difference in free energy between the two stable prolyl bond conformers is small, thus causing a significant amount of cis isomer of the proline-containing molecule in solution. However, the enthalpic difference between fully or partially unfolded polypeptide chains containing non-native prolyl bonds and the final folded state remains unknown. EXPERIMENTAL SECTION Bovine pancreatic RNase A was purchased from Serva (Heidelberg, Germany). Ac-(Pro)13-NH2 was synthesized in-house, whereas all other peptides were obtained from Bachem (Weil am Rhein, Germany). Ultrapure guanidine hydrochloride (GdmCl) was obtained from ICN Biochemicals (Cleveland, OH). GdmCl concentrations were determined by refractive index.16 All other chemicals, buffers, and salts were of the highest purity commercially available and were purchased from Sigma Chemical Co. or Merck Eurolab (Darmstadt, Germany). Calorimetric Measurements. All experiments were carried out using a VP-ITC microcalorimeter from MicroCal (Northampton, MA) at the temperatures indicated. The correct function of the calorimeter was checked by applying electrically generated heat pulses, as suggested by the manufacturer. Due to the design of the calorimeter, in particular, the open construction of the injection syringe, it was not possible to obtain a steady baseline when the difference in GdmCl concentration between the syringe and the sample cell was too big. The following procedure was chosen to perform the jump experiments: all solutions were degassed for at least 5 min at the temperature used for the experiment. Before filling the sample cell, the solutions were cooled to at least 5 °C below the temperature of the conducted (12) Garel, J. R.; Nall, B. T.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 1853-1857. (13) Garel, J. R.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 33473351. (14) Houry, W. A.; Rothwarf, D. M.; Scheraga, H. A. Biochemistry 1994, 33, 2516-2530. (15) Fanghanel, J.; Fischer, G. Front. Biosci. 2004, 9, 3453-3478. (16) Nozaki, Y. Methods Enzymol. 1972, 26 PtC, 43-50.

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experiment. The stirring rate for all experiments was set to 510 rpm, and the feedback mode was set to “high”. All functions of the calorimeter were started manually. The injection syringe of the ITC was filled with the same buffer as the sample cell. After the system had been assembled and the desired temperature had been reached, the measurement was started with a data resolution of 2 s. The system was allowed to attain a steady baseline, which is crucial for the experiment. To ensure that no slow baseline drift occurred during the measurement, the baseline was recorded for at least 20 min. To start the reaction, the measurement was stopped, the injection syringe was removed, and the sample was manually injected into the sample cell using a gastight syringe. The system was then quickly reassembled, and stirring and recording was started again. Normally, the system required 200400 s to reequilibrate. The heat recorded after reequilibration was due to the reaction occurring within the sample cell. All data were recorded with the software provided by the calorimeter manufacturer. All further data analysis was performed with SigmaPlot for Windows Version 8. All data were fitted by either a single- or double-exponential equation, depending on the type of experiment. The subsequent integrations were carried out in the interval between the dead time of the method (usually 300 s) and the infinity value given by the exponential fits. Generally, low temperatures are to be preferred, because they slow the reactions and, thus, diminish the disadvantage of the dead time. Calorimetric Measurements of RNase A Refolding. Single jump refolding experiments were started by diluting 100 µL of the indicated amounts of unfolded RNase A in 5 M GdmCl and 50 mM glycine, pH 2.1, into the sample cell (1.45 mL) of the calorimeter that contained various refolding buffers, giving final RNase A concentrations between 43.5 and 290 µM. In the case where L.p.Mip was added to the folding reaction, the syringe of the calorimeter was filled with the PPIase (240 µM) diluted in refolding buffer. The indicated amounts of the enzyme were injected into the sample cell 250 s after the refolding had been initiated. The exact concentration of RNase A was determined spectroscopically after the experiment using the extinction coefficient 280 ) 9800 M-1 cm-1. The refolding phases observed in the thermogram were fitted to either single or double exponential equations by nonlinear regression. We used the determined concentrations of RNase A, the sample cell volume of 1.45 mL, and the area between the baseline and the single- or doubleexponential fit to calculate the enthalpy of folding, ∆Hfolding, at the respective temperature. Calorimetric Measurements of Peptidyl Prolyl Cis/Trans Isomerization. Either Ala-Pro or Ala-Ala-Pro was dissolved in water up to a concentration of ∼30 mM; the pH was adjusted with HCl or NaOH to the final pH values of 2.1 or 7.5. To perform solvent jump experiments, Ala-Ala-Pro was dissolved in anhydrous 0.47 M LiCl/TFE. The final concentrations of all peptide stock solutions were determined by NMR spectroscopy. All samples were allowed to equilibrate for at least 2 h before starting the experiment. The equilibrium constant (Keq) for the isomers was determined by NMR spectroscopy as described earlier.17 To perform the pH jump experiments, 100 µL of the peptide stock (17) Zhang, Y.; Fussel, S.; Reimer, U.; Schutkowski, M.; Fischer, G. Biochemistry 2002, 41, 11868-11877.

