© Copyright 1997 by the American Chemical Society
VOLUME 101, NUMBER 6, FEBRUARY 6, 1997
LETTERS Hydration of Gas Phase Proteins: Folded +5 and Unfolded +7 Charge States of Cytochrome c Ju1 rgen Woenckhaus, Yi Mao, and Martin F. Jarrold* Department of Chemistry, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208 ReceiVed: October 30, 1996X
Free energy changes for the first steps in the hydration of the +5 and +7 charge states of gas-phase cytochrome c have been determined from equilibrium constant measurements at 271 K. Previous ion mobility studies (Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1996, 118, 10313) indicate that the +5 charge state is tightly folded in the gas phase, while the +7 charge state is partially unfolded under the conditions employed. Free energy changes have been determined for the adsorption of the first nine water molecules on the folded +5 charge state and the first five water molecules on the unfolded +7 charge state. The free energy changes for transfer from bulk water to the hydration shell of the protein are remarkably small, between -2 and -7 kJ mol-1, indicating that the charge is very effectively shielded in both the unfolded and folded conformations. The free energy changes for initial hydration of the folded +5 charge state are significantly more negative than for the unfolded +7 state. This may indicate that the charge is more effectively shielded in the unfolded +7 charge state, or the incorporation of some of the adsorbed water molecules as structural water molecules in the folded +5 charge state.
Introduction The conformations, and ultimately the physiological properties, of biological molecules result from a delicate balance between intramolecular interactions and solvation. For example, estimates of the folding free energy of cytochrome c in Vacuo1-3 range from -2182 to -3497 kJ mol-1, but in solution the folding free energy2 is -37.1 kJ mol-1. This enormous difference results because solvation effects compensate for the intramolecular interactions that dominate in the gas phase. Clearly, understanding the interaction of proteins with their solvent is central to understanding their properties, and an enormous effort has been devoted to this topic.4 With the recent development of new ionization techniques that make it possible to obtain large biological molecules in the gas phase5 there has been growing interest in examining their geometries in this X
Abstract published in AdVance ACS Abstracts, January 15, 1997.
S1089-5647(96)03389-5 CCC: $14.00
environment.7-14 Studies of unsolvated proteins can provide important information about their intramolecular interactions. However, gas-phase studies also offer new opportunities to study solvation. Starting with an unsolvated protein ion in the gas phase, it is in principle possible to sequentially hydrate it and study the hydration process one water molecule at a time. In this letter, we report a step toward achieving this goal. We have determined free energy changes for adsorption of the first few water molecules on the +5 and +7 charge states of cytochrome c generated by electrospray ionization. These charge states were selected because gas-phase ion mobility measurements14 have indicated that they have dramatically different conformations under our conditions. The +5 charge state has a compact, tightly folded geometry in the gas phase. However, between the +5 and the +7 charge states, a sharp unfolding transition occurs. This unfolding transition is believed to be driven by Coulomb repulsion, and it is somewhat analogous to acid denaturation in solution. © 1997 American Chemical Society
848 J. Phys. Chem. B, Vol. 101, No. 6, 1997
Figure 1. Drift time distributions recorded for the +5 and +7 charge states of cytochrome c in helium at 271 K. The injection voltage, the voltage between the exit of the desolvation region and the entrance of the drift tube, was 300 eV. The drift time distributions have been scaled to a helium buffer gas pressure of 5.0 Torr, and they are plotted against a reduced time scale obtained by multiplying the measured drift time by the charge state.
