Cosolvent and Crowding Effects on the ... - ACS Publications

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Cosolvent and Crowding Effects on the Temperature and Pressure Dependent Conformational Dynamics and Stability of Globular Actin Paul Hendrik Schummel,† Andreas Haag,‡ Werner Kremer,‡ Hans Robert Kalbitzer,‡ and Roland Winter*,† †

Physical Chemistry I − Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, D-44227 Dortmund, Germany ‡ Institute of Biophysics and Physical Biochemistry, Centre of Magnetic Resonance in Chemistry and Biomedicine (CMRCB), University of Regensburg, Universitätsstrasse 31, D-93047 Regensburg, Germany S Supporting Information *

ABSTRACT: Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular functions. The polymerization process of actin has been found to be among the most pressure sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), and, to mimic a more cell-like environment, crowding agents on the pressure and temperature stability of globular actin (G-actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon unfolding, such as enthalpy and volume changes, have been determined by fluorescence spectroscopy over a wide range of temperatures and pressures, ranging from 10 to 80 °C and from 1 to 3000 bar, respectively. Complementary high-pressure NMR studies revealed additional information on the existence of native-like conformational substates of G-actin as well as a molten-globule-like state preceding the complete pressure denaturation. Different from the chaotropic agent urea, TMAO increases both the temperature and pressure stability for the protein most effectively. The Gibbs free energy differences of most of the native substates detected are not influenced significantly by TMAO. In mixtures of these osmolytes, urea counteracts the stabilizing effect of TMAO to some extent. Addition of the crowding agent Ficoll increases the temperature and pressure stability even further, thereby allowing sufficient stability of the protein at temperature and pressure conditions encountered under extreme environmental conditions on Earth.

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INTRODUCTION Actin is one of the main components of the cytoskeleton in eukaryotic cells.1 The skeletal muscle isoform, the so-called α− actin, can be found in cells in two principal forms: the monomeric state, with its globular shape (G-actin) and, by adding cations to G-actin, it is able to polymerize to filaments (F-actin).2,3 These two morphological states exist in dynamic equilibrium, which is controlled by actin binding proteins as well as the properties of the monomer itself.4 Actin is responsible for many different cellular functions, including cytokinesis, endocytosis, muscle contraction, transport of organelles or generation of forces.5 G-actin has been shown to have a lower thermostability than F-actin, which depends on the ionic strength and the bound nucleotide.4 Conversely, about the pressure dependent stability of G-actin is much less known, despite the fact that high hydrostatic pressure (HHP) conditions on Earth are widely spread. For example, pressures up to the 1 kbar (100 MPa) level and beyond are encountered in the ocean trenches and deep subsurface,6 and the average pressure in the deep sea is 380 bar. To survive under such extreme conditions, both microorganisms and macroorganisms have to undergo adaptions to be able to rescue proteins from denaturation.7−9 Generally, the stability of biomacromolecules, such as proteins, is also sensitively affected by the composition of the bathing solution.10−15 Solvation of proteins depends not only © XXXX American Chemical Society

on the concentration of added salt, but also on the specific ions that make up the solution (the Hofmeister-Effect). Generally, the stability of proteins is also dramatically influenced by cosolvents, inside the biological cell by the cellular milieu, i.e., by metabolic synthesis of cosolvents. Depending of the environmental stresses encountered (such as desiccation, high temperature, freezing, and high pressure), different cellular milieus have been identified. Compared to the effects of temperature and osmotic stress upon regulation of the internal milieu, very little is yet known about mechanistic aspects behind the adaptation of the cellular milieu to external pressure stress, and our corresponding knowledge about water-cosolutebiomolecule interactions is also largely incomplete. Such cosolvents may also be distinguished between chaotropic and kosmotropic ones, which is related to their effect on the water structural properties, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), which are known to destabilize or are able to stabilize the native folded state of proteins, respectively. Furthermore, the free energy landscape of proteins is affected by macromolecular crowding, i.e., the presence of a high concentration of macromolecules, which is largely, but not exclusively, due to the excluded volume Received: May 10, 2016 Revised: June 15, 2016