solution was injected into the sample cell of the calorimeter containing 50 mM sodium phosphate buffer at either pH 7.5 or 2.1. The solvent jumps were carried out by injecting 50 µL of the peptide dissolved in LiCl/TFE into the cell containing 50 mM sodium phosphate buffer at pH 7.5. The apparent first-order rate constant (kobs) was calculated from the thermogram by nonlinear regression. This rate constant is the sum of the microscopic rate constants for the cis-to-trans isomerization (kc/t) and the reverse reaction (kt/c) (eq 1). With help of eq 2, the rates of both constants kc/t and kt/c were determined.

kobs ) kc/t + kt/c

(1)

Keq ) kc/t/kt/c

(2)

The rate of cis/trans isomerization determined with the method described here and the equilibrium constant determined by NMR spectroscopy were measured between 5 and 25 °C. The respective rates of the cis-to-trans isomerization were subsequently calculated, and the resulting values were fitted to the Arrhenius equation (eq 3) to determine the thermodynamic parameters.

( ) (

ln

)

()

kc/t - ∆Hqc/t 1 ∆Sqc/t kb + + ln ) T R T R h

(3)

The temperature dependency of the equilibrium constant was used to calculate the difference in enthalpy between the cis and the trans conformer of the respective peptide (eq 4).

ln Kc/t )

(

)

- ∆H0c/t 1 ∆S0c/t + R T R

(4)

To observe the conformational transition from the polyproline I to the polyproline II form, 5 mg of Ac-(Pro)13-NH2 polyproline peptide was incubated overnight in 50 mL of 2-propanol; under this condition, it adopts the all-cis polyproline I conformation. The solvent was then evaporated, and the peptide was dissolved in 100 µL of 50 mM sodium phosphate buffer at pH 7.5. After the peptide had dissolved completely (time zero), the solution was quickly injected into the sample cell containing 50 mM sodium phosphate buffer, pH 7.5, thus starting the slow conversion from polyproline I to polyproline II conformation. All peptide solutions were analyzed by analytical HPLC and mass spectrometry to check for possible degradation or modification under the conditions used. The final concentrations of the Ala-Pro and Ala-Ala-Pro stock solutions were determined by NMR spectroscopy. RESULTS AND DISCUSSION Refolding of RNase A under Conditions Marginally Stabilizing the Native State. To determine the kinetic and thermodynamic parameters of the refolding of RNase A, experimental conditions which simplify the refolding pathway of this protein were used. By using low-pH buffer (pH 5.3) in combination with higher concentrations of GdmCl (2 M), possible folding intermedi-