Experimental Methods Experiments were performed using an injected ion drift tube apparatus equipped with an electrospray ionization source.15 Unacidified solutions of ∼1.5 × 10-3 M bovine cytochrome c (Sigma Chemical Company) in a 75:25 mixture of water and acetonitrile were electrosprayed at 5 kV in air. Ions enter the apparatus through a small aperture, pass through a desolvation region, and are then focused into a quadrupole mass spectrometer. After passing through the quadrupole, the ions are focused and injected into the drift tube. The voltage between the source exit plate and the entrance of the drift tube was 300 V, and so the injection energies were 1500 eV for the +5 charge state and 2100 eV for the +7. No fragmentation of the cytochrome c ions was observed. The drift tube was operated with a drift field of 13.2 V/cm and at a total pressure of water vapor and helium of 5 Torr. The drift tube was temperature regulated with a precision of (0.3 K to 271 K. At the end of the drift tube the ions exit through a small aperture and they are then focused into a second quadrupole mass spectrometer. After being mass analyzed, the ions are detected by an off-axis collision dynode and dual microchannel plates. Drift time distributions were recorded by injecting 50 µs pulses of ions into the drift tube and recording arrival time distributions at the detector with a multichannel scaler. Results Figure 1 shows drift time distributions measured for the +5 and +7 charge states of cytochrome c in pure helium under the conditions used to study the hydration reactions. The distributions are plotted against a reduced time scale given by the measured drift time multiplied by the charge state. This removes
Letters
Figure 2. Mass spectra measured for the +5 and +7 charge states of cytochrome c with no water vapor in the drift tube (parts a and c) and with approximately 0.6 Torr of water vapor (parts b and d). The points are the measured data, and the lines are simulations discussed in the text.
the effect of the charge on the drift time and facilitates comparison of drift time distributions measured for different charge states. Dashed lines labeled N and E show the drift times determined using an exact hard spheres scattering model16,17 for the native conformation (using crystal structure coordinates18) and a fully extended form of cytochrome c.15 For the +5 charge state there is a single peak at a drift time slightly shorter than expected for the native conformation. For the +7 charge state there are two peaks with drift times between those expected for the native conformation and a fully extended conformation. Mass spectra recorded for the +5 and +7 charge states of cytochrome c with no water vapor in the drift tube are shown in parts a and c of Figure 2, respectively, and mass spectra recorded with approximately 0.6 Torr of water vapor are shown in parts b and d of Figure 2. Several peaks are apparent in the spectra recorded without water vapor. The smaller, higher-mass peaks are due to protonated cytochrome c ions where Na+ or K+ has substituted for one of the protons (sodium and potassium are impurities in the cytochrome c sample). Although the resolution of our quadrupole mass spectrometers is around 1 amu/charge, the effective mass resolution is degraded by the broad (8 amu fwhm) isotope distribution of cytochrome c, which is unresolved here because the ions carry more than one charge. It is possible to separate the ions containing Na+ and K+ from the fully protonated cytochrome c ions with the first quadrupole mass spectrometer. However, this was not done because with the relatively low intensities available from the electrospray source, operation of both quadrupoles at high mass resolving power results in signals that are too small to perform measurements. In most of the experiments, the first quadrupole was set to transmit all ions. As can be seen from Figure 2, spectra recorded with approximately 0.6 Torr of water vapor in the drift tube show peaks due to the addition of water molecules onto the protein ions. The propensity for water adsorption is clearly
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J. Phys. Chem. B, Vol. 101, No. 6, 1997 849
much larger for the +5 charge state than for the +7 charge state. At room temperature, with the same water vapor pressure, no significant amount of water adsorption was observed. Equilibium constants for the hydration reactions
Cytcm+(H2O)n + H2O T Cytcm+(H2O)n+1
(1)
were then derived from
K)
I(Cyctm+(H2O)n+1) I(Cyctm+(H2O)n)P(H2O)
(2)
where P(H2O) is the water vapor pressure in the drift tube in atmospheres and I(Cytcm+(H2O)n+1) and I(Cytcm+(H2O)n) are the intensities from the mass spectra. To account for the presence of the Na+ and K+ peaks, the spectra recorded without water were fit, using a least-squares procedure, to a function consisting of a sum of Gaussian functions. Two Gaussian functions were used to represent each peak in the mass spectrum so that it was possible to account for the asymmetry in the peaks that often occurs with quadrupole mass spectrometers. The solid lines in parts a and c of Figure 2 show the fits. The good fits obtained here indicate that in the absence of water vapor in the drift tube the protein ions contain no water molecules. Intensities were then obtained from the mass spectra recorded with water vapor in the drift tube by summing together a series of the Gaussian functions separated by 18 amu/charge, using a least-squares procedure to adjust their intensities and fit the data. The solid lines in parts b and d of Figure 2 show the resulting fits. In using this procedure we assume that substitution of Na+ or K+ for a proton in the protein ions does not dramatically affect the propensity to adsorb water. Small differences will not significantly influence our results because the relative abundances of ions that contain Na+ or K+ are small. Equilibrium constants were determined for water vapor partial pressures ranging from 0.125 to 0.75 Torr. Equilibrium constants determined for hydration reactions (eq 1) with small n were independent of the water vapor pressure over the 0.1250.75 Torr range examined. For hydration reactions with larger n, which were only observed at high water vapor pressures, the equilibrium constants also appeared to be independent of water vapor pressure over the limited range accessible. Water vapor pressures significantly larger than 0.75 Torr are difficult to access at the temperature employed because of condensation onto the cooled metal surfaces of the drift tube. With the high injection energies used, the ions are collisionally excited as they enter the drift tube, so there is a concern that incomplete cooling could influence the results. However, the ions experience (39) × 106 collisions in the drift tube with helium and water, which should be more than enough to equilibrate their internal degrees of freedom. As a further test that equilibrium had been established, mass spectra were recorded with the drift voltage reduced by a factor of 2 so that the ions spend twice as long in the drift tube. These mass spectra were not significantly different from those recorded with the higher drift field. Although the results described above provide a strong indication that equilibrium has been established under the conditions employed, they do not completely rule out the possibility that we are observing a series of steady states involving protein ions with slightly different conformations. For the +7 charge state, it is clear from the drift time distributions that there are two significantly different, partially unfolded conformations present. The equilibrium constants determined for this charge state are a weighted average.
