Anal. Chem. 1994,66, 706-711
Conformation of Cytochrome c Studied by Deuterium Exchange-Electrospray Ionization Mass Spectrometry David S. Wagner and Robert J. Anderegg' Glaxo Research Institute, Five Moore Drive, Research Triangle Park, North Carolina 27709
Electrospray is a soft ionization technique that produces multiply charged gas-phase ions from a protein s o l ~ t i o n . ~ - ~ ~ The distribution of observed charge states is believed to be related to the number, accessibility, and proximity of ionizable groups on the protein's surface. Therefore, it has been implied that the conformation of a protein in solution can be probed by observing the extent of protonation in the ESI mass ~ p e c t r u m . ~ , ~ JDenaturing solution conditions (increasing the pH, temperature, or percent organic solvent) have a dramatic effect on the observed ESI mass spectrum, causing a shift to higher charge states and consequently to lower m / z . This shift is rationalized in conformation terms by predicting that, in the tightly folded native state, ionizable groups may be close together or buried and therefore inaccessible, leading to fewer charges in the ESI mass spectrum (i.e., a distribution Proteins are among the most abundant and most important of charge states at higher m / z ) . As the protein denatures, of biopolymers. The folding of a linear string of amino acid buried ionizable groups become accessible and can move residues into a functioning enzyme, receptor, or structural further away from each other, leading to a greater degree of protein is an exquisitely tuned but incompletely understood charging and a shift to lower m/z. However, most of the process. Subtle changes in conformation are frequently used evidencein support of this conformation/spectrum relationship in nature to modulate or regulate the function of enzymes and is circumstantial and does not take into account that altered receptors.'T2 Experimental evidence for a protein's folded solution conditions (pH, surface tension, rate of desolvation, conformation generally comes from circular d i c h r ~ i s m , ~ , ~ concentration) and instrumental variables (gas flows, voltage NMR,2,3X-ray cry~tallography,23~ and more recently, liquid settings) can also affect the charge-state d i s t r i b ~ t i o n . ~ , ~ J ~ J ~ chromatography fast atom bombardment4 and electrospray One recent study concluded that the field polarity was more important in determining the charge state of ions than was ionization (ESI) mass ~pectrometry.~,~ the solution charge of the functional groups.I5 Circular dichroism (CD) spectroscopy measures the difDeuterium exchange studies have been used in combination ference in absorption of left and right circularly polarized with NMR to characterize the fluctuations between different light. The technique is sensitive to the secondary structure protein conformations, to monitor the changes in the protein's of proteins and may be used to determine the relative conformation due to ligand binding, or to monitor the proportions of a-helicity, @-sheet,and random CD is denaturation transition. Recently, deuterium exchange of performed in solution, thus allowing proteins to be analyzed amide hydrogens has been observed with electrospray ionin their aqueous environment. This permits one to monitor ization mass ~pectrometry.~,'~ These studies show that, in the changes in the conformation of proteins resulting from addition to the observed shift in the charge-state distribution, changes in temperature, pH, salt, solvent composition, ligand the measured rate of deuterium exchangevaries when analyzed binding, and quaternary structure. Problems often arise when under denaturing conditions, faster exchange being indicative the relative proportions of the three structure types are being of a more open structure, slower exchange associated with a determined, possibly because of spectral contributions from (9) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. K.; Whitehouse, C. M. Mass the side chains; however, CD continues to be one of the most Specrrom. Reu. 1990, 9, 377. (10) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. RapidCommun. Mass sensitive probes for protein backbone conformation.
Deuterium exchange of bovine cytochromechas been monitored by electrospray ionization mass spectrometry. Different charge-state distributions in the mass spectrum appear to represent different protein conformations, but rapid interconversion of the conformations can lead to a coincidence of the deuterium exchange rates. When interconversionis blocked, the conformation correspondingto higher m / z (lower charge) exchanges more slowly, indicating a tightly folded state. Furthermore, the data suggest that at least two conformations can have identical charge-state distributions,but have different exchange rates. Thus, neither charge-state distribution nor deuteriumexchange rate alone is a sufiicientindicator of protein conformation.
