Equilibrium and Kinetic Unfolding of GB1: Stabilization of the Native

Sep 6, 2018 - NMR spectroscopy allows an all-atom view on pressure-induced protein folding, separate detection of different folding states, determinat...
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Cite This: J. Phys. Chem. B 2018, 122, 8846−8852

Equilibrium and Kinetic Unfolding of GB1: Stabilization of the Native State by Pressure Matthias Dreydoppel, Paul Becker, Heiner N. Raum, Stefan Gröger, Jochen Balbach, and Ulrich Weininger* Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, D-06120 Halle (Saale), Germany

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S Supporting Information *

ABSTRACT: NMR spectroscopy allows an all-atom view on pressure-induced protein folding, separate detection of different folding states, determination of their population, and the measurement of the folding kinetics at equilibrium. Here, we studied the folding of protein GB1 at pH 2 in a temperature and pressure dependent way. We find that the midpoints of temperature-induced unfolding increase with higher pressure. NMR relaxation dispersion experiments disclosed that the unfolding kinetics slow down at elevated pressure while the folding kinetics stay virtually the same. Therefore, pressure is stabilizing the native state of GB1. These findings extend the knowledge of the influence of pressure on protein folding kinetics, where so far typically a destabilization by increased activation volumes of folding was observed. Our findings thus point toward an exceptional section in the pressure−temperature phase diagram of protein unfolding. The stabilization of the native state could potentially be caused by a shift of pKa values of glutamates and aspartates in favor of the negatively charged state as judged from pH sensitive chemical shifts.

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INTRODUCTION Protein folding can be studied by shifting the equilibrium between folded, unfolded, and possible intermediate states. This is routinely done by changing temperature, pH, or denaturant concentration.1 Pressure is a favorable alternative to affect the equilibrium2 because it does not rely on changing the composition of the sample (pH, denaturant) and is, in contrast to temperature induced unfolding, often fully reversible.3,4 One drawback is that in most cases the change in pressure is not sufficient to change the equilibrium from a completely folded to a completely unfolded state. Therefore, this transition is difficult to be studied by ensemble methods (e.g., fluorescence, ultraviolet, or circular dichroism) at equilibrium. Recently, high-pressure NMR spectroscopy has advanced to a more established NMR method.2,5−9 Studying protein folding with NMR spectroscopy has several advantages. First, different states (like folded, unfolded, or intermediate) give rise to separate signals,10 if they do not exchange on the very fast NMR time-scale where signals of different states are averaged. Protein folding is in almost all cases slower and therefore resulting in separated signals, even for fast (millisecond timescale) folding proteins.11,12 Following the different states separately allows to reliably extract populations, even if folding transitions have not reached completeness. Second, highresolution two-dimensional NMR facilitates an all-atom view of protein folding. This enables a straightforward identification and characterization of possible intermediates10 and largely © 2018 American Chemical Society

increases the amount of data points, if all of the reporters are fitted globally. Further, NMR spectroscopy can be used for studying kinetics on various timescales,13,14 allowing the characterization of transient states15 and energy barriers.16 Recently, pressure-jump experiments to study protein folding have been introduced.17,18 GB1 is a well-studied small 56 residue model for protein folding.19−21 The pH dependence of the here used QDD mutant (T2Q, N8D, and N37D) is structurally identical to the wild type and22 has been studied in terms of stability,22 pKa values,23 and protonation kinetics of Asp and Glu side chains.24 At pH 2 and below this GB1 variant is still folded at low salt concentration, but signals of the unfolded state at a low population, as well as unfolding kinetics could be detected by NMR.24 This makes QDD-GB1 an ideal candidate for pressure-induced folding studies monitored by NMR. At pH 2, all ionizable groups are fully protonated, except for D22, D47, and E56 which reach only about 80% protonation. This has two further advantages. First, small inconsistencies in pH have little (higher pH) to none (lower pH) effect on QDDGB1 in general and only very minor on the protonation state of D22, D47, and E56. Additionally, these residues can be used to internally report the apparent pH of the sample at different Received: July 18, 2018 Revised: September 5, 2018 Published: September 6, 2018 8846

DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852

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The Journal of Physical Chemistry B temperatures and pressures. Second, QDD-GB1 is highly positively charged, which helps to prevent aggregation and ensures reversible thermodynamic measurements. Combining temperature and pressure variations with atomic resolution allows for a detailed investigation of the multidimensional protein folding landscape.4,25,26 This landscape can be further explored by characterizing local minima and probing energy barriers by kinetic experiments. Combining information from thermodynamic and kinetic experiments allows furthermore establishing linear free-energy relationships.24,27

1

Int =

MATERIALS AND METHODS Protein Samples. 15N13C-labeled QDD-GB1 was expressed and purified as described elsewhere.22 It was dissolved to a concentration of 5 mM in 90% H2O/10% D2O with addition of small amounts of DSS and NaN3. The pH was adjusted to 2.0 directly in the sample. Differential Scanning Calorimetry (DSC) Measurements. DSC measurements were performed with a VP-DSC MicroCalorimeter (MicroCal) between 278 and 363 K with a heating rate of 1 K/min at ambient pressure. QDD-GB1 was dissolved and dialyzed in ddH2O, and pH values of 1.74, 2.26, 2.59, and 3.10 were adjusted. Baseline subtraction and calculation of the enthalpy were performed by the MicroCal DSC Data Analysis tool in Origin, assuming a two-state model for unfolding. The heat capacity change upon unfolding, ΔCp, dΔH was calculated via linear regression of ΔCp = dT with

ΔG(p , T ) =

M

transition midpoint, TM, and van’t Hoff enthalpy, ΔH (Supporting Information Figure S1). NMR Spectroscopy. All experiments were performed on a Bruker AVANCE III NMR spectrometer at a static magnetic field strength of 14.1 T in a commercial 3 mm ceramic cell28 (Daedalus Innovations LLC), connected to a home-built pressure generator. At various combinations of temperature (288, 293, 298, 303, 308, 313, and 318 K) and pressure (0.1, 50, 100, 150, and 200 MPa) 1H−15N heteronuclear single quantum coherence spectroscopy (HSQC), ct 1H−13C aromatic HSQC, ct 1H−13C aliphatic HSQC, and Hx2(Cx)CO29 experiments were recorded. Additionally, 15N Carr− Purcell−Meiboom−Gill (CPMG) relaxation dispersion experiments30 were acquired at 298 and 308 K for 0.1, 100, and 200 MPa. Pressure-dependent CPMG experiments have been limited to one magnetic field by a single pressure device installed at one magnetic field. 15N CPMG relaxation dispersion experiments comparing pH 2.0 and 1.8 were acquired on a Varian Direct Drive at a static magnetic field strength of 11.7 T. Spectra were processed with NMRPipe31 and analyzed with NMRView32 and PINT.33 Data Analysis. Data modeling utilized the Levenberg− Marquardt algorithm34 implemented in MATLAB. Errors were estimated by Monte-Carlo simulations.34 Temperature Transitions. Intensities of as many as 130 well-resolved signals of the native state and 14 of the unfolded state were fitted globally to derive the transition midpoint (TM) for each pressure according to eq 1 (native state) or eq 2 (unfolded state)

1+e

M

TM TM T + ln( T ))

TM

1

+ ln(

TM T ))

TM T ))

(2)

Δβ (p − p0 )2 + Δα(p − p0 )(T − T0) 2 ΔCp (T − T0)2 + ΔV0(p − p0 ) − 2·T0 − ΔS0(T − T0) + ΔG0

(3)

pH/pKa Monitoring. Hx2(Cx)CO correlation spectra29 were recorded at 288, 298, 308, 318 K, and 0.1, 100, and 200 MPa, and chemical shifts were referenced to the signal of DSS. Known 13CO versus pH correlations24 were used to monitor the change in protonation of Asp and Glu side chains. These could be interpreted as changes in pH or pKa values with temperature or pressure. CPMG Relaxation Dispersions. 15N CPMG relaxation dispersion data were fitted to the Carver−Richards equation36,37 using fixed populations derived from equilibrium transitions. All dispersion curves could be fitted well in a global approach by a two-state model.