Figure 1. a: Representative thermogram for the refolding of RNase A. The measurement was started after manual injection of 100 µL of 5 M GdmCl/50 mM glycine, pH 2.0 (‚‚‚‚) and 100 µL of 1.3 mM RNase A in 5 M GdmCl/50 mM glycine, pH 2.0, into the sample cell (2 M GdmCl and 100 mM sodium acetate buffer, pH 5.3) at 10 °C. b: Section of the thermogram shown in Figure 1a that represents the slow refolding phase of RNase A. A 100-µL portion of 1.3 mM denatured RNase A (5 M GdmCl/50 mM glycine, pH 2.0) was injected into the calorimeter cell containing 2 M GdmCl/100 mM sodium acetate buffer, pH 5.3, at 10 °C (0), leading to a final RNase A concentration of 87 µM. The nonlinear regression to a first-order kinetic (-, k1 ) 1.77 × 10-3) is shown in the upper panel; the residuals between fit and experimental data are presented in the lower panel.

ates of RNase A are destabilized and cannot be populated.18 Lyophilized RNase A was denatured using 5 M GdmCl and 50 mM glycine at pH 2 and manually injected into the preequilibrated ITC sample cell. The final GdmCl concentration in the sample cell was 2 M; the final pH was 5.3. A control experiment under identical conditions, but without RNase A, was also performed (Figure 1a). Within the first 200-400 s, the calorimeter reequilibrates from the thermal disturbances that arise from reassembling the calorimeter as well as the heat exchanged by dissolving a high concentration of GdmCl and the heat produced by stirring the sample cell. No meaningful data could be extracted from the thermograms before the system returned to thermal equilibrium. After reequilibration, the sample containing RNase A, but not the control, showed an observable heat exchange. It can be concluded that the exothermic reaction results directly from processes linked to the refolding of the protein. To (18) Schmid, F. X. FEBS Lett. 1986, 198, 217-220.

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Figure 2. Thermogram of the slow folding phase recorded by isothermal calorimetry with 290 (‚‚‚‚, k1 ) 1.97 × 10-3), 145 (-‚‚-, k1 ) 2.02 × 10-3), 87 (-‚-, k1 ) 1.78 × 10-3), 43.5 (- -, k1 ) 1.70 × 10-3), and 0 µM (-) unfolded RNase A (5 M GdmCl/50 mM glycine, pH 2) refolded in 2 M GdmCl/100 mM sodium acetate, pH 5.3, at 10 °C. The data were fitted to a single-exponential function, and integration was performed as described in the Experimental Section. The insert shows the measured heat of refolding for the different amounts of RNase A used in the experiment.

extract the rate constants of refolding, the signal within the dead time of the method, which corresponds to the first 400 s of the thermogram, was omitted from the analysis. The kinetic trace can be described sufficiently by a single-exponential equation with a time constant τ ) 541 ( 50 s (Figure 1b). This time constant is in good agreement with the value of 500 s measured by intrinsic fluorescence and UV spectroscopy, as well as by a specific unfolding assay to measure the kinetics of Pro93 trans to cis isomerization of RNase A under similar conditions.18 Under these conditions, the refolding kinetics of RNase A is simplified through destabilization of certain folding intermediates and, thus, fits to a single-exponential equation.18 By extrapolating the observed refolding kinetics to the start of the reaction and calculating the heat evolved during the entire process, the enthalpy change of folding could be determined for the refolding of the slow folding species (US) to the native protein as ∆Hfolding ) -12 kcal/mol at 10 °C. To evaluate whether the observed signal depends linearly on the RNase A concentration and to determine the detection limit of the method, we performed single jump experiments with decreasing amounts of RNase A. Kinetic traces shown in Figure 2 result from heat evolved by refolding of 290, 145, 87, and 43 µM RNase A solutions in the sample cell of the calorimeter. The calculated ∆Hfolding values under these conditions were -9.9, -9.1, -12.0, and -12.3 kcal/mol, respectively, giving a mean value of -10.9 ( 1.5 kcal/mol. From the linear dependency of the evolved heat to the protein concentration (Figure 2 insert) it can be inferred that ∆Hfolding corresponds to the amount of RNase A in the sample cell. What is the source of the observed exothermic reaction? Under marginally stabilizing conditions, we only observe the conformational changes involved in the restructuring of the US species to the native protein; as mentioned above, possible folding intermediates are not populated under these conditions. The refolding of 4520

