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Defining Protein Ensembles with Native-state NH Exchange: Kinetics of Interconversion and Cooperative Units from Combined NMR and MS Analysis
Article No. jmbi.1998.2338 available online at http://www.idealibrary.com on
J. Mol. Biol. (1999) 285, 1265±1275
Defining Protein Ensembles with Native-state NH Exchange: Kinetics of Interconversion and Cooperative Units from Combined NMR and MS Analysis Cammon B. Arrington1,2, Lynn M. Teesch3 and Andrew D. Robertson1* 1
Department of Biochemistry
2
Medical Scientist Training Program and 3The Highresolution Mass Spectrometry Facility University of Iowa, Iowa City IA 52242, USA
Previous studies of native-state peptide hydrogen atom (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen atoms (NHs). Unfolding rate constants and free energies for nine of the NHs are very similar, suggesting that these NHs exchange during a single cooperative unfolding event. Electrospray ionization mass spectrometry (ESI-MS) has been used to test this hypothesis. ESI-MS data and MS peak simulations suggest that this hypothesis is incorrect: in spite of the similarity in their unfolding rate constants, only three to ®ve of the nine residues exchange in a cooperative manner. Thus, residues with similar thermodynamics and kinetics of exchange are probably involved in multiple conformational equilibria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins. # 1999 Academic Press
*Corresponding author
Keywords: ensembles; protein unfolding; hydrogen atom exchange; electrospray mass spectrometry; ovomucoid third domain
Introduction Recent results from experimental and computational studies suggest the possibility of multiple unfolding pathways for native proteins (Zaidi et al., 1997; Bhuyan & Udgaonkar, 1998; Li & Daggett, 1996; Lazaridis & Karplus, 1997). This view of the kinetics of unfolding is consistent with the emerging picture of proteins as conformational ensembles (Brandts, 1969; Dill & Chan, 1997). Native-state peptide hydrogen atom (NH) exchange is proving to be one of the most informative experimental probes of equilibrium ensemble properties (Hilser & Freire, 1996; Bahar et al., 1998). Recent NMR studies suggest that kinetic ensemble properties can also be examined by native-state NH exchange (Arrington & Robertson, 1997). Here, combined NMR and mass spectral (MS) analysis of Abbreviations used: NH(s), peptide hydrogen atom(s); OMTKY3, turkey ovomucoid third domain; ESI-MS, electrospray ionization mass spectrometry; MS, mass spectral; amu, atomic mass unit. E-mail address of the corresponding author: [email protected] 0022-2836/99/031265±11 $30.00/0
native-state NH exchange demonstrates that the most slowly exchanging NHs in turkey ovomucoid third domain (OMTKY3) report on multiple conformational equilibria. Previous NMR studies of NH exchange were used to determine the thermodynamics (SwintKruse & Robertson, 1996) and kinetics (Arrington & Robertson, 1997) of unfolding at multiple sites throughout native OMTKY3. For the 14 most slowly exchanging NHs, exchange-derived free energies, GHX, were similar to the overall free energy of unfolding, Gu, suggesting that global unfolding precedes exchange at these sites. For ten of these NHs, unfolding rate constants were also similar and overlapped at two standard deviations, with a mean rate constant for unfolding of 0.008 (0.004) secondÿ1 at 30 C. The simplest hypothesis to explain these results is that exchange at these ten NHs results from the cooperative unfolding of OMTKY3. The present study uses electrospray ionization mass spectrometry (ESI-MS) to address this hypothesis. The rationale for using ESI-MS to assess the cooperativity of NH exchange at multiple NHs is based on the well-established kinetic mechanism # 1999 Academic Press
1266
Ensembles from NH Exchange
for slow NH exchange in native proteins (Hvidt, 1964): kop
kch
NH closed NH open * exchanged kcl
1
Each slowly exchanging NH participates in an equilibrium between a closed conformation, NH (closed), from which exchange cannot occur, and an open solvent-exposed state, NH(open). The rate constants for interconversion between closed and open conformations are kcl and kop, respectively. From the open state, NH exchange can occur at the intrinsic chemical rate constant, kch, known from model compound studies (Bai et al., 1993). The exchange reaction is effectively irreversible in the presence of a vast excess of 2H2O. The resulting substitution of peptide hydrogen atoms for deuterium can be followed by ESI-MS. Under conditions favoring the native state, i.e. kcl4kop, the following relation describes the observed rate constant of exchange, kobs, for an individual NH: kobs kop kch = kcl kch
2
Depending on the relative magnitudes of kcl and kch, two limiting cases can be derived from equation (2) (Hvidt & Nielsen, 1966). When kch4kcl, equation (2) simpli®es to: kobs kop
3