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RESULTS Temperature Transitions. The population of the native state of QDD-GB1 could be monitored from the intensities of as many as 130 well-resolved NMR signals (Figure 1 and Supporting Information Figure S2), corresponding to 30−34 amides, 75−90 aliphatic side chains, and 4−6 aromatic side chains depending on the respective pressure. Additionally, the unfolded state could be followed by 5−14 well-resolved NMR signals for different temperatures and pressures (Supporting Information Figure S3). In general, all signals from the native state disappear with increasing temperatures and signals of the unfolded state arise, in agreement with unfolding in the slow chemical exchange regime (Supporting Information Figure S4). All monitored resonances could be analyzed by one twostate unfolding process (Figure 1). Only minor differences were observed for exchangeable amide protons compared with

A + a·T −ΔH / R ·( T1 − T1 ) −ΔCp / R ·(1 −

1

1 + e−ΔH / R·( T − TM ) −ΔCpP / R·(1 − T

+ ln(

where Int is the signal intensity, T the temperature in K, A and B are the intensity of the native and unfolded states at 0 K, respectively, a and b are the temperature dependences of the native and unfolded state intensity, respectively, TM the transition midpoint, ΔH the van’t Hoff enthalpy at the transition midpoint, and ΔCp the heat capacity difference between native and unfolded states. Note that the unfolded baseline of native state signals and the native baseline of unfolded state signals are 0 when analyzing well-resolved NMR resonances. Because ΔH and especially ΔCp are not very well described by the NMR-detected temperature transitions, additional DSC experiments at 0.1 MPa have been performed to determine them properly. These values have been set fixed during fitting of the NMR transitions. Temperature and Pressure Phase Diagram of Protein Stability. Free-energy changes upon unfolding, ΔG(p,T), were calculated from the populations of the equilibrium transitions at the corresponding temperatures and pressures. Estimations of the change of thermal expansion Δα, isothermal compressibility Δβ, and heat capacity ΔCp as well as the change of entropy ΔS, and volume ΔV upon unfolding were obtained by global fitting to the Hawley equation35

■

Int =

TM

1

(B + b·T ) ·e−ΔH / R·( T − TM ) −ΔCp/ R·(1 − T

(1) 8847

DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852

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Figure 1. Temperature-induced unfolding transitions of QDD-GB1 at different static pressures. Four exemplary positions (E19α, A20α, K31ε, and A34β) are shown as fraction of native state (f N) at 0.1 MPa (blue), 100 MPa (magenta), and 200 MPa (red) static pressurederived from the native state resonances in 2D NMR correlation spectra. Lines correspond to individual fits according to eq 1.

carbon-bound protons. This is in line with extremely slowed down amide exchange at pH 2. Therefore, unfolding transitions derived from all NMR probes at one pressure were included in a final global analysis (Figure 2A). Because the required difference in heat capacity between the folded and unfolded state, ΔCp, is not accurately described by the NMR detected transitions, it was determined by DSC (Supporting Information Figure S1) to 2.34 kJ/(mol·K), which is comparable to 2.96 kJ/(mol·K) of wt GB1,38 and kept fixed during global fitting. In the individual fittings of the temperature transitions, the difference in enthalpy, ΔH, was determined to be values between 166 and 212 kJ/mol. For the global analysis, we used the fixed value of 192 kJ/mol, determined by DSC (Supporting Information Figure S1), which is very close to the average (189 kJ/mol) derived from the individual NMR transitions. Pressure Dependence. Transition midpoints from temperature-induced unfolding QDD-GB1 increase with increased pressure. This holds for individually fitted (Figure 1) as well as for the globally (in respect to TM) fitted temperature transitions (Figure 2A). This unexpected result suggests that QDD-GB1 resides under these conditions in an exceptional area of the pressure−temperature phase diagram.4 To illustrate this result, we roughly estimated this phase diagram (Figure 2B). From the temperature transitions between 288 and 318 K at 5 different pressures between 0.1 and 200 MPa, we sampled the free-energy change, ΔG, of QDD-GB1 unfolding within the gray section in Figure 2B by 35 values for ΔG(p,T) at the corresponding pressure and temperature values. To estimate the temperature width, the midpoint of cold denaturation at ambient pressure (blue in Figure 2B) was calculated from the DSC-derived ΔCp and ΔH(TM) values assuming ΔCp to be temperature independent. The latter is an oversimplification given the broad temperature range of the phase diagram together with negative pressures, which have no physical meaning. Figure 2B shows the result of a fit of eq 3 to these ΔG(p,T) values by plotting the black line for ΔG(p,T) = 0 showing the typical elliptic shape of the pressure−temperature phase diagram.4 The inside of this ellipse corresponds to the pressure and temperature range for positive ΔG(p,T) values, which favor the native state of QDD-