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the fast folding species (UF) cannot be recorded because of the dead time of the method. The heat of thermal unfolding of RNase A has been determined by DSC as 153 kJ/mol (36.6 kcal/mol) at 25 °C under conditions similar to those we used here (see ref 19 for details). Taking into account the temperature-independent ∆Cp value of 6.6 kJ/mol/K (1.57 kcal/mol/K) estimated in the same work, we calculated the enthalpy of unfolding at 10 °C as ∆Hunfolding ) 54 kJ/mol (12 kcal/mol).19 Both enthalpies can be compared, since reversible folding processes were found to obey the equation ∆Hunfolding ) -∆Hfolding. The enthalpy of folding determined by the method described here is -10.9 ( 1.5 kcal/ mol; however, only ∼80% of the unfolded RNase A molecules belong to the US species and are accounted for by this method. Under the prerequisite that the refolding enthalpy of all unfolded RNase A species are similar, we can estimate the molar folding enthalpy for RNase A as -13.6 ( 1.9 kcal/mol. This assumption is justified by the observation that all unfolded species do not show any long-range interaction.12 We can, therefore, reason that the different unfolded RNase A species are energetically equivalent. The corrected value of -13.6 ( 1.9 kcal/mol must be used for comparison with the value calculated from the DSC experiments of 12 kcal/mol. The similarity of the two values underlines the validity of the method presented here. Folding under Conditions Strongly Favoring the Native State of RNase A. To demonstrate that this method can also be used to observe the refolding of proteins under conditions strongly favoring the native state, the refolding kinetics of RNase A in 0.4 M GdmCl/100 mM sodium acetate (pH 5.3) at 5 °C was investigated. Refolding of RNase A under these conditions is faster and more complex. At least two slow refolding phases associated with the two slow refolding species USII and USI are detectable by UV-vis and fluorescence spectroscopy.14 Refolding of RNase A monitored by isothermal calorimetry resulted in a biphasic thermogram (Figure 3a). The refolding kinetics fit to a doubleexponential function with time constants of τUsII ) 112 ( 5 s and τUsI ) 454 ( 4.5 s. Houry and Scheraga reported time constants for the refolding of RNase A under conditions similar to those used in our experiments as τ1 ) 29.5 s and τ2 ) 311 s;14 these values, however, were obtained by spectroscopic means. Comparing the constants from these two methods, it becomes clear that the differences are more pronounced than under conditions in which no folding intermediates are stabilized. This might be partly due to the fact that it is still being debated up to now how many slow refolding phases are observable under these conditions, as reflected by several models employing either two or three exponential functions to fit the observed refolding kinetics.20 Associated with the signal of the first and the second calorimetrically observable slow folding phases are exothermic reactions of -16.8 ( 1.3 kcal/mol and -3 ( 0.13 kcal/mol, respectively. Hence, heat production of the entire ensemble of the slow folding species was assigned to the sum of ∆H of the two phases, totalling -19.8 kcal/mol. The experiment was repeated three times to estimate the uncertainty of these values. It must be noted that even though the constants show a low standard deviation, the results might contain an inherent systematic error for two reasons: first, the faster reaction has a time constant too small to (19) Makhatadze, G. I.; Privalov, P. L. J. Mol. Biol. 1992, 226, 491-505. (20) Lin, L. N.; Brandts, J. F. Biochemistry 1987, 26, 3537-3543.