Spectrom. 1988, 2 , 243.
(11)Chowdhury,S.K.;Katta,V.;Chait,B.T.J.Am.Chem.Soc.1990,112,9012. ( I ) Schulz, G. E.; Schirmer, R. H. Principles of Protein Structure; SpringerVerlag: New York, 1978. ( 2 ) Creighton,T. E. ProfeinStrucfuresandMolecularProperties; W. H. Freeman and Co.: New York, 1984. (3) Creighton, T. E. Biochem. J . 1990, 270, 1. (4) Zhang, A.; Smith, D . L.Protein Sci. 1993, 2, 522. (5) Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1991, 5 , 214. (6) LeBlanc, J. C. Y.; Beuchemin, D.; Siu, K. W. M.: Guevremont, R.; Berman, S . S. Org. Mass Spectrom. 1991, 26, 831. (7) Johnson, W. C. Annu. Rev. Biophys. Chem. 1988, 17, 145. (8) Chen, Y. H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350.
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(12) Guevremont, R.; Siu, K. W. M.; Le Blanc, J. C. Y.; Berman, S . S. J. Am. SOC. Mass Spectrom. 1992, 3, 216. (13) Loo, J. A.; Loo,R. R. 0.;Udseth, H. R.;Edmonds,C.G.;Smith, R. D.Rapid Commun. Mass Spectrom. 1991, 5, 101. (14) Ding, J. M.; Anderegg, R. J.; Kassel, D. B. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics; Washington DC, 1992: p 1671. (15) Kelly, M.; Vestling, M.; Fenselau, C.; Smith, P. B. Org. Mass Spectrom. 1992, 27, 1143. (16) Stevenson,C. L.;Anderegg,R. J.; Borchardt, R. T. J.Am. Soc. MassSpecrrom., in press.
0003-2700/94/0366-0706$04.50/0
0 1994 American Chemlcai Society
tightly folded structure. The presence of H-bonding is the major factor of slow exchange because preexisting H-bonds must be broken for exchange to occur. However, the rate of deuterium exchange of model compounds is known to be affected by the chemical environment, as well as the threedimensional s t r u ~ t u r e . ~ , ~The + ' ~exchange -~~ of protons between the solvent and the protein is catalyzed by both OHand H+ ions. The pH profile of the exchange rate for amide protons is U-shaped, having a minimum at pH 3 where the acid- and base-catalyzed processes are equal in rate.3 Above pH 3 the deuterium exchange rate is governed by the activity of the OH- ion, and below pH 3 the exchange is dominated by H+ ion activity.2' Thus the pH of the protein solution has a major influence on the exchange process. Each unit change in pH produces a IO-fold increase in the exchange rate. The deuterium exchange rate is also sensitiveto temperature and addition of organic solvents. Both alter the water dissociation constant, K,, which in turn affects the activity of OH- and H+ ions.21 In addition, the exchange rate of an amide is affected by the local chemical environment, by inductive and charge effects on the amide.22 The same principles govern the exchange rates of the polar side chains. Most freely exposed side chains exchange on a fast time scale relative to backbone amides, except for Arg and Lys at low pH and Asn and Gln at high pH, where their rates are similar to those of peptide amide^.^ We undertook this study to see whether direct evidence for multiple solution conformations could be obtained using deuterium exchange followed by ESI-MS. Because the mass spectrometric experimentsperformed to date monitor exchange rates for the native and denatured states of proteins in different solution environments, it is not always clear whether the variation is due to the protein's conformational change, to the change in the chemical environment, or to a combination of both. In order to accurately compare the exchange rates for two different conformations, both the native and denatured states must be analyzed in the same chemical environment. We present data for bovine cytochrome c in which the deuterium exchange rates of different charge-state distributions are measured. If interconversionbetween conformational states is blocked, two distinct rates of exchange are observed for different charge-state envelopes, thus indicating the presence of a native and a denatured species in the same chemical environment. The lower charge state (higher m/z) experiences slow exchange, indicative of a tightly folded state, while the higher charge state (lower m / z ) experiences a faster rate of exchange, indicating a denatured protein. Furthermore the data demonstrate that using the charge-state distribution of the ESI mass spectrum of a protein or the spectrum obtained by CD is not sufficient for determining a protein's solution conformation. However, deuterium exchange information complements the ESI and/or the CD data and permits the conformation of the protein to be more precisely understood. (17) Englander, J. J.; Rogero, J. R.; Englander, S. W. Anal. Biochem. 1985,147. 234. (18) Englander, S. W.; Poulsen, A. Biopolymers 1969, 7 , 379. (19) Englander, S. W.; Mayne, L. Annu. Rev. Biophys. Biomol. Struct. 1992,2J, 243. (20) Roder, H.; Wagner, G.; Wuthrich, K. Biochemistry, 1985, 24, 7407. (21) Englander, S. W.; Kallenbach, N. R. Q.Reu. Biophys. 1984, 521. (22) Molday, R. S.; Englander, W. S.; Kallen, R. G. Biochemistry 1972, J J , 150.