Figure 3. Free energy changes for adsorption of a water molecule onto the +5 and +7 charge states of cytochrome c plotted against the number of adsorbed water molecules. Free energy changes for adsorption of water onto H3O+ (from ref 23) and GlyH+ (from ref 24) are shown for comparison. The dashed line shows the free energy change for adsorption onto bulk water (see text).
Gibbs free energy changes were determined from the measured equilibrium constants using ∆G° (271 K) ) -RT ln K. The free energy changes obtained in this way are plotted in Figure 3 against the number of water molecules. The results shown in the figure are an average of two completely independent sets of measurements performed four months apart, and the absolute values are believed to be reliable to (1 kJ mol-1. For the +7 charge state, free energy changes are shown for the first five water molecules adsorbed and for the first nine water molecules adsorbed on the +5 charge state. For the +7 charge state the free energy changes increase slightly from around -15 to around -14 kJ mol-1 as the number of water molecules increases. For the +5 charge state the hydration free energies increase nearly linearly from around -19 to around -15 kJ mol-1 as the number of water molecules increases. The larger free energy changes for the +5 charge state are a reflection of its larger propensity to adsorb water, which is apparent from the mass spectra shown in Figure 2. Discussion The drift time distribution for the +5 charge state shows a single, relatively narrow peak with a drift time slightly shorter than expected for the native (crystal structure) conformation. In solution or in a crystal there is an effective force field that prevents the protein from packing tightly. In the gas phase the protein becomes more compact, by 5-8% according to molecular dynamics simulations for BPTI (bovine pancreatic trypsin inhibitor).19,20 The +5 charge state has a drift time close to that expected for a contracted native conformation. However, this does not indicate that the +5 charge state has this geometry. With the transient heating that occurs as the ions are injected into the drift tube21 it is possible that the +5 charge state
850 J. Phys. Chem. B, Vol. 101, No. 6, 1997 rearranges to a lower-energy, gas-phase conformation. For the +7 charge state, a peak with a drift time similar to that for the +5 charge state is observed at low injection energies. But with the high injection energies employed here, collisional heating as the ions enter the drift tube causes the +7 charge state to unfold.15 This unfolding is irreversible, at least on the time scale of our experiments. For the +7 charge state, two peaks are present in the drift time distribution. Both peaks are shifted to substantially longer drift times than expected for the native protein, indicating unfolded structures. However, both peaks occur at a drift time substantially less than expected for a completely extended geometry, suggesting that the observed conformations retain some elements of secondary structure.13,14 The unfolding transition that occurs between the +5 and +7 charge states is believed to be driven by Coulomb repulsion, which is much more important in the gas phase than in a solvent, such as water, with a high dielectric constant. The free energy changes determined from the equilibrium constants can be compared with the free energy change for adsorption of a water molecule onto a bulk water surface (which is equivalent to adding a water molecule into the bulk liquid). From the vapor pressure of water22 at 271 K this free energy change is -11.9 kJ mol-1. This value is represented by the dashed line in Figure 3. The difference between the free energy change for adsorption onto bulk water and the free energy change for adsorption onto the protein ions represents the free energy required to transfer a water molecule from the bulk liquid to the hydration shell of the protein. These free energies range from around -4 to -3 kJ mol-1 for the +7 charge state and from around -7 to -3 kJ mol-1 for the +5 charge state. The fact that these values are negative indicates hydrophilic interactions; positive values would indicate hydrophobic interactions. There have been a number