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DOI: 10.1021/acs.jpcb.6b04738 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B effect.11,16 Typically, soft-matter crowding reagents such as Ficoll or dextran can be used in vitro to simulate crowding in cellular environments in an appropriate way. Next to significant changes in protein conformation as observed upon unfolding and denaturation of a protein, more subtle changes in conformer population form a generic mechanism for high fidelity biological functions. The thermal energy triggers proteins to sample different conformations around their average structure, which are known as conformational substates (CSs) and lead to the concept of a multidimensional potential energy landscape for proteins.17,18 In fact, to study the conformational subspace of proteins, identification and characterization of these high-energy conformations is a central task of molecular biophysics. Owing to its many different functions and interaction partners, a multitude of conformational substates might also be expected for G-actin. Due to low fractional populations, such conformers may be difficult to detect, however. Hence, chemical or physical perturbations may be employed to shift conformational equilibria and characterize the otherwise rare conformational substates. To this end, pressure provides an elegant and efficient means to redistribute the population via volume differences.19−26 By favoring states with smaller partial molar volume, pressure shifts the equilibrium toward a system with smaller overall volume (according to Le Châtelier’s principle). Pressure perturbation, in particular coupled with NMR spectroscopy,21−23 can be effectively used to explore these CSs by acting on the volumes of the protein conformers, thus providing novel information on the structure, thermodynamic properties and conformational dynamics of proteins with atomic resolution, and hence is employed here as well. Earlier studies have shown, that urea and TMAO affect the polymerization kinetics of actin. Urea is able to decelerate and TMAO accelerates the kinetics of the G-to-F transformation.27,28 Here, we show how particular solution conditions, such as the most potent compatible osmolyte TMAO and the presence of macromolecular crowders, are able to lift deleterious effects of HHP on the stability and conformational dynamics of F-actin’s basic monomeric unit, G-actin. We used Ficoll PM 70 as crowding agent, which has a molecular mass of 70 kDa and is obtained by copolymerization of sucrose with epichlorhydrin. To this end, pressure and temperature dependent fluorescence and nuclear magnetic resonance spectroscopy (NMR) spectroscopies were carried out, yielding information about concomitant changes in structural and thermodynamic properties of the protein.

The G-actin-TMAO sample was prepared by directly adding an appropriate amount of perdeuterated TMAO-d9 (trimethylamine-N-oxide) up to a final concentration of 1 M. After addition of TMAO, the final pH value was readjusted to 7.85 by adding appropriate quantities of DCl or NaOD to sample. The pH was measured using a Hamilton Spintrode attached to a Beckman Coulter pH-meter. The pH values were not corrected for the deuterium isotope effect. Fluorescence Spectroscopic Measurements. A solution of 5 μM G-actin was incubated in G-buffer containing the appropriate amount of osmolytes or crowding reagent. Intrinsic Trp fluorescence spectra were recorded using a Multifrequency Cross-Correlation Phase and Modulation fluorometer K2 from ISS (Champaign, IL, USA) operating in the photon-counting mode. For the high pressure experiments, a high-pressure cell from ISS were used, which is equipped with sapphire windows, allowing pressure dependent measurements up to 4 kbar.30 The sample solution is placed in a small quartz-glass bottle, which is separated from the pressurizing medium by a Dura Seal film. The pressure was generated by a hand pump. The Trp fluorescence of G-actin was excited at 295 nm using a Xenon arc lamp. Fluorescence spectra on N-acetyl-L-tryptophanamide (NATA) were measured under the same temperature and pressure conditions for calibration purposes. The fluorescence spectra were normalized to their area using I(λ , x)Norm =

I (λ , x )

∫ I (λ , x )d λ

(1)

where x stands for pressure, p, or temperature, T. The area normalized intensities at 323 nm emission wavelength were used for thermodynamic analysis. The intrinsic pressure effect of tryptophan was corrected by subtracting the area normalized intensities of NATA at the same wavelength. Assuming a twostate process for unfolding, a Boltzmann function can be fitted to the pressure or temperature dependent unfolding curve, respectively:31 I= I=

1+e

IF − IU −(p − pu )·(ΔV / RT )

+ IU

1+e

IF − IU −( T1 − T1 )·(ΔH / R )

+ IU

m

(2)

(3)

IF and IU are the area-normalized fluorescence intensities of the folded and unfolded protein, respectively. The points of inflection, pu and Tm, describe the transition pressure and transition temperature, respectively. These fits allow the direct determination of the standard volume and enthalpy changes, ΔV° and ΔH°, respectively.31 NMR Spectroscopic Measurements. All NMR experiments were performed on an 800 MHz Bruker Avance spectrometer (Bruker, Biospin GmbH, Rheinstetten, Germany) equipped with a TCI cryoprobe. If not stated otherwise, the experiments were performed at 298 K, with temperature calibration after each sample change by measuring the difference of the proton resonance of the hydroxyl and the methyl group in 100% methanol as described by Raiford (1979).32 1H NMR spectra were directly referenced to the methyl resonances of internal DSS (4,4-dimethyl-4-silapentane1-sulfonic acid). 1H chemical shifts were obtained from highly resolved 1D spectra with a typical digital resolution of the time domain data of 0.055 Hz. For the determination of chemical shifts, a Lorentzian-to-Gaussian transformation was applied to

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EXPERIMENTAL SECTION Materials and Sample Preparation. Rabbit skeletal muscle α-actin was obtained from HYPERMOL (Bielefeld, Germany). The natural osmolytes TMAO and urea as well as the crowding reagent Ficoll PM 70 were purchased from Sigma-Aldrich (Seelze, Germany). G-actin was dialyzed against G-buffer containing 2 mM Tris-Cl pH 8.2, 0.4 mM ATP, 0.5 mM DTT, and 0.1 mM CaCl2 and subsequently centrifuged for 3 h (100 000 g, 4 °C) before the experiments started. The concentration of G-actin was determined by the Biuret method.29 The final NMR sample containing 25 μM of the G-actin was dissolved in 2 mM Tris-HCl (tris(hydroxyl-methyl-d3)aminod2-methane), 0.4 mM ATP, 0.5 mM DTT, 0.08 mM CaCl2, and 25 μM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) with a ratio H2O:D2O of 90:10. The pH was adjusted to 8.2. B