EXPERIMENTAL SECTION Materials. Bovine cytochromec was purchased from Sigma Chemical Co. (St. Louis MO). The purity was checked by HPLC, and a single peak was observed, so the protein was used without further purification. The deuterated solvents deuterium oxide, methanol, and acetic acid used in this study (D2O 99.9%, MeOD 99.9%, and acetic acid44 99.8%) were obtained from Cambridge Isotopes (Woburn, MA). A 100 pM stock solution of bovine cytochrome c was prepared by dissolvingthe protein in H2O. The mass spectrometry samples were prepared by lyophilizing 500-pmol aliquots from the stock solution to dryness in an Eppendorf tube. This ensured the deuterium exchange experiments did not have competition from residual solvent protons. The deuterium exchange experiments proceeded by dissolving the dried sample in 100 r L of the appropriate deuterated solvent, to give a final concentration of 5 r M , and then introducing the sample into the mass spectrometer as quickly as possible (10-20 s). Electospray Ionization Mass Spectrometry. Electrospray ionization mass spectra were collected using a Sciex API-I11 mass spectrometer (Sciex, Thornhill, ON, Canada) in the positive ion mode. A Harvard Model 22 syringe pump (Harvard Apparatus, South Natick, MA) was used to infuse the samples into the instrument at a rate of 2 pL/min. The ion spray needle was maintained at 5300 V, and the orifice potential was 80 V. The degree of deuterium incorporation was deduced from the protein's mass shift during the course of an experiment. The ionization chamber was enclosed and continuallyflushed with nitrogen to prevent the fast exchanging deuterium atoms from reexchanging with hydrogens in the laboratory's air. A small mass range (-30 Da) encompassing an ion of a selected charge state was scanned continuously with a 0.1-Da step size and a 10-ms dwell time, leading to a total scan time of 3 s. The small mass range and fast scan times were used during these exchange experiments to ensure high quality and accurate data, particularly for early time points when the mass was changing rapidly. In order to monitor all charge states in a single experiment, a larger mass range (800-1800 Da) was employed and used a step size of 0.3 Da at a dwell time of 1 ms, leading to a total scan time of 3.3 s. Thecalculated molecular weight and hence thedegree of deuterium incorporation for each time point could then be obtained for individual charge states. The pH measurements were taken with a standard glass electrode standardized with aqueous solutions. Circular Dichroic Spectroscopy. CD spectra were obtained with an Aviv Model 62DS CD spectrophotometer using quartz cells with a 1-mm path length. The measured ellipticity data were corrected by a blank, averaged, and subjected to a fivepoint smoothing algorithm. CD spectra are presented as a plot of mean molar ellipticity [e] (in deg cm2/dmol) versus wavelength in 0.5-nm steps. Protein concentration was determined by quantitative amino acid analysis of a stock solution. RESULTS AND DISCUSSION Cytochrome c contains 104 amino acid residues with a covalently attached heme group and mainly consists of a-helices arranged in a spherical shape.2 Circular dichroism spectra of cytochrome c at pH 6, 2.7, and 2.5 (Figure 1) Ana!ytical Cbrnistry, Vol. 66, No. 5, March 1, lQQ4
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Flgure 1. Clrcular dichroic spectra of bovine cytochrome c in H20 (30 pM) with 0% acetic acM, pH 6.0 (O),with 2 % acetic acid, pH 2.7 (0), and with 4 % acetic acM, pH 2.5 (0)and the spectrum of bovine cytochrome c after heating at 90 OC for 3 h In H20, lyophilizing to dryness, and then dissolving In H20 with 0% acetic acid, pH 6.0 (A).