Figure 3. a: Thermogram of the slow refolding phase observed after injection of 100 µL of 4.4 mM denatured RNase A (5 M GdmCl/ 50 mM glycine, pH 2.0) (290 µM final protein concentration) into 100 mM sodium acetate buffer, pH 5.3, at 5 °C (0). The nonlinear regression to a double exponential equation (-, k1 ) 9.4 × 10-3, k2 ) 2.44 × 10-3) is shown in the upper panel. The residuals presented in the lower panel correspond to either a single- (b) or double-exponential (0) fit of the experimental data. b: Thermograms recorded at 5 °C of the refolding kinetics of 290 µM denatured RNase A (5 M GdmCl/50 mM glycine pH 2.0) in 0.53 M GdmCl/35 mM HEPES, pH 7.8, at various L.p.Mip concentrations. The curves represent the time traces in the presence of 0 (-, k1 ) 7.2 × 10-3, k2 ) 1.74 × 10-3), 0.7 (-‚-, k1 ) 1.2 × 10-2, k2 ) 2.02 × 10-3), and 3.8 µM (- -, k1 ) 1.2 × 10-2, k2 ) 1.93 × 10-3) L.p.Mip and an experiment in which 3.8 µM L.p.Mip and 32 µM rapamycin where added prior to the folding reaction (9 9, k1 ) 1.0 × 10-2, k2 ) 2.32 × 10-3). The thermogram indicated with the dotted line represents a control experiment in which no RNase A was present. The residuals between fits and experimental data are presented in the lower panel.

be assessed with high accuracy because of the long dead time of the experiment; and second, the amplitude of the slower phase is rather small and, in combination with the slow reaction, the error caused by small drifts in the baseline becomes more prominent. Comparing the total amount of enthalpy detected with this method (-19.8 kcal/mol) with the enthalpy of thermal unfolding observed previously by DSC (∆Hunfolding(5 °C) ) 42 kcal/mol),19 we can conclude that about one-half of the total enthalpy of folding can

be accounted for by using our method. Again, the observed heat change is generated exclusively by the slow folding species of RNase A, because this method cannot detect any heat generated from fast folding species, which might explain the differences observed with these two calorimetric approaches. Earlier results obtained by spectroscopic measurements indicated that within the first milliseconds of refolding of the slow folding species USII, an early intermediate (I1) is populated. This intermediate is considered to have a well-defined hydrogen bond network and some of the secondary structural features of the native protein.18,21,22 I1 then folds to the nativelike intermediate (IN) which possesses already many properties of the native protein, such as enzymatic activity, binding to the specific inhibitor 2′-CMP, and a similar tyrosine absorbance. IN is further characterized by at least one non-native peptidyl prolyl bond. The slow refolding of the intermediate IN to the native protein N involves the cis/trans isomerization of nonnative peptidyl prolyl bonds. Judging from the exothermic reaction, none of the calorimetrically observed refolding phases can arise solely from peptidyl prolyl cis/trans isomerization of a nonnative prolyl isomer, since in the reference peptides, the enthalpic differences between cis and trans conformers of peptidyl prolyl bonds are small (Table 1). Hence, exothermic conformational changes within the protein must take place concomitant or after the cis/trans isomerization, leading to the observed heat that accompanies the slow folding phases. One possible origin of the observed enthalpy could be the folding of the second slow folding species USI. It was proposed that prior to its conformational interconversion to a more structured intermediate, a slow peptidyl prolyl cis/trans isomerization step must take place.23 Since the ratio between UF, USII, and USI is considered to be 20:65:15,13,14,21 the refolding of the USI species alone cannot account for all the calorimetrically observed enthalpy of refolding. Only ∼6 kcal/ mol of the total refolding enthalpy can theoretically originate from this species. The folding enthalpy ratio between USII und USI should be ∼13:3.13,21 In our experiments, a ratio of ∼16:3 between two refolding events was observed. We must, therefore, conclude that the slower refolding phase characterized by τUsI originates from the refolding of the USI species to either another folding intermediate or the native protein, and the τUsII-controlled reaction derives from the slow restructuring of the folding intermediate IN of the USII species to the native protein (N). The exothermic processes accompanying this folding step could have their source in major structural changes associated with the reequilibration of non-native peptidyl prolyl bonds in the protein, which have not been observed by other methods. Acceleration of RNase A Refolding by Peptidyl Prolyl Cis/ Trans Isomerases. It has been reported that PPIases have an accelerating effect on slow refolding phases caused by peptidyl prolyl bonds in proteins.24 Furthermore, it has been shown that L.p.Mip efficiently accelerates the refolding of carboxymethylated RNase T1 Ser54Gly/Pro55Asn.25 Therefore L.p.Mip was added to the reaction to examine whether the slow kinetic phases (21) Schmid, F. X. Biochemistry 1983, 22, 4690-4696. (22) Schmid, F. X.; Blaschek, H. Eur. J. Biochem. 1981, 114, 111-117. (23) Mui, P. W.; Konishi, Y.; Scheraga, H. A. Biochemistry 1985, 24, 44814489. (24) Lang, K.; Schmid, F. X.; Fischer, G. Nature 1987, 329, 268-270. (25) Kohler, R.; Fanghanel, J.; Konig, B.; Luneberg, E.; Frosch, M.; Rahfeld, J. U.; Hilgenfeld, R.; Fischer, G.; Hacker, J.; Steinert, M. Infect. Immun. 2003, 71, 4389-4397.