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The Journal of Physical Chemistry B

wavelength was chosen, which corresponds to the emission maximum of folded G-actin. As a model compound for correcting the effect of pressure on the intrinsic fluorescence properties of Trp, NATA was used.37,38 With increasing pressure, the area-normalized fluorescence intensity at 323 nm decreases slightly, only (−3.87 × 10−7 bar−1, Figure S1), and is essentially independent of the addition of cosolvents.38 In Figure 1a, we show as a representative example the areanormalized fluorescence spectra of G-actin in the presence of 2 M urea at 25 °C. With increasing pressure, a red-shift of the emission band of the four Trp residues39 as well as a decrease of the intensity at 323 nm emission wavelength is observed, which indicates the onset of pressure-induced denaturation of G-actin around 2 kbar. The area-normalized and with NATA corrected intensity at 323 nm emission wavelength is plotted as a function of pressure in Figure 1b, together with the results obtained for further solution conditions. The maximum pressure reached by the pressure cell is not sufficient for recording complete unfolding of G-actin in pure buffer solution. However, upon addition of urea, the unfolding pressure is markedly reduced so that the full unfolding process of G-actin can be recorded. Due to the fact that urea does not markedly affect the value of the volume change of unfolding,38,40−42 ΔV°u of G-actin can be estimated to −97 ± 3 mL mol−1 using eq 2 from the ureadependent unfolding data. From the intensity data obtained, we can also determine the standard Gibbs energy of unfolding using43

the FID to obtain signals as narrow as possible. Data acquisition and processing were performed with Bruker TopSpin 3.2. Data evaluation and fitting were done with the software Origin 6.0 (OriginLab Corporation). The high pressure NMR system, especially the autoclave holding the ceramic cell, was described in detail by Koehler et al.33 Pressure was applied to the NMR sample via pressurized water contained in high-pressure lines. For generating the pressure, a manually operated piston compressor and an air-toliquid pressure intensifier (Barocycler HUB440, Pressure BioSciences Inc., South Easton, MA, USA), which is controlled by the spectrometer, was used. The ceramic cell was purchased from Daedalus Innovations LLC (Aston, PA, USA) with a maximum pressure limit of 250 MPa. The autoclave holding the ceramic cell is similar to the original autoclave provided by Daedalus Inc.34 but has an integrated safety valve, similar to the security valve described by Beck Erlach et al.22 Evaluation of High Pressure NMR Data. For an equilibrium between n states i (i = 1, ..., n) of the protein, under fast exchange conditions, i.e. |Δωij τc| ≪ 1, the observed chemical shift is a combination of the chemical shifts δi of the states (for details see Kalbitzer35) and can described as n

δ=

∑i = 1 δi e−ΔG1i / RT n

∑i = 1 e−ΔG1i / RT

(4)

The Gibbs free energy change ΔG1i is given as ΔG1i(T0 , p) = ΔG1i0 + ΔV1i0(p − p0 ) −

36

Δβ1i0′ 2

(p − p0 )2

ΔGu°(p) = −RT ln K = −RT ln (5)

VT(p) n ∑i = 1 e−ΔGli(p)/ RT

(6)

⎛ ∂ΔGu° ⎞ ⎜ ⎟ = ΔV u° ⎝ ∂p ⎠

m

n

I (p) − I f IU − I(p)

(9)

i.e., the slope of the linear plot of ΔGu°(p) is equal to the volume change of unfolding. We obtain a value of ΔV°u = −98 mL mol−1, in good agreement with the results obtained from global fits using eq 2, and with literature data.44 Using a partial specific volume of 0.749 mL g−1 and a molecular mass of 42 348 Da for G-actin,31,45 we calculate this volume change to correspond to ∼0.3%, which is equivalent to the volume of about 5 water molecules. Both, the standard Gibbs energy of unfolding at 1 bar, ΔG°u (p0) (determined by extrapolation to p0 = 1 bar), as well as the unfolding pressure, pu (obtained from fits to eq 2), decrease drastically with increasing urea concentration (Figure 2, Table S1). The unfolding pressure of G-actin in pure buffer solution is equal to the intercept, where c(urea) = 0, and amounts to 2700 ± 40 bar, which is, within experimental error, in good agreement with literature data using FTIR spectroscopy.31,44 The standard Gibbs energy of unfolding at ambient pressure, ΔG°u (p0), decreases by ∼4 kJ per mol urea (Table S1).