500 750
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m/z
display overlapping curves at pH 6 and 2.7 and a distinctly different spectrum at pH 2.5. At pH 6 and 2.3 the CD spectra show a double minima at 208 and 222 nm, indicating a significant proportion of the residues are in the a-helical conformation. The CD spectrum at pH 2.5 displays a significant decrease in the signal strength of the 222-nm minimum and a disappearance of the minimum at 208 nm, indicating a significant loss of a-helicity. The CD data suggest that the protein has the same conformation at both pH 6 and 2.7 and that a marked conformational change occurs when the pH is lowered to 2.5. Altering the acidity of a solution containing bovine cytochrome c also has a dramatic effect on the charge-state distribution observed in the corresponding ESI mass spectrum (Figure 2). A sample of bovine cytochrome c (average MW = 12 23 1) dissolvedin H2O containing no acid (pH 6.0) mainly produces an abundant charge-statedistribution (CSD) ranging between +7 ( m / z 1748.3) and +11 ( m / z 1112.9), with a less abundantCSDranging between+12(1020.2) and+l6 (765.4) (Figure 2a). It is useful to describe these distributions using the parameters of a Gaussian curve ( p , a) obtained by curve fitting a plot of ion abundance versus charge.23 The center of the Gaussian curve can be used to monitor shifts in chargestate distribution caused by changes in the instrumental and solution parameters. For Figure 2a, a peak center of +9.2 is determined. In a 2% acetic acid solution (pH 2.7) the ESI mass spectrum gives rise to two distinct CSDs (a “bimodal distribution”), one ranging from +7 to +10 charge states and the second ranging between +11 (m/z 1112.9) and + 18 ( m / z 680.5) (Figure 2b). Curve fitting the CSDs gives two Gaussian functions with p = +8.7 and p = +15.4. The center of the high m / z (lower charge state) distribution shifted slightly to a lower value due to the increase in the +8 ion abundance. Increasing the acid content to 4% (pH 2.5) leads to the dominance of the higher CSD (Figure 2c). The Gaussian curve fits for the two distributions indicate that the higher (23) Anderegg, R. J.; King, G . Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics: Washington DC, 1992, p 623.
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Ana&ticalChemistry, Vole66, No. 5, March 1, 1994
al
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+a 500 750
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mlz Flgurr 2. ESI mass spectra of bovine cytochrome c In H20 (a) wlth 0% acetic acM, pH 6.0, (b) wlth 2 % acetic acid, pH 2.7, showlng the “bimodal dlstribution”, and (c) with 4% acetic acid, pH 2.5.
CSD (lower m / z ) shifted to p = +16.0 and the lower CSD shifted t o p = +9.0. These data show that, in addition to the change in relative abundance of the ions observed, the center of the CSDs varies as the solution pH changes, consistent with recent reports.13 Similar to pH effects on the ESI mass spectrum, increasing the percent organic~olvent’~ or temperaturez4of a cytochrome c sample also shifts the CSD from a lower number of charges (high m / z ) to a higher number of charges (low m/z). The changes in the appearance of the ESI mass spectrum have been attributed to changes in the protein conformation, with the tightly folded native states appearing as an ion distribution at high m / z (low charge) and a loose denatured state represented by the distribution at lower m/z (high charge). However, many processes can contribute to the shape of the ion series profile, including protein concentration, aqueous solution chemistry, desorption, desolvating, and declustering processes, ionization efficiencies, and ion transmission into the vacuum. If the bimodal distribution observed in the ESI mass spectrum represents two different conformationsof cytochrome c, then the two conformations might be expected to exchange (24) Mirza, U. A.; Cohen, S. L.; Chait, B. T. Anal. Chem. 1993,65, 1.