of studies of the hydration of protein films.4 Hydration free energies determined from those studies can be compared directly with the free energy changes reported here. At low levels of hydration, the average free energy determined for transfer from the bulk solvent to the hydration layer is around -7 kJ mol-1. The water is believed to interact with charged groups in this regime. At slightly higher levels of hydration, the average free energy required for transfer from the bulk solvent to the hydration layer is around -1 kJ mol-1. The water is believed to interact mainly with polar groups on the surface of the protein in this regime. The free energy changes determined here lie between those expected for solvation of charged groups and polar groups. Free energy changes measured for the hydration of H3O+ and GlyH+ (protonated glycine) are shown in Figure 3. The free energy changes for hydration of H3O+ were obtained from the values for ∆H° and ∆S° determined by Lau et al.23 The free energy changes for GlyH+ were obtained from values determined by Klaissen et al.24 at room temperature (293 K) and adjusted to 271 K assuming25 that ∆S ) 104 J K-1 mol-1. Charge solvation is responsible for the large negative values for the free energy changes for adsorption of the first few water molecules on H3O+ and GlyH+, and the free energy changes become less negative as the number of water molecules in the solvation shell increases. Klaissen et al. have also measured free energies for hydration of (Gly)nH+ (n ) 2-4). The free energy changes for the adsorption of the first water molecule on (Gly)nH+ at 293 K are -41, -37, -28, and -24 kJ mol-1 for n ) 1-4. The substantial difference in the free energy changes between n ) 2 and 3 has been attributed to cyclization of the larger oligomers through hydrogen bonds to carbonyl oxygens. This intramolecular charge “solvation” decreases the free energy change for adsorption of the first water molecule
Letters by effectively shielding the charge. If the charged sites in the cytochrome c ions examined here were exposed on the surface of the protein, initial hydration free energies comparable to those found for H3O+ and GlyH+ would result. Thus, the small free energy changes determined for adsorption of the first few water molecules on the cytochrome c ions indicates that the charge is effectively shielded, presumably by interaction with a number of carbonyl oxygens from different parts of the protein. Recent molecular dynamics simulations of protonated bradykinin, a peptide containing nine amino acids,12 show an intramolecular charge “solvation” shell around the charge site. The results presented here suggest that larger, multiply charged protein ions possess a similar structural motif with each charge site surrounded by an intramolecular charge “solvation” shell that shields the charge. That the free energy changes for initial hydration of the +7 charge state are significantly less negative than for the +5 charge state is surprising because the more highly charged ion would be expected to have more negative hydration free energies. One interpretation of these results is that the increased freedom in the unfolded +7 charge state allows for more effective intramolecular charge “solvation” so that the charge is more effectively shielded. An alternative explanation is that the more negative hydration free energies observed for the +5 charge state result from adsorbed water molecules that enter the interior of the protein as structural water molecules. For native cytochrome c in solution, six structural water molecules have been identified.26 However, it is not clear that structural water molecules can be accommodated in the tightly packed structure that the +5 charge state appears to have in the gas phase. The determination of ∆S° from equilibrium constants measured as a function of temperature may be able to resolve this issue because structural water molecules are rotationally constrained. In addition to addressing this issue, in the future we intend to perform ion mobility measurements for hydrated protein ions to determine how many water molecules are required for the unfolded, higher charge states to fold. Acknowledgment. J. Woenckhaus gratefully