Figure 2. Global analysis of temperature-induced unfolding at different static pressures. (A) Midpoints of temperature unfolding (TM) plotted against static pressure. Error bars derived from the S/N ratio are within the symbol size. (B) Pressure−temperature phase diagram of QDD-GB1 derived from global fitting of all NMR derived populations within the gray section. The black line illustrates ΔG(p,T) = 0 according to eq 3. Red circles represent direct experimental midpoints of the temperature transition at given pressures. The blue circle is the midpoint for cold denaturation at 0.1 MPa calculated from DSC-derived ΔH and ΔCp. Axis for 0 MPa and 273 K are shown as light gray lines. Lines for ΔV = 0 and ΔS = 0 are shown in green and orange, respectively.

GB1. Two lines corresponding to ΔS = 0 and ΔV = 0 divides the diagram into four segments revealing that the experimental data points are located in the segment of ΔS > 0 and ΔV > 0. This location is independent from the extrapolated midpoint of cold denaturation (Supporting Information Figure S5). This implies that the folded state of QDD-GB1 under our experimental conditions has a smaller volume compared with the unfolded state. pH Effects Monitored by Internal pH Meter. Under the here explored experimental conditions (pH 2.0), all ionizable groups are fully (>95%) protonated, except for D22, D47, and E56 which are protonated 77, 80, and 88% protonated, respectively,24 in agreement with previous studies.39 D22 is in the N-terminal helix cap and the transition of E56 is severely broadened because two negative charges (side chain and Cterminus) can coexist at this residue. Thus changes in the NMR chemical shifts of these residues can be directly interpreted as a change in the sample pH (if they display a homogenous behavior with temperature or pressure) or in their local pKa values (if they display a heterogeneous behavior). Independent from this interpretation changes in 8848

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ature, eventually followed by loss of signal intensity due to the unfolding process (Figure 4) above 310 K. Because the line

the NMR chemical shifts reflect a slight change in charge in these positions. These effects were monitored by side chain carbonyl/carboxyl shifts that are very sensitive to changes in charge24,40 at 288, 298, 308, and 318 K and 0.1, 100, and 200 MPa. With temperature increase, all indicative resonances show a decrease in pH of about 0.5 units (for D22) (Figure 3A). This is in line with an increased ionization constant of

Figure 4. Temperature profile of NMR intensities of aromatic F30δ (red), Y45δ (blue), and Y45ε (green) at 0.1 MPa. Intensities have been normalized to match the relative intensities with the unfolding curve derived from the global analysis (black line) at 0.1 MPa and 308 K.