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at different rates upon exposure of the protein to deuterated solvents. The lower charge-state distribution, representing a tightly folded protein, would be expected to exchange more slowly than a more denatured protein, represented by the high CSD. In order to determine whether the two CSD represent different conformations of the same protein, deuterium exchange experiments were performed on the different chargestate envelopes in 2%acetic acid, conditions shown in Figure 2 to produce a bimodal distribution. Bovine cytochrome c contains 190 labile hydrogens and 2 exchangeable hydrogens on the heme group. If all 192 exchangeable hydrogens were replaced with deuterons, the average molecular weight would increase from 12 23 1 to 12 423. An initial analysis was performed on a wide mass range (700-1800 Da) to ensure the bimodal distribution was observed utilizing deuterated solvents. After the bimodal distribution was confirmed, we chose to monitor the change in m / z as a function of time for the +8 and +15 charge states, representing the two charge-state envelopes. Individual experiments were performed by analyzing each charge state separately over a 30-Da range. The change in m / z as a function of time for the two charge states was tabulated and converted to the protein's molecular mass. The rate of deuterium incorporation can be depicted graphically by plotting the molecular mass as a function of time. Figure 3 represents such a plot for the +8 and +15 charge states of cytochrome c at pH 2.7. Surprisingly, the plots for the two charge states overlap one another, thus indicating that both CSDs have the same rates of deuterium exchange. Identical experiments were performed on the +8 and 15 ions at different solution pH's and with the addition of organic solvent. Altering the pH and/or organic solvent concentration of the solution changed the observed exchange rate; however, the two CSDs always exhibited overlapping exchange profiles. Similar results were observed for studies performed on ubiquitin (data not shown). The unexpected coincidence of the exchange rates for the different CSDs can be interpreted in two ways. One might conclude that the two CSDs do nor represent different protein conformations, but rather result from some instrumental condition. Such an interpretation, however, would contradict
+
Flguro 4. ESI mass spectrum of bovine cytochrome c atter heating at 90 O C for 3 h in H20, lyophlllrlng to dryness, and then dlssolvlng In H20 wlth 0% acetic acld, pH 6.0.
the large body of evidencethat suggests a relationship between CSD and conformation. An alternative explanation is that there are, in fact, two conformations, but that they are interconverting rapidly. During the time a protein is in the Ydenatured"state, it would exchange rapidly. While folded, it would exchange more slowly. However, if interconversion were fast, all protein molecules would spend a significant proportion of their time in an unfolded state, exchanging at the faster rate. In the ESI-MS experiment, all protein molecules, regardless of their conformation at the instant of ion formation, would appear to exchange at the faster rate. In order to confirm this hypothesis, we blocked the interconversion between the open and closed forms of the protein by thermal denaturation. Samples of cytochrome c in H2O were partially denatured in an irreversible manner by heating at 95 O C for 3 h.24 One of the samples was lyophilized to dryness and then dissolved in H2O containing no acid, conditions shown for the native protein to produce a single Gaussian distribution with 1.1 = +9.2. The corresponding mass spectrum exhibited a bimodal distribution, with the low and high CSDs having 1.1 = 8.7 and 1.1 = 14.9, respectively (Figure 4). The high CSD presumably represents a denatured state that cannot refold on the time scale of our experiment. A conformational change is also indicated by the CD spectrum of the heated sample (Figure 1) at pH 6.0. The spectrum shows a double minima for 208 and 222 nm, denoting a-helicity. However, the signal strengths are significantly weaker than in thespectrumof the unheated sample, indicating that the protein's conformation has been altered. In order to determine whether the two CSDs represent different conformations, deuterium exchange experiments similar to those described above were performed on heatdenatured samples. Here we scanned between the +8 and +15 charge states in order to monitor all charge states simultaneously. Lyophilized samples of the heat-denatured protein were dissolved in D20 only, and the change in protein's molecular weight as a function of time was ascertained by monitoring the change in m / z for the different charge states. The molecular mass versus time for the +8 and + 15 charge states clearly shows two distinct rates of deuterium exchange for the two CSDs (Figure 5). The lower charge-state ions represent a tightly folded structure as evidenced by the slow rate of exchange, while the higher charge-state ions undergo rapid exchange, indicating a more denatured structure. This AnaEytcal Chemistry, Vol. 86, No. 5, March 1, 1994