VT(p) ∑i = 1 e−ΔG1i(p)/ RT ∑i = 1 e−ΔG1i(p)/ RT

= −RT ln

where the equilibrium constant, K, for protein unfolding is given by the ratio of the mole fractions of unfolded and folded species, f U and f F, respectively, with f F + f U = 1, and I(p) = f FIF + f UIU. IF and IU are the plateau values of the folded and unfolded protein state, respectively, which have been obtained from the sigmoidal fits to the experimental data using eq 2 (Figure 1b). The plots of ΔG°u(p) are shown in Figure 1c, and allow also calculation of the volume change of unfolding, as41

with VT(p) the total cross peak volume (in 1D spectroscopy, the area below a resonance line) corresponding to the sum of all states at pressure p. When the integral, V(p), of the observed peak is derived from m states 1, ..., m in fast exchange, eq 4 can be generalized to yield V (p) =

ff

(8)

where ΔV01i and Δβ01i′ are the differences of the partial molar volumes and of the partial molar compressibility factors between states 1 and i at temperature T0 and pressure p0, respectively. Since Δβ01i′ is usually rather small, we have neglected it in the fit of the data. In case of slow exchange, the cross peak volume of a peak 1, V1, is given by V1(p) =

fu

(7)

All data were fitted with these equations using the program Origin. The intensities (integrals) were corrected for the pressure dependent increase of concentration on the basis of the integral of the Tris-HCl signal.

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RESULTS AND DISCUSSION Pressure-Induced Unfolding of G-Actin in the Presence of Urea and TMAO. To follow the unfolding process of G-actin by fluorescence spectroscopy, intrinsic Trp fluorescence spectra were recorded. Unfolding of the protein is indicated by a red-shift of the emission band of Trp.37,38 For thermodynamic analysis, the fluorescence intensity at 323 nm emission C


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Figure 2. Pressure of unfolding, pu, of G-actin as a function of urea concentration.

and 2 M TMAO are depicted in Figure 3. Whereas unfolding of pure G-actin solution starts at ∼2000 bar, the onset of

Figure 3. Pressure dependent area-normalized fluorescence intensities at 323 nm of G-actin in the absence and presence of 1 and 2 M TMAO at 25 °C.

unfolding is shifted to ∼2400 bar in the presence of 1 M TMAO. Upon addition of 2 M TMAO, G-actin is even stable up to at least 3 kbar, i.e., TMAO imposes a strong stabilizing effect on the protein. It is well-known that in deep sea organisms the combination of chaotropic and kosmotropic osmolytes prevents osmotic shrinkage.46 To explore the combined effect of chaotropic and kosmotropic agents on the piezostability of G-actin, TMAO and the metabolic waste product urea were also measured in different concentrations and ratios in a similar way. As clearly seen in Figure 4, a counteracting effect of these two osmolytes is observed. A TMAO:urea ratio between 1:1 and 1:2 is able to abolish the deteriorating effect of urea on the stability of Gactin. NMR measurements. Three different G-actin preparations were measured in the pressure range from 1 to 2500 bar at different temperatures. Figure 5 shows the upfield part of the 800 MHz spectrum of G-actin at selected pressures. The

Figure 1. (a) Selected area-normalized fluorescence spectra of G-actin in 2 M urea over the pressure range from 1 to 3000 bar at 25 °C. (b) Pressure dependent area-normalized fluorescence intensities at 323 nm of G-actin and also of G-actin solutions in the presence of various urea concentrations as a function of pressure. (c) Standard Gibbs energy of unfolding of G-actin solutions in the presence of 1, 2, 3, and 4 M urea as a function of pressure.

The pressure-induced unfolding of G-actin in the presence of 1 and 2 M TMAO has been studied in a similar way. The areanormalized and with NATA corrected intensity data at 323 nm emission wavelength of G-actin as well as in the presence of 1 D


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Figure 4. Pressure dependent area-normalized fluorescence intensities at 323 nm of G-actin in the presence of different TMAO and urea concentrations at 25 °C.

resonances used for the quantitative thermodynamic analysis are labeled A−E. All of them probably represent methyl groups but only signal E had been identified earlier as the signal of the N-acetyl group still visible after actin polymerization.47,48 Unfortunately, further assignments are not available since recombinant actin with 15N, 13C enrichment is not available. At ambient pressure, the NMR spectrum represents a typical structured spectrum of a well-folded protein with many highfield shifted methyl resonances. As to be expected, many resonances shift with pressure. However, also the intensity of the highfield shifted resonances changes with pressure. At 2500 bar, the pressure where according to the fluorescence data unfolding of G-actin has largely proceeded (pu ≈ 2700 bar), the intensities of the high field shifted resonances are reduced by approximately 80% of the initial value. Concomitantly, the signal intensity increases in the range between 0.8 and 1.1 ppm, where methyl resonances in unstructured proteins are to be expected. The application of pressures does not lead to a complete unfolding of the protein with NMR signals typical for highly mobile random-coil structures. Even at the highest pressure, where only 20% of the protein is in its native state, narrow random-coil signals can barely be detected and may represent only a few percent of the total signal. This denaturation process is only partly reversible under our experimental conditions. When going back to ambient pressure and waiting several hours, the initial concentration of the wellfolded structure is only partially recovered; the spectral features in the range from −0.9 to 0.7 ppm are identical to those observed in the initial spectrum but with reduced intensity (Figure 5). The integration of the highfield shifted methyl resonances reveals that after pressure denaturation approximately 40% of the protein is in its initial well-folded state. In fact that is to be expected since proteins that contain substrates important for their structure, usually do not refold after release of the substrate. The intensities of some methyl resonances (line A in Figure 5b) first increase up to a pressure of ∼850 bar (even after correcting for the compression of the solvent and thus a small increase of the actin concentration). Figure 6 shows the pressure dependence of the high field shifted resonances A and B. These resonances have been selected for a more quantitative analysis because they can be followed without larger overlap