width of these signals decreases simultaneously, it can be safely assumed that aromatic ring flips16 of these aromatic side chains occur in the intermediate exchange regime at lower temperatures causing severe line broadening and loss of intensities. At higher temperatures, they reach the fast exchange regime only displaying one sharp averaged signal which is the typical case for many proteins. At further elevated temperature unfolding of QDD-GB1 takes place. Temperature and Pressure Dependence of Native State Chemical Shifts. To identify possible low populated states, that are interconverting with the native state on the fast NMR timescales,5 the pressure dependence of the chemical shifts for different groups has been monitored. Most resonances only show a very minor dependence, and the dependence is linear (Supporting Information Figure S7). Even more, there are no systematic trends within the different groups (e.g., amides, methyl). Finally, we mapped the most affected resonances on the structure. No hotspots were identified, they are spread all over the structure. Therefore, we concluded the absence of any detectable alternative state in fast exchange with the native state. This observation confirms earlier findings and conclusions for wild-type GB1,42 where nonlinear pressure dependent chemical shift changes have been ascribed to nonuniform compression of the protein structure. This behavior of amide proton shifts is confirmed in this study, with exception of residues close to the QDD mutation. Kinetic Unfolding Monitored by CPMG Relaxation Dispersion Experiments. 15N CPMG relaxation dispersion experiments were recorded for 18 to 30 well-resolved amide signals for 298 and 308 K and three pressures (0.1, 100, and 200 MPa). In general, the dispersion steps were significantly increased at 308 K compared with 298 K (Figure 5A and Supporting Information Figure S8). This is caused by the much higher fraction of unfolded protein at 308 K (Figure 1). 15 N CPMG relaxation dispersions can only be observed at low pH for 25 °C,24 in agreement with the reduced stability of the protein under such conditions. Because no signals from a possible intermediate species could be detected and the

Figure 3. Side chain chemical shift changes of QDD-GB1 at different temperatures and pressures. Indicative 13CO chemical shift differences of all Asp and Glu carboxyl groups are plotted against temperature (A) and pressure (B). D22 is colored blue and E56 is colored red.

water at higher temperature.41 Because QDD-GB1 is already protonated near to completion at pH 2.0, this has no consequences on stability, which is nicely demonstrated by identical NMR relaxation dispersions describing the unfolding at pH 2.0 and 1.8 (Supporting Information Figure S6). Higher pressure should also increase the ionization constant of water41 and therefore cause lower apparent pH. However, here we observe the opposite, an apparent increase in pH, again monitored by side chain carbonyl/carboxyl shifts (Figure 3B). Using the chemical shift changes of D22 this would translate into an apparent pH increase of 0.6 units according to previous work.24 Because this finding is not uniform for all probes, and in contrast with the behavior of water, it is more likely that the pKa values of certain groups (like D22 and E56) shift to lower values, thus resulting in a reduced level of protonation and an increased level of negative charge at high pressure. Ring Flips Affect Transitions Monitored by Aromatic Side Chains. The isolated resonances of residues Y45 and F30 in the aromatic HSQC spectrum show an abnormal behavior. Their intensity dramatically increases with increasing temper8849

DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852

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The Journal of Physical Chemistry B relaxation dispersion profiles match expectations from the temperature transitions, the dispersions were analyzed as a two-state exchange process between native and unfolded protein. Furthermore, populations derived from the temperature transitions were used in the CPMG relaxation dispersion fittings, to compensate for the lack of data from a second magnetic field. Thus, one can extract an apparent exchange rate (kex) constant from the global analysis which can be decomposed into intrinsic folding (kUN) and unfolding (kNU) rate constants according to the populations. All relaxation dispersion profiles can be described well by this approach, resulting in very similar (10%) individual Δδ and R2,0 values in all six conditions. Thereby, the applied two-state model of unfolding by temperature transitions is validated by kinetic data. Folding rates both for 298 and 308 K show almost no pressure dependence, while unfolding is slowed down with increasing pressure (Figure 5B). Thus, the stabilizing effect of pressure can be mainly allocated to stabilization of the native state while unfolded and transition states remain unaffected (Figure 6).

Figure 6. Schematic energy diagram of unfolding of QDD-GB1 at pH 2.0. Protein states at low pressure are shown in solid blue lines, at high pressure in dashed red lines. U illustrates the unfolded state, N is the native state, and * is the transition state.