709
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givesunequivocalevidencethat two different CSDs correspond to two different conformations in solution. Data obtained for charge states ranging from +7 to 15 of the partially denatured protein indicate a clear distinction between the two conformations, shown in Figure 5 . The +8, +9, and +10 chargestate ions display exchange at the slower rate, presumed to be associated with the folded protein. The 1 1 to + 15 states represent the denatured protein exchanging at the faster rate. Somewhat surprisingly, there was no overlap observed between the charge-state distributions. The +10 and +11 charged ions fell consistently in the slower and faster exchanging populations, respectively, regardless of changes in pH and addition of organic solvent. Local unfolding or breathing is generally used to explain the exchange of interior hydrogens (protons that are buried in the protein or involved in intramolecular bonding). In order
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kex
deuterated protein
for the folded state to be stable, the rate of unfolding (kl) must be much less than the rate of refolding (k-1). A number of open states must exist to explain the broad range exchange rates at different sites on the protein. There are two possible rate-limiting steps in the exchange of interior hydrogens: (1) opening or unfolding of the protein, the so-called EX1 mechanism or (2) the exchange reaction, the EX2 mechan i ~ m . ~ The ~ ' ~ EX1 ~ ' ~ mechanism requires the deuterium exchange rate to be faster than the rate of refolding (kex > k-1). In this case themeasured exchangerate yields kl directly, the rate of opening the protein. The EX2 mechanism requires the exchange rate to be slower than the rate of refolding (kex < k-1). Here the measured exchange rate corresponds to only the putative equilibrium constant [(kl/k-l)k,,] for the breathing process. Under most conditions proteins exhibit the EX2 types of exchange, except at high pH and high
temperature^.^,*^-^^ A comparison of the exchange behavior for the native and thermally denatured samples of cytochrome c reveals that the high and low CSDs of native cytochrome c and the low CSD (25) Roder, H.;Wagner, G.; Wuthrich, K.Biochemistry 1985, 24, 7396.
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Flgure 6. Comparison of the change in molecular mass as a function of time, computed from observing the change in m/z of the 4-15 and +8 charge states of a unheatedsample and a heatdenaturedsample of cytochrome c dissolved In D20 with 0% acetlc acid, pH 6.0.
of the heat-denatured protein undergo exchange at similar rates, as evidenced by the similarity of their exchange profiles at pH 6.0 (Figure 6). However, the high CSD of the heatdenatured protein indicates a markedly faster rate of exchange. Because all of the exchanges were conducted under identical solution conditions, the intrinsic exchange rate for cytochrome would be the same in all cases and can be no slower than the rate for the high CSD of the thermally denatured sample. The relative slowness of the exchange of conformations from both CSDs in the unheated sample must result from protection of some exchangeable hydrogens, presumably by hydrogen bonds resulting from stable secondary structure. As mentioned earlier, the native and denatured states of the unheated protein are interconverting on a fast time scale; thus only one rate is observed. In the heat-treated sample, the denatured conformation is more fully unfolded, so the observed exchange rate is closer to the intrinsic exchange rate. The two conformations represented by the two CSDs in the heated sample are not interconverting, at least not on the time scale of our experiments, as evidenced by the two distinct, measurable exchange rates. The structural basis for this irreversible denaturation is uncertain, but probably involves the cis to trans isomerization of the four proline residues. The influence of pH on the exchange rate (i.e., the structure of the protein) of both the unheated and heat-treated proteins is depicted in Figure 7. The data indicate that the exchange rate for the high CSD of the thermally treated sample does not vary between 2.5 and 6. This is characteristic of an EX1 mechanism, where the rate is not susceptible to factors known to affect the intrinsic exchange reaction. This response is expected because the protein is locked into an open form, thus, k-1