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Table 1. Thermodynamic Parameters for the cis/trans Isomerization of Ala-Pro, Ala-Ala-Pro, and Ac-(Pro)13-NH2 Determined at pH 2.1 and 7.5 peptide

∆Hqc/ta (kcal mol-1)

T∆Sqc/ta (kcal mol-1)

∆Gqc/ta (kcal mol-1)

∆H0c/t,calorimetrya (kcal mol-1)

∆H0c/t, NMR (kcal mol-1)

APpH 7.5b APpH 2.1c AAPpH 7.5b AAPpH 2.1c AAPpH 7.5d Ac-(Pro)13-NH2e

22.4 ( 0.5 17.2 ( 1.1 24.8 ( 0.2 17.5 ( 1.3 19.9 ( 0.6 20.6 ( 0.5

1.3 -3.0 4.1 -2.8 -1.0 -1.6

21.1 ( 0.5 20.2 ( 1.1 20.7 ( 0.2 20.3 ( 1.3 20.9 ( 0.6 22.2 ( 0.5

-2.1 ( 0.4 -1.3 ( 0.2 -2.1 ( 0.4 -1.2 ( 0.3 nd nd

-1.67 ( 0.05 -1.29 ( 0.05 -1.36 ( 0.05 -0.78 ( 0.06 nd nd

a Indicated error limits were calculated from the linear regression of the Arrhenius plot shown in Figure 5 or were calculated using eq 3 based on the temperature dependency of the equilibrium constant of the peptides as measured by NMR spectroscopy. The standard deviation of T∆Sqc/t was below 1%. b Jumping from pH 2.1. c Jumping from pH 7.5. d Jumping from 0.47 M LiCl/TFE. e Experiments were performed at pH 7.5.