Figure 5. 1H NMR spectra of G-actin at various pressures. (a) Downfield part of a 800 MHz spectrum of 25 μM G-actin in a buffer containing 2 mM Tris-HCl, 0.4 mM ATP, 0.5 mM DTT, 0.08 mM CaCl2 in H2O/D2O of 90/10, pH 8.2. Temperature: 298 K. (b) Zoomed regions. The labels A−E define resonances used for the thermodynamic analysis.

with other signals. The biphasic changes of the peak integral of line A (denoted intensity in the following) can only be explained by a three-state model with NMR line A representing native folded state 1 being in slow exchange on the NMR time scale with the same group in native folded state 2 and a denatured state 3. Such behavior appears to be typical for many signals in the range from 0 to 0.7 ppm. The differences of the Gibbs free energies ΔG12 and ΔG13 are 0.9 and 10 kJ mol−1, respectively (Table S1). The corresponding differences of the partial molar volumes, ΔV12 and ΔV13, are 86 and −58 mL mol−1, respectively. Line B represents another isolated highfield E


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−38 mL mol−1, respectively (Figure 7). Line E at 2.035 ppm at ambient pressure has been assigned earlier as the methyl group

Figure 6. Pressure-induced spectral intensity changes in G-actin. Experimental conditions as described in Figure 5. The data shown are the mean of two different samples, the error bars correspond to the standard deviation. Data were fitted with a three-state model (eqs 6 and 7, respectively), with the parameters given in Table S1. (a) Pressure dependence of line B, (b) pressure dependence of the peak area of signal A.

Figure 7. Pressure-induced chemical shift changes of the methyl resonances B, C, D, and E in G-actin. Temperature: 298 K. Data were fitted with eq 1 using a two-state model with the parameters given in Table S1. Error bars correspond to two different measurements. (a) Line B and (b) lines C, D, and E.

shifted methyl resonance, but does not show such a biphasic behavior. The intensity changes can be fitted with a two-state model with Gibbs free energy and partial molar volume differences that are within the limits of error equal to those obtained for the 1 to 3 transition for line A. However, line B is strongly shifting with pressure, by more than −0.1 ppm at 2500 bar. This indicates the existence another equilibrium in fast exchange on the NMR time scale. The pressure where half of the protein is in state 3 is about 1900 bar (calculated relative to the maximum values of lines A and B). This pressure is clearly smaller than the value pu of 2700 bar obtained from the fluorescence data for unfolding of G-actin. This indicates that the two methods probably detect two different transitions. Since even at the highest pressure measured by NMR only very weak random-coil signals are detected by NMR, NMR may detect the transition to a moltenglobule (MG)-like state occurring prior to complete unfolding of the protein as detected by fluorescence spectroscopy. There are three relatively sharp resonance lines, C, D, and E, with line widths of approximately 3.6 Hz (Figure 5). It is possible to fit the chemical shift changes of lines C and D together with line B with a ΔG and ΔV of 4.1 kJ mol−1 and

of the N-terminal N-acetyl modification of actin.48 Its chemical shift change with pressure can only be fitted with a different set of parameters (ΔG and ΔV of 5.6 kJ mol−1 and −85 mL mol−1), indicating that it is involved in a different transition than lines B, C, and D (Figure 7). Line E is part of the Nterminal domain from amino acid 1 to 22 that is also visible and still highly mobile after polymerization of actin to F-actin in the so-called M-(mobile) state in equilibrium with the I(immobile) state. From the fast exchange condition, an upper limit for the exchange correlation time, τe, can be estimated as 55 ms. The thermodynamic parameters obtained here may describe the same equilibrium in G-actin. Lines C and D disappear after polymerization of actin. It is not clear if they represent a doublet resonance with a coupling of 4.1 Hz or two singlet signals. In the latter case, they could represent methyl groups of methionines with random-coil values of 2.109.49 Since they are not assigned to specific residues, it is not possible yet to associate a specific transition with their shift changes. In F


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The Journal of Physical Chemistry B addition, one has to note that state 1 in the fitting procedure of different resonance does not have to correspond exactly to the same state since not all lines have to sense all possible transitions of the protein. G-Actin in the Presence of TMAO. Perdeuterated TMAO-d9 was added in the appropriate quantity to a Gactin sample prepared as described before up to a total concentration of 1 M, a concentration similar to that found in deep sea fish. The general spectral features of the G-actin spectra were unchanged. However, spectral details and chemical shifts are different for many easily identifiable resonance lines (Figure 8).