(low pH) is stabilized by pressure (Figure 6). Generally, pressure effects on proteins due to volume changes typically include solvation of hydrophobic groups, reduction of water inaccessible cavities, and electrostriction of polar and charged groups.4,43 Wild-type GB1 at pH7 is a very stable and wellpacked protein, and therefore a major shift toward its unfolded state is not expected within the here studied pressure range and was experimentally not observed.42 The QDD variant at pH 2 revealed a strongly reduced thermodynamic stability making it sensitive to pressure-induced stability changes. At pH 2, this variant is highly positively charged. Therefore electrostriction of water molecules, which corresponds to a contraction of this dipolar solvent in the electric field of exposed charges, is one very likely explanation for the here observed pressure induced stabilization of QDD-GB1. The same phenomenon might explain pressure induced drops in pKa values of D22 and E56 because electrostriction favors the deprotonated side chain at elevated pressure. As a result from our equilibrium and kinetic results, directly looking at the native state structure should reason why pressure has a stabilizing effect. In general, we find ΔV and ΔS to be >0 under the here applied conditions. There are no large cavities in the structure of GB1,42 so the native state can be seen as relatively compact. In order for ΔV > 0, the unfolded state needs to be more extended and thus occupying a larger volume compared with the folded state. This can be explained by the highly positively charged (+7) unfolded state at pH 2. In other words, repulsive positive charges are forcing the unfolded state to be more extended. However, this is just an explanation for the stabilization of N relative to U and does not explain a stabilization of N in absolute terms. Thus, one has to consider additional mechanisms of stabilization by pressure than volume effects alone. Underlying Folding Kinetics in a Temperature Pressure Phase Diagram of Protein Stability Are

Figure 5. 15N R2 CPMG relaxation dispersion experiments of QDDGB1. (A) Relaxation dispersion profiles of four exemplary amides (I6, V21, A34, D40) are shown at 298 K (blue) and 308 K (red). (B) Natural logarithm of derived rate constants of unfolding (triangles) and folding (circles) plotted against static pressure for 298 K (blue) and 308 K (red). Fits revealing the activation volumes of unfolding (solid lines) and folding (dashed lines) are shown.

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DISCUSSION Native State of QDD-GB1 is Stabilized by Pressure. From combining results of equilibrium (higher TM with increased pressure) and kinetic (lower rate constants of unfolding with increased pressure) experiments, we find that the native state of QDD-GB1 under the applied conditions 8850

DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852

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The Journal of Physical Chemistry B Complex. Our findings, that pressure is stabilizing the native state of a protein, appears to be in contradiction to observations from NMR-detected ZZ-exchange data and fluorescence-derived folding kinetics under pressure, where pressure was typically found to destabilize proteins by slowing down the folding reaction.9,44,45 However, both findings are a direct reflection of the behavior of the respective proteins in different sections (ΔV > 0, ΔS > 0; ΔV < 0, ΔS > 0; ΔV < 0, ΔS < 0) of the pressure−temperature phase diagram of protein stability.4,9 This is pointing toward complex changes in the underlying folding kinetics and physical principles for the influence of pressure on proteins. While in some cases a large increase in the activation volume of folding was observed,46 in the QDD-GB1 case we monitor an increase in the activation volume of unfolding at elevated pressure and hardly any influence on the activation volume of folding. The physical principles for these different locations in pressure−temperature phase diagrams of proteins remain so far elusive. Possible Change in pKa Values Could Explain Stabilization of the Native State. When monitoring the change in the level of protonation in Asp and Glu side chains in QDD-GB1, we make the following observations. With increased temperature their carboxyl groups become more protonated and thereby less charged. This is in line with the behavior of water with temperature. The pKw values decrease with temperature and the water is apparently more acidic.41 The same change is expected with pressure. Note that this is a pure effect of water because no additional buffers are used that could affect the pH.9 However, we monitor the opposite behavior, certain Asp and Glu become less protonated and more negatively charged. Because this finding is opposite to the theory and less uniform among the residues, we interpret it as a change in the pKa values instead. Under pressure, distinct pKa values of Asp and Glu are shifted to lower values and therefore the carboxyl groups become less protonated and more negatively charged at a constant pH. If this observation is true, pressure changes the charged state of QDD-GB1 in the same way as a higher pH would. At significantly higher pH values, QDD-GB1 becomes stabilized.22 A change of pKa values of Asp and Glu in favor of the negatively charged state therefore might be the explanation of the stabilizing effect of pressure on the native state of QDD-GB1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49 345 55 28555. Fax: +49 345 55 27161. ORCID

Ulrich Weininger: 0000-0003-0841-8332 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Sara Linse (Lund University) for kindly providing an expression plasmid of QDD-GB1. This work has been supported by grants from the Deutsche Forschungsgemeinschaft (WE5587-1, SFB TRR102) and the European Fund for Regional Development by the European Union.