This rate-limiting case is referred to as EX1 exchange. In this case exchange occurs with every opening event. In the case of cooperative unfolding, exchange occurs at different NHs simultaneously. Cooperativity of exchange at multiple NHs can be determined by ESI-MS (Miranker et al., 1993; Yi & Baker, 1996; Chung et al., 1997). Under EX1 conditions, cooperative unfolding and subsequent correlated exchange is detected as a bimodal mass spectrum: the lower mass peak represents a protonated molecular population that has not yet opened to exchange, while the higher mass peak represents deuterated molecules that have undergone the exchange reaction. The mass separation of the two peaks indicates the number of cooperatively exchanging NHs. If exchange is purely cooperative, the mass of each peak remains constant with time while the lower and higher mass peaks decrease and increase in intensity, respectively. The second limiting case, known as EX2 exchange, occurs when kcl4kch. In this case, equation (2) simpli®es as follows: kobs kch kop =kcl
4
This rate-limiting case is characterized by numerous visits to the open state prior to exchange. Under EX2 conditions, exchange happens in a random or uncorrelated manner at different NHs, regardless of whether cooperative unfolding occurs. Resulting mass spectra contain a single
peak that shifts to higher mass as exchange proceeds. The same spectral pattern will also exist under EX1 conditions when molecular ¯uctuations are uncorrelated or non-cooperative. Native-state NH exchange in OMTKY3 has been monitored by ESI-MS under the same solution conditions as used in the NMR studies, where G0u is about 7 kcal/mol. To discern the molecular cooperativity of exchange, the experiments have been conducted under EX1 conditions, 30 C and pH > 9.5 (Arrington & Robertson, 1997). Experimental results have been interpreted using a new program for the simulation of MS peak shapes resulting from NH exchange.
Results and Discussion Residue-specific unfolding rate constants in native OMTKY3 NH exchange has been monitored at the 14 slowest-exchanging NHs in OMTKY3 by NMR spectroscopy. In the context of this study, the rationale for the NMR measurements is twofold: (1) to obtain residue-speci®c values of kobs for interpretation of the MS exchange data and (2) to test the extent to which MS exchange samples were prepared under EX1 conditions. The pH dependence of kobs for 13 of the 14 slowly exchanging NHs has been reported previously (Arrington & Robertson, 1997). For nine of these NHs, values of kobs were determined above pH 9.5 and a switch from EX2 to EX1 exchange was detectable. The remaining ®ve NHs (Cys24, Ser26, Asn28, Asn33, and Ser51) exchanged too rapidly to measure above pH 9.5 and a switch from EX2 to EX1 exchange was not detected. To obtain estimates of kobs for these residues at high pH, a quench protocol has been employed to measure more rapid exchange. Updated plots of log(kobs) versus pH are shown in Figure 1 for 12 of the slowly exchanging NHs (data not shown for Ser26 and Asn33). Values of kobs obtained from the quench experiment are displayed as open circles. The data for Ser26 are similar to those for Cys24, whereas NH exchange for Asn33 is still too rapid to detect using the quench protocol; lower limits of kobs have been estimated for Asn33 at high pH. Fits for nine of the NHs in Figure 1 are very similar to those shown previously (Arrington & Robertson, 1997). With the additional data point at high pH, more accurate simulations of log(kobs) versus pH have been performed for Cys24, Ser26, Asn28, Asn33 and Ser51. From the ®tted or simulated curves, residuespeci®c values of kop and kcl as well as exchangederived free energies, GHX, have been estimated (Table 1). In the absence of the high pH data point, Asn28 was previously grouped with the nine NHs with relatively low kop values. Present results clearly demonstrate that Asn28 belongs to the more rapidly exchanging group.
1267
Ensembles from NH Exchange
ent with a switch from EX2 to EX1 exchange, where the plateau value of kobs asymptotically approaches kop. For the majority of residues, the results in Figure 1 indicate that EX1 exchange predominates above pH 9.5. In Figure 2, residue-speci®c values of kop (Table 1) are mapped onto the three-dimensional structure of OMTKY3. The values of kop are derived from the plateaux described by the data in Figure 1. The nine NHs hypothesized to exchange during a single cooperative unfolding event are colored blue-green, with kop 0.008 secondÿ1: Leu23, Gly25, Lys29, Tyr31, Phe37, Cys38, Asn39, Ala40, and Val41. Figure 1. The pH dependence of kobs for 12 of the 14 NHs monitored in this study. The data point at pH 9.65 (open circle) is derived from the manual mixing experiment described in this study while all other data points are from previous work (Arrington & Robertson, 1997). The observed rate constants for all residues except Cys24, Ser26 (not shown), Asn28, Asn33 (not shown) and Ser51 were subjected to non-linear least-squares analysis using equation (2) (Johnson & Faunt, 1992). Plateaux in the data for Cys24, Ser26, Asn28, Asn33 and Ser51 were insuf®ciently de®ned for ®tting, so simulations of equation (2) were performed to obtain lower limits for kop and kcl. Continuous and broken lines represent the ®tted or simulated curves for each NH. The exchange data for Ser26 are similar to those for Cys24, while the data for Asn33 show little deviation from pure EX2 exchange.