Figure 9. Pressure induced spectral intensity changes in G-actin. Experimental conditions as described in Figure 8. The data shown are the mean of two different samples, the error bars correspond to the standard deviation. Data were fitted with a three-state model (eqs 6 and 7, respectively), with the parameters given in Table S1. (top) Pressure dependence of the peak area of signal B, (bottom) pressure dependence of line A.

This indicates that the denaturation pressure increases upon addition of 1 M TMAO, which is in accord with the fluorescence spectroscopy data. For quantification, the pressure dependence of lines A and B was analyzed. For line A, the ΔG12 and ΔV12 values obtained for the transition from substate 1 to 2, describing the increase in peak intensity are (within the limits of error) identical to those obtained in the absence of TMAO (Table S1). This is also true for ΔG13 and ΔV13. The pressure where the intensity is reduced by 50% of its maximum value is with approximately 2200 bar not much larger than 1900 bar in the absence of TMAO. In the presence of TMAO, line B shows a different behavior at low pressures to that observed in the absence of TMAO. Its intensity first increases with pressure before it decreases again. The pressure where the intensity is reduced by 50% of its maximum value is with approximately 2700 bar much larger than 1900 bar in the absence of TMAO and closer to the unfolding pressure pu ≥ 3200 bar observed by fluorescence spectroscopy. The thermodynamic parameters obtained are also very different, with ΔG13 and ΔV13 values of 25 kJ mol−1 and −94 mL mol−1, respectively. Line C and D cannot be separated because they are slightly shifted and broadened in the presence of TMAO. Again, the chemical shifts of line B and the

Figure 8. Effect of TMAO on the 1H NMR spectra of G-actin. The regions containing signals A and B of G-actin in the presence of TMAO is shown.

As an example, the chemical shifts of those described above change significantly by −0.004 ppm (A), −0.008 ppm (B), 0.011 ppm (C, D), and −0.005 ppm (E) after addition of TMAO. Lines C and D are not separated because they are broadened in the presence of TMAO. These effects may be partially due to direct interactions with TMAO but probably also due to shifts of local and/or global conformational equilibria. As seen in the absence of TMAO, the intensity of the highfield shifted resonance lines A and B first increase and finally decrease with pressures (Figure 9). At 2500 bar,their intensities are decreased by about 20% compared to the intensity at ambient pressure. Again, the reduction of intensities typical for a denaturation process is only partially reversible upon decompression from 2500 bar to ambient pressure. Approximately 10% of the protein seems to be irreversibly denatured as concluded from the reduced intensity of the highfield shifted methyl resonances typical for well-folded G-actin. G


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The Journal of Physical Chemistry B average of lines C and D can be fitted simultaneously with the same thermodynamic parameters, yielding with ΔGxy and ΔVxy of 3.0 ± 0.6 kJ mol−1 and −36 ± 4 mL mol−1, respectively (Figures S1 and S2). Within the error limits they are identical to those obtained in the absence of TMAO. Macromolecular Crowding Effects on the Pressure Stability of G-Actin. To create a more cell-like environment, Ficoll was added as macromolecular crowding agent. The pressure stability of 5 μM G-actin solutions was determined using Ficoll PM 70 at concentrations from 5 up to 20 wt%. Figure 10 shows the pressure dependent area-normalized and with NATA corrected fluorescence intensity at 323 nm

Figure 10. Pressure dependent area-normalized fluorescence intensities at 323 nm of G-actin and also of G-actin solutions in the presence of 10 and 20 wt% Ficoll PM 70 as a function of pressure at 25 °C. Due to scattered background, the intensities have been normalized with Imax.

Figure 11. a) Temperature dependent area-normalized fluorescence intensities at 321 nm of 5 μM G-actin and also of 5 μM G-actin solutions in the presence of 1 and 2 M urea as a function of temperature at 1 bar. b) Corresponding data in the presence of 1 and 2 M TMAO.

emission wavelength of G-actin as well as in the presence of various Ficoll concentrations. As it can be clearly seen, an increase in Ficoll concentration leads to a stabilization of the folded state against pressure-induced unfolding. Hence both, the compatible osmolyte TMAO as well as the macromolecular crowding agent lead to a stabilization of the folded state of the protein with increasing concentration. Temperature-Induced Unfolding of G-Actin. The effect of cosolvents and crowding have also been studied on the temperature stability of G-actin. As described before, the Trp fluorescence was recorded and the fluorescence intensity of the emission maximum at 321 nm was used for thermodynamic analysis. The area-normalized intensity data at 321 nm emission wavelength of 5 μM G-actin in the absence and presence of urea and TMAO are shown as a function of temperature in Figure 11. Plateau values are seen for G-actin from 10 to 40 °C, which represent the folded state of G-actin. At higher temperatures, unfolding of the protein commences, which is indicated by a marked red shift. Fits of the fluorescence decays, using eq 3, allow calculation of the temperature of unfolding, T m , as well as the accompanying enthalpy change, ΔH°u (Tm),which amount to 49.4 ± 0.3 °C and 262 ± 19 kJ mol−1, respectively, for G-actin in pure buffer solution. The entropy change of unfolding ΔSu°(Tm), is given by ΔSu°(Tm) = ΔHu°(Tm)/Tm, and is 0.81 ± 0.06 kJ mol−1K−1 at Tm (Table S2).