acknowledges the Alexander von Humboldt Stiftung for a fellowship, and we thank the National Science Foundation (Grant No. CHE9306900) for partial support of this work. References and Notes (1) A value for the free energy of folding of -3497 kJ mol-1 is obtained using the enthalpy (-5261 kJ mol-1) and entropy (-5919 J K-1 mol-1) of folding in Vacuo determined by Makhatadze and Privalov2 from solution values using the accessible surface area model of solvation. A value of -2182 kJ mol-1 for the free energy of folding in Vacuo is obtained using the enthalpy of folding (3946 kJ mol-1) determined by Lazaridis et al.3 by molecular mechanics modeling with the CHARMM force field and the entropy determined by Makhatadze and Privalov.2 (2) Makhatadze, G. I.; Privalov, P. L. AdV. Protein Chem. 1995, 47, 307. (3) Lazardis, T.; Archontis, G.; Karplus, M. AdV. Protein Chem. 1995, 47, 231. (4) For reviews see the following. Kuntz, I. D.; Kauzmann, W. AdV. Protein Chem. 1974, 28, 239. Rupley, J. A.; Careri, G. AdV. Protein Chem. 1991, 41, 37. (5) Monagham, J. J.; Barber, M.; Bordolim, R.; Sedgewick, E.; Taylor, A. Org. Mass Spectrom. 1982, 17, 596. Whitehouse, C. M.; Dreyer, R. N.; Yamashuta, M.; Fenn, J. B. Anal. Chem. 1985, 57, 675. Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (6) Winger, B. E.; Light-Wahl, K. J.; Rockwood, A. L.; Smith, R. D. J. Am. Chem. Soc. 1992, 114, 5897. (7) Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 90, 790. Wood, T. D.; Chorush, R. A.; Wampler, F. M.; Little, D. P.; O’Connor, P. B.; McLafferty, F. W. Proc. Natl. Acad. Sci U.S.A. 1995, 92, 2451.
Letters (8) Gross, D. S.; Schnier, P. D.; Rodriguez-Cruz, S. E.; Fagerquist, C. K.; Williams, E. R. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3143. Williams, E. R. J. Mass Spectrom. 1996, 31, 831. (9) Covey, T. R.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1993, 4, 616. Collings, B. A.; Douglas, D. J. J. Am. Chem. Soc. 1996, 118, 4488. (10) Cox, K. A.; Julian, R. K.; Cooks, R. G.; Kaiser, R. E. J. Am. Soc. Mass Spectrom. 1994, 5, 127. (11) Quist, A. P.; Ahlbom, J.; Reiman, C. T.; Sundquist, B. U. R. Nucl. Instrum. Methods Phys. Res. 1994, 88, 164. Sullivan, P. A.; Axelsson, J.; Altman, S.; Quist, A. P.; Sundquist, B. U. R.; Reinmann, C. T. J. Am. Soc. Mass Spectrom. 1996, 7, 329. (12) Wyttenbach, T.; von Helden, G.; Bowers, M. T. J. Am. Chem. Soc. 1996, 118, 8355. (13) Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141. (14) Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1996, 118, 10313. (15) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Chem. Soc., in press. (16) Shvartsburg, A. A.; Jarrold, M. F. Chem. Phys. Lett. 1996, 261, 86. (17) Drift times estimated using the exact hard sphere scattering model employed here are significantly longer than those estimated using the simple projection approximation used in previous ion mobility studies (see for
J. Phys. Chem. B, Vol. 101, No. 6, 1997 851 example, refs 12-14). The projection approximation ignores the details of the scattering between the ion and buffer gas atom and underestimates the collision integral. (18) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214, 585. The coordinates were obtained from the Protein Data Bank: http: //www.pdb.bnl.gov. (19) van Gunsteren, W. F.; Karplus, M. Biochemistry 1982, 21, 2259. (20) Levitt, M.; Sharon, R. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7557. (21) Jarrold, M. F.; Honea, E. C. J. Am. Chem. Soc. 1992, 114, 459. (22) Handbook of Chemistry and Physics; Weast, R. C., Ed.; CRC Press: Boca Raton, FL, 1985. (23) Lau, Y. K.; Ikuta, S.; Kebarle, P. J. Am. Chem. Soc. 1982, 104, 1462. (24) Klassen, J. S.; Blades, A. T.; Kebarle, P. J. Phys. Chem. 1995, 99, 15509. (25) For the addition of the first six water molecules to H3O+ the values for ∆S determined by Lau et al are in the range 91-109 J K-1 mol-1. Most of this entropy change is due to the loss of translational degrees of freedom. An average value of 104 J K-1 mol-1 was used to estimate the change in the free energy for GlyH+ on going from 293 to 271 K. The change is only 2.2 kJ mol-1, and so it is not very significant. (26) Qi, P. X.; Urbauer, J. L.; Fuentes, E. J.; Leopold, M. F.; Wand, A. J. Struct. Biol. 1994, 1, 378.