Plots of log(kobs) versus pH can be used to distinguish between EX2 and EX1 exchange. For example, the exchange behavior of Cys24 is characteristic of EX2 exchange wherein log(kobs) increases linearly with a slope of 1. However, systematic deviation from a unit slope is evident in the data for Cys38 above pH 8.5. This deviation is consist-
ESI-MS test for cooperative exchange ESI-MS has been used to investigate the cooperativity of NH exchange for the most slowly exchanging NHs in native OMTKY3. ESI-MS exchange samples were prepared under EX1 conditions (pH > 9.5). Under these conditions, 87 of the 100 exchangeable protons in OMTKY3 are deuterated in the time required to initiate exchange and quench the ®rst sample (®ve seconds). The remaining 13 slowly exchanging NHs complete the exchange process in approximately 12-25 minutes. At high pH Asn33 exchanges too rapidly to detect in these experiments. The MS results in Figure 3 (thin line) re¯ect the time-course of NH exchange for OMTKY3 at 30 C, pH 9.79. To determine whether cooperative exchange of nine residues can account for these results, simulated spectra are superimposed upon the experimental spectra (Figure 3, upper panels). For the simulations, nine residues were assumed to exchange cooperatively with an average rate constant of 0.008 secondÿ1. The remaining four NHs were assumed to exchange in an uncorrelated manner with residue-speci®c values of kobs ranging
Table 1. Values of kop and kcl for native OMTKY3 at 30 C Residue Leu23 Cys24b Gly25 Ser26b Asn28b Lys29 Tyr31 Asn33b Phe37 Cys38 Asn39 Ala40 Val41 Ser51b
kop 102 (secondÿ1) 0.3 (0.2, 58 0.8 (0.6, 54 55 1.0 (0.7, 0.7 (0.5, 530 1.0 (0.5, 0.6 (0.4, 2.0 (1.0, 0.8 (0.5, 0.6 (0.4, 57
0.6) 1.2) 1.6) 1.2) 2.5) 0.9) 6.2) 2.3) 1.0)
kcl (secondÿ1)
GHXa (kcal/mol)
1.6 (0.5, 4.3)102 52104 2.0 (1.0, 3.7)103 55103 54103 1.6 (0.8, 3.0)103 0.9(0.4, 1.8)103 53104 1.0 (0.3, 3.3)103 4.3 (2.0, 8.3)103 1.6 (0.5, 5.8)104 6.0 (2.2, 20)103 7.5 (3.8, 15)102 55103
6.6 7.4 7.5 7.1 6.8 7.2 7.1 6.9 7.0 8.2 8.2 8.1 7.1 6.7
Values for all residues except Cys24, Ser26, Asn28, Asn33 and Ser51 were determined from non-linear regression analysis using equation (2). Numbers in parentheses denote lower and upper limits corresponding to a 95 % con®dence level. The ®tting error was determined using F analysis (Bevington & Robinson, 1992). a Estimated error for GHX is 0.5 kcal/mol (Swint-Kruse & Robertson, 1996). b Simulations of equation (2) were used to obtain lower limits for Cys24, Ser26, Asn28, Asn33 and Ser51.
1268
Ensembles from NH Exchange
Figure 2. Structural distribution of kop for the most slowly exchanging NHs in native OMTKY3. The magnitude of the rate constants has been color-coded onto balls representing individual NHs. The ribbon diagram was generated in part with MOLSCRIPT (Kraulis, 1991) and PDB ®le 2OVO (Bode et al., 1985).