Neglecting the heat capacity change upon unfolding,31 the temperature dependence of ΔGu°(T) is given by50 ⎛ ∂ΔGu° ⎞ ΔGu°(T ) = ΔGu°(Tm) + ⎜ ⎟ ·(T − Tm) ⎝ ∂T ⎠ p = −ΔSu°(Tm) ·(T − Tm)

(10)

For the pure buffer solution at 25 °C, we obtain ΔG°u = 10.8 ± 1.5 kJ mol−1. Here, a two-state kind of analysis has been used for deriving the thermodynamic data, which also relies on full reversibility of the process. Whereas all pressure dependent studies have been fully reversible, the temperature dependent unfolding curves have been partially reversible, only, when highest temperatures have been reached. Moreover, deviations from a simple twostate unfolding model in temperature-induced unfolding have been discussed in some works.51,52 Hence, the temperature dependent thermodynamic data have to be taken with some care. This is not a major drawback, however, as we are essentially interested in relative changes upon addition of cosolvents and crowding agents. Not only the pressure, but also the temperature of unfolding of G-actin is drastically reduced by urea. The presence of 2 M H


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The Journal of Physical Chemistry B urea leads to a reduction of Tm from 49.4 ± 0.3 to 43.6 ± 0.2 °C. It is well-known, that urea, unlike TMAO, shows preferential accumulation in the neighborhood of protein backbones and side chains, i.e., destabilizes proteins due to a direct mechanism without largely influencing the solvent structure.14,53,54 Conversely, in the presence of TMAO, the unfolding transition is significantly shifted to higher temperatures. The presence of 2 M TMAO leads to an increase of Tm from 49.4 ± 0.3 to 57.5 ± 0.3 °C. The standard Gibbs energy of unfolding at 25 °C, ΔGu°, increases from 10.8 ± 1.5 to 31.5 ± 3.0 kJ mol−1 for 2 M TMAO (see Table S2). Several studies have shown, that TMAO is able to enhance the protein stability without direct interaction with protein groups, but rather due to attenuation of the strength of the H-bonds formed between water and polar goups,14,55 which could also be the explanation for the similar enthalpy changes of unfolding for the various Gactin solutions (Table S2). To study the combined effect of chaotropic and kosmotropic agents on the thermostability of G-actin, TMAO and urea were measured in different concentrations. Similar to the pressure dependent studies, a counteracting effect of the two osmolytes can be seen here as well (Figure 12).

Figure 13. Temperature-induced denaturation of 5 μM G-actin and also of 5 μM G-actin solutions in the presence of 5, 10, and 20 wt% Ficoll PM 70 as a function of temperature at 1 bar. The Trp fluorescence intensity at 321 nm has been measured and rescaled to cover the range from 0 to 1.

60 °C, the protein is almost completely unfolded. The temperature of unfolding increases markedly with increasing Ficoll concentration (see also Table S2), similar to the effect of the compatible osmolyte TMAO. In fact, it is known, that macromolecular crowding agents, such as Ficoll, are able to stabilize the folded state indirectly due to a strong destabilization effect on the unfolded state by the excluded volume effect,56−58 which seems to play a major role here as well. This also indicates that destabilizing enthalpic effects due to soft interactions with the crowding agent seem largely absent, here. In the presence of 20 wt% Ficoll, representing cell-like conditions, the temperature of unfolding has increased by ∼10 °C, i.e., has almost the same effect as the addition of 2 M TMAO.

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CONCLUSIONS In this study, we explored the pressure- and temperatureinduced unfolding reaction of G-actin as well as of G-actin solutions containing different concentrations of the chaotropic cosolvent urea, the kosmotropic osmolyte TMAO, mixtures thereof, and of the crowding agent Ficoll to explore if the cellular milieu is able to alleviate deleterious effects of extreme environmental conditions (Figure 14). G-actin unfolds at ∼49 °C at ambient pressure (1 bar =0.1 MPa). At ambient temperature (25 °C), G-actin unfolds completely at a pressure of ∼2700 bar, only, which is accompanied by a volume change of unfolding of −97 mL mol−1 as determined by fluorescence spectroscopy. Both, the temperature and pressure stability is reduced by the addition of urea, and increases markedly in the presence of the compatible solute TMAO, which is reflected in an increase of the Gibbs energy of unfolding at 25 °C by ∼5 kJ per mol TMAO. In mixtures of these two disjunct cosolvents, TMAO is found to counteract the deleterious effect of urea. At a TMAO:urea molar ratio of 1:2, the stabilizing effect of TMAO and the destabilizing effect of urea on the folded state of G-actin are almost completely ameliorated. Similar to the effect of TMAO, the presence of the macromolecular crowding agent Ficoll, at cell-like high concentrations (∼20 wt%), is able to stabilize the protein even more than by 1 or 2 M TMAO. In

Figure 12. Temperature dependent area-normalized fluorescence intensities at 321 nm of 5 μM G-actin and of 5 μM G-actin solutions in the presence of different TMAO and urea mixtures as a function of temperature at 1 bar.