observed in the thermogram of RNase A refolding are limited by the rate of reequilibration of non-native cis/trans isomers of peptidyl prolyl bonds present in the unfolded state of the protein. This experiment was performed to further prove that the reaction observed calorimetrically is, indeed, associated with the slow conformational changes during the refolding of RNase A. High concentrations of denaturant and low pH values, as normally used to observe the refolding kinetics, inhibit PPIase activity. We therefore performed single jump refolding experiments to strongly native conditions and neutral pH values (0.32 M GdmCl, 35 mM HEPES, pH 7.8) at 5 °C. In the absence of the PPIase, the reaction shows a biphasic time course with time constants of 104 and 384 s. As can be seen in Figure 3b, the addition of increasing amounts of L.p.Mip leads to a change in amplitude of the first phase. The presence of an ∼10-fold excess of rapamycin, a tightly binding inhibitor of L.p.Mip, abolishes the effect of the PPIase and restores the thermogram to the shape that was observed when no L.p.Mip was added, thus indicating that the accelerating effects are due to the PPIase activity of L.p.Mip. The observed change in amplitude indicates that, due to the accelerating influence of the PPIase on the peptidyl prolyl cis/trans isomerization, a greater amount of RNase A is refolded within the dead time of this method. This shows that the observed enthalpy of folding associated with this refolding event must originate from steps concomitant or after peptidyl prolyl cis/trans isomerization. Peptidyl Prolyl Cis/Trans Isomerization of ProlineContaining Peptides. The method introduced here can be applied not only to investigate the slow refolding of RNase A, but also to observe the peptidyl prolyl cis/trans isomerization in proline-containing di-, oligo-, and polypeptides. The enthalpic difference between the cis and trans prolyl isomers in peptides is very small. To directly observe the cis/trans conversion, it is necessary to ensure that the difference in concentration between both isomers is as large as possible. A number of techniques have been developed to alter transiently the cis/trans ratio of peptide bonds. The most commonly used methods involve pH and solvent jump experiments. The method presented here allows the use of both jumps techniques. The cis content of the dipeptide Ala-Pro decreases from ∼39% at pH 7.5 to 9.9% at pH 2.1 or, as in the case of Ala-Ala-Pro, decreases from 37% when dissolved in 0.47 M LiCl/TFE at 25 °C to ∼19% in phosphate buffer at pH 7.5.17 This shift in equilibrium can be used to observe the heat of isomerization from cis to trans and vice versa. The change in the ionization state of the peptides following the pH jump is very fast 4522 Analytical Chemistry, Vol. 78, No. 13, July 1, 2006

Figure 4. Thermograms of the cis/trans isomerization of Ala-AlaPro after a pH jump from pH 2.1 to 7.5 (O, k1 ) 3.05 × 10-3) at 23 °C and from pH 7.5 to 2.5 (0, k1 ) 3.52 × 10-3) at 12 °C. A 100µL portion of a 33 mM stock solution of Ala-Ala-Pro was injected into the sample cell preequilibrated with 50 mM sodium phosphate buffer. The solid lines represent the fit to a single-exponential rate equation. The residuals between fits and experimental data are presented in the lower panel.

and, just as the solvent jump, completed within the dead time of the method. All thermograms of the investigated peptides fit to a single-exponential function, as exemplified in Figure 4 for AlaAla-Pro after a pH jump from 2.1 to pH 7.5 and vice versa. The reaction is exothermic if the cis content of the peptide is higher at the beginning of the experiment than at equilibrium, and vice versa. On the basis of (i) the observed rate constant kobs obtained using the described jump experiments and (ii) the cis/trans equilibrium constant determined independently by NMR spectroscopy, the rates of the cis to trans isomerization (kc/t) and the reverse reaction (kt/c) were calculated using eqs 1 and 2. Figure 5 shows the temperature dependency of the rate constant for the cis-to-trans isomerization of Ala-Pro, Ala-Ala-Pro, and the polyproline I-to-polyproline II transition of Ac-(Pro)13-NH2. The corresponding activation enthalpies for the cis to trans isomerization of Ala-Pro, Ala-Ala-Pro, and the polyproline I-to-II transition of Ac(Pro)13-NH2 are summarized in Table 1. In all cases, the free

Figure 5. Arrhenius plot of the cis-to-trans isomerization of AlaPro (pH jump from 2.1 to 7.5, 1), Ala-Ala-Pro (pH jump from 2.1 to 7.5, O; solvent jump from 0.47 M LiCl/TFE to 50 mM sodium phosphate buffer, pH 7.5, 2) and Ac-(Pro)12-NH2 (2-propanoltreated peptide injected into 50 mM sodium phosphate buffer, pH 7.5, 0).