from 0.03 to 0.05 secondÿ1. Due to the more rapid uncorrelated exchange at four NHs, a four atomic mass unit (amu) shift in the peak maximum occurs early in the simulation. This mass shift is complete by approximately 30 seconds and two peaks, separated by 9 amu, are observed subsequently as a result of cooperative exchange at nine NHs. The comparison of simulated and experimental spectra demonstrates that cooperative unfolding at nine NHs cannot explain the experimental results. To assess whether completely uncorrelated exchange can account for the experimental results, simulated spectra have been generated in which the 13 NHs were assumed to exchange independently of each other (Figure 3, lower panels). Residue-speci®c values of kobs were obtained from NMR experiments and ranged from 0.003 to 0.05
secondÿ1. Although the mass shifts of the simulated and experimental spectra are similar, differences in peak shape are apparent. For example, the ®rst experimental spectrum (®ve seconds) is broadened (mostly at the base) and skewed towards higher mass. In addition, the experimental peak shape at 1.5 minutes is much broader than the simulated peak. These results and the simulations discussed below suggest that a subset of the most slowly exchanging NHs in OMTKY3 may exchange in a cooperative manner. Cooperative protein fluctuations from deconvolution of NH exchange mass spectra In MS studies of NH exchange, two distinct mass distributions contribute to spectral peak
Figure 3. Time dependence of NH exchange at 30 C and pH 9.79 as monitored by mass spectrometry. Time of exchange is denoted in the upper right corner of each panel. The experimental data (®ne lines) in the top three panels are superimposed on simulated spectra generated by assuming that nine NHs exchange cooperatively and four NHs exchange in an uncorrelated manner. In the bottom three panels, data are superimposed on simulations of completely uncorrelated exchange at 13 NHs.
1269
Ensembles from NH Exchange
tations) are summed to generate the overall probability at a given deuteration level or mass outcome. Lastly, the probability at each deuteration level is multiplied by the natural mass distribution of a molecule and the resulting peaks are summed to generate a full mass spectrum. The algorithm will be illustrated in more detail by simulating spectra for a hypothetical molecule with three exchangeable NHs (Figure 4(a)). Using NMR-derived values of kobs, the program calculates the probability of an amide being protonated, PROBH, or deuterated, PROBD:
Figure 4. Diagrammatic representation of the approach used to simulate MS exchange results. (a) Simulated results for a hypothetical molecule with three exchangeable NHs. All possible combinations of protons (H) and deuterons (D) are listed and separated by color into groups with the same number of deuterons. The histogram represents the molecular probability at each deuteration level after one minute of exchange and assuming the values of kobs listed in the text. The probability at each deuteration level is multiplied by the natural mass distribution of OMTKY3 to generate the colored peaks. The continuous black line is the full mass spectrum obtained by summing the colored peaks. (b) Simulated results for a hypothetical molecule with six exchangeable NHs, four of which exchange cooperatively. The number 4 in parentheses represents the group of NHs that exchange cooperatively.
shapes. First, mass spectra of pure proteins exhibit ®nite line widths characteristic of the protein's molecular formula and the presence of naturally occurring isotopes (e.g., 13C, 15N, etc.). Second, the molecular population has a distribution of protons and deuterons as a result of NH exchange. A computer algorithm has been developed to simulate mass spectra by accounting for the two mass distributions. In outline, the program ®rst de®nes all possible molecular species in terms of proton/deuteron occupancy and then calculates the probability of each species using NMR-derived NH exchange rate constants. Next, the probabilities of all species with the same number of deuterons (i.e., indistinguishable proton/deuteron permu-
PROBH i exp ÿkobs i t
5
PROBD i 1-PROBH i
6
where i represents an exchangeable NH and t is the exchange time. Let us assume that, kobs(l) 0.5 minuteÿ1, kobs(2) 1.2 minuteÿ1, and kobs(3) 0.3 minuteÿ1. After one minute of exchange, the site-speci®c probabilities are as follows: PROBH(1) 0.61, PROBD(1) 0.39; PROBH(2) 0.30, PROBD(2) 0.70; PROBH(3) 0.74, PROBD(3) 0.26. The total number of molecular species distinguished by different patterns of protonation and deuteration equals 2N, where N is the number of exchanging NHs. For the sample molecule with three NHs there are eight possible molecular species (Figure 4(a), top). If exchange at the three NHs is uncorrelated, then the probability of each molecular species is the product of the site-speci®c probabilities for the three sites. Probabilities for each molecular species in the present example are as follows: P HHH PROBH 1 PROBH 2 PROBH 3 0:14
7a
P DHH PROBD 1 PROBH 2 PROBH 3 0:09
7b
P HDH PROBH 1 PROBD 2 PROBH 3 0:31
7c
P HHD PROBH 1 PROBH 2 PROBD 3 0:05 P DDH PROBD 1 PROBD 2
7d
PROBH 3 0:20 P DHD PROBD 1 PROBH 2
7e
PROBD 3 0:03
7f
P HDD PROBH 1 PROBD 2 PROBD 3 0:11
7g