The destabilizing effect of 2 M urea is almost completely counteracted by 1 M TMAO. Hence, the stabilizing effect of TMAO on the thermostability of G-actin is higher than the destabilizing effect of the chaotropic osmolyte urea. The different mixtures do not significantly influence the enthalpy change of unfolding, which is also reflected in the similar slopes at the points of inflection. We also added Ficoll to explore the effect of macromolecular crowding on the temperature stability of G-actin. Figure 13 depicts the folded fraction of G-actin in the absence and presence of various amounts of Ficoll PM 70. The areanormalized fluorescence intensity data at 321 nm emission wavelength are converted to the folded fraction f F here for better comparison. Between 10 and 40 °C, no significant changes in the folded fraction are observed for the different solutions. Beyond T ≈ 43 °C, G-actin begins to unfold, and at temperatures higher than I


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Figure 15. Schematic of the effect of pressure on the free energy landscape of G-actin. Upon pressure perturbation, excited conformational substates can be reversibly populated. At sufficiently high pressures (at ∼2700 bar), protein unfolding takes place, which is preceded by a molten-globule (MG) like state (at ∼1900 bar). The native ground state of a globular protein contains always cavities, some of which are partially occupied by water molecules, and some of which are empty. The basic ground state therefore has always a larger partial molar volume than the unfolded state, which is fully hydrated and contains no internal cavities anymore. Hence, pressure generally destabilizes the ground state compared to partially unfolded higherenergy states. Owing to the relatively high compressibility of void volume, the nonhomogeneous packing of the amino acids in the protein and the differential pressure stability of secondary structure elements, pressure also affects the population of the various conformational substates, which have been identified by NMR spectroscopy in this study, e.g., the transition from the ground state (I-state) to the more mobile M-state both sensing the nucleotide-metal complex (at ∼650 bar).

Figure 14. Temperature of unfolding, Tm, of 5 μM G-actin at different concentrations of cosolvents and the crowding agent Ficoll at 1 bar.

mixtures of TMAO and urea, TMAO counteracts the destabilizing effect of pressure on the structure and stability of the protein. At a TMAO:urea molar ratio of 1:1 to 1:2, the two cosolvents completely override their effects on the stability of G-actin. Such effect on the stability of the protein may be largely due to indirect, i.e., solvent-mediated interactions of the two agents.59 High-pressure NMR spectroscopy can be used for technical reasons only in the pressure range up to 2500 bar, where complete denaturation of G-actin does not occur, yet. However, in the lower pressure range additional conformational transitions could be observed. Highfield shifted methyl resonances typical for a compactly folded hydrophobic core disappear at a pressure of ∼1900 bar without appearance of spectral features typical for random coil structures. This suggests that in this pressure range, a MG-like structure is formed prior to unfolding of the protein. In the presence of 1 M TMAO, the transition pressure to the MG-like state is significantly increased, but this increase of stability differs for different regions of the protein, ranging from 300 to 900 bar. Actin has to operate in many different conformational (sub)states, for example in the ADP- and ATP-bound states and the conformations induced by polymerization or upon complex formation with a multitude of different interacting proteins. Accordingly, a rather complicated pressure response of actin is to be expected and has indeed been observed here. Amino acids 1 to 22 of the N-terminal domain are also visible and still highly mobile after polymerization of actin to F-actin in the so-called M-(mobile) state in equilibrium with the I(immobile) state (Figure 15). The equilibrium between the two states in F-actin is shifted specifically to the I-state by replacing the physiologically bound Mg2+-ions by Ca2+-ions or unspecifically by high concentrations of monovalent ions such as K+. The mobile N-terminal domain contains Asp11 that is coordinated to the bound Mg2+ ion as well as Gly12, Ser14, Gly15. Leu16, and Lys18 that are bound to the phosphate groups of the nucleotide.60 Ser14 initiates the nucleotide dependent structural changes and forms a hydrogen bond to the γ-phosphate of ATP and to the β-phosphate group of ADP, respectively, and transmits this information to the surface.61 As reported by the N-terminal acetyl group, the equilibrium can be shifted by pressure to one of the states, probably the more open M-state. This functional equilibrium is not influenced significantly in the presence of TMAO, although small chemical shift changes indicate small local conformational changes all over the actin surface.

To conclude, by adjusting the level of compatible osmolytes such as TMAO and the intracellular crowding conditions, deleterious effects of extreme environments, including high hydrostatic pressures as encountered in the deep sea, on the stability of G-actin can be largely abrogated without significantly perturbing the conformational equilibria of the functional native states.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b04738. Additional figures and tables (thermodynamic and NMR data) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Telephone: +49 231 755 3900. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Deutsche Forschungsgemeinschaft (DFG FOR 1979) and HFSPO is gratefully acknowledged. REFERENCES

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