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REFERENCES

(1) Buchner, J.; Kiefhaber, T. Protein Folding Handbook; WileyVCH: Weinheim, 2005. (2) Akasaka, K.; Kitahara, R.; Kamatari, Y. O. Exploring the folding energy landscape with pressure. Arch. Biochem. Biophys. 2013, 531, 110−115. (3) Akasaka, K.; Tezuka, T.; Yamada, H. Pressure-induced changes in the folded structure of lysozyme. J. Mol. Biol. 1997, 271, 671−678. (4) Luong, T. Q.; Kapoor, S.; Winter, R. Pressure-A Gateway to Fundamental Insights into Protein Solvation, Dynamics, and Function. ChemPhysChem 2015, 16, 3555−3571. (5) Kalbitzer, H. R. High Pressure NMR Methods for Characterizing Functional Substates of Proteins. High Pressure Bioscience: Basic Concepts, Applications and Frontiers; Springer Netherlands, 2015; Vol. 72, p 179. (6) Vajpai, N.; Nisius, L.; Wiktor, M.; Grzesiek, S. High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, E368−E376. (7) Nisius, L.; Grzesiek, S. Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network. Nat. Chem. 2012, 4, 711−717. (8) Munte, C. E.; Erlach, M. B.; Kremer, W.; Koehler, J.; Kalbitzer, H. R. Distinct Conformational States of the Alzheimer β-Amyloid Peptide Can Be Detected by High-Pressure NMR Spectroscopy. Angew. Chem., Int. Ed. 2013, 52, 8943−8947. (9) Roche, J.; Royer, C. A.; Roumestand, C. Monitoring protein folding through high pressure NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc. 2017, 102-103, 15−31. (10) Low, C.; Weininger, U.; Neumann, P.; Klepsch, M.; Lilie, H.; Stubbs, M. T.; Balbach, J. Structural insights into an equilibrium folding intermediate of an archaeal ankyrin repeat protein. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3779−3784. (11) Zeeb, M.; Balbach, J. NMR spectroscopic characterization of millisecond protein folding by transverse relaxation dispersion measurements. J. Am. Chem. Soc. 2005, 127, 13207−13212. (12) Weininger, U.; Respondek, M.; Akke, M. Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion. J. Biomol. NMR 2012, 54, 9−14.

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CONCLUSIONS Here, we studied the thermodynamic and kinetic folding behavior of QDD-GB1 at pH 2. Temperature-induced unfolding transitions at five different pressures (0.1, 50, 100, 150, and 200 MPa) have been monitored. Additionally, folding kinetics were derived by 15N CPMG relaxation dispersion measurements at 298 and 308 K for three different pressures (0.1, 100, and 200 MPa). We find under these conditions that QDD-GB1 gets thermodynamically stabilized by elevated pressure. In addition, high pressure slows down the unfolding kinetics. Taking these findings together, increasing pressure stabilizes the native state of QDD-GB1 and we speculate that protonation of a few side chains might be a reason for this stabilization.

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DSC-derived ΔHv versus TM, individual temperature induced unfolding transitions, example sections from NMR spectra, full 1H15N spectra at different temperatures, pressure−temperature phase diagrams of QDDGB1 for different midpoints of cold denaturation, 15N CPMG relaxation profiles at pH 1.8 and 2.0, pressure dependence of amide proton shifts, and individual 15N R2 CPMG relaxation dispersion profiles (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b06888. 8851

DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852

Article

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DOI: 10.1021/acs.jpcb.8b06888 J. Phys. Chem. B 2018, 122, 8846−8852