enthalpy of activation of the isomerization is ∼20 kcal/mol, which is in good agreement with the barriers of activation reported in the literature for the same process.17,26-28 Under acidic conditions, the enthalpic fraction of the free enthalpy is smaller than at pH 7.5. This effect does not have a strong influence on the free enthalpy because it is compensated by a smaller entropic contribution. The small temperature-dependent changes in the cis/trans equilibrium constant of Ala-Pro and Ala-Ala-Pro determined by NMR spectroscopy were used to calculate the differences in the ground-state enthalpy ∆H° of both peptides at pH 2.1 and 7.5 (Table 1). With the method presented here, we can measure the amount of heat exchanged during the isomerization reaction by simply integrating the obtained single-exponential fits of the thermograms and then extrapolating to the start of the reaction. Using the cis/trans equilibrium constants determined by NMR, the observed values can then be normalized to the pure isomer (Table 1). For all investigated peptides, the calorimetrically determined difference in ground-state enthalpy was slightly more negative than the enthalpy difference calculated by using the temperature dependency of the equilibrium constant. For the dipeptide Ala-Pro, this difference was found to be within the limits of error. For the tripeptide Ala-Ala-Pro, the difference between both ground-state enthalpies is more pronounced. To explain this difference, one has to consider that, in contrast to the RNaseA experiments, the final concentration of the peptides in the sample cell could not be determined spectroscopically. The amount of (26) Fischer, G.; Bang, H.; Berger, E.; Schellenberger, A. Biochim. Biophys. Acta 1984, 791, 87-97. (27) Schoetz, G.; Trapp, O.; Schurig, V. Electrophoresis 2001, 22, 2409-2415. (28) Grathwohl, C.; Wu ¨ thrich, K. Biopolymers 1981, 20, 2623-2633.

peptide present during the experiment was, therefore, determined on the basis of 1H NMR spectra that were collected with a diluted sample of the used stock solution. As a consequence, the cis contents calculated from the signal ratios in the NMR spectra are accompanied by a rather large uncertainty; i.e., a small deviation of the cis content in the calorimetric cell from the NMR-derived value leads to considerable changes in the difference in groundstate enthalpy. To our knowledge, this is the first time that the heat of cis/ trans isomerization of a peptidyl prolyl bond has been determined directly. The values obtained with this direct method are in agreement with the values obtained indirectly from the temperature dependence of the equilibrium constant of these peptides. This corroborates our hypothesis that the kinetic and thermodynamic information extracted from the thermograms are useful parameters characterizing slow conformational processes. CONCLUSION The method described here allows the kinetic and thermodynamic characterization of slow conformational changes in peptides and proteins. Under conditions marginally stabilizing the native state of RNase A, the same rate of folding and enthalpy of folding as estimated by other methods was observed. Interestingly, under folding conditions strongly stabilizing the native state of RNase A, we could observe a much higher change in enthalpy than predicted by the recent molecular models of the refolding pathway of this enzyme. This allowed us to conclude that exothermic restructuring events concomitant or after the cis/trans isomerization of non-native peptidyl prolyl bonds must take place. The method is also capable of detecting the small change in enthalpy between the cis/trans isomers in short proline-containing peptides and is, therefore, useful to thermodynamically and kinetically characterize such peptides. Kinetic and thermodynamic parameters determined for the cis/trans isomerization of Ala-Pro, Ala-Ala-Pro, and Ac-(Pro)13-NH2 are in agreement with published values or values we obtained with other methods. In addition to the advantage of not requiring chromophores for the observation of folding/isomerization events, this method allows the determination of kinetic and thermodynamic reaction parameters simultaneously. The major drawback of the method is its rather long dead time, which prevents the observation of faster reactions, but this limitation might be overcome with newer generations of calorimeters. A reduced dead time would also allow the determination of a more complete set of thermodynamic parameters for prolyl isomerization-limited protein folding reactions, as exemplified here by means of the peptides. We postulate that this method is also suitable to characterize native-state isomerization of prolyl bonds directly, provided that conditions can be found to overpopulate either of the native isomer states in order to perform jump experiments. Received for review November 17, 2005. Accepted April 3, 2006. AC052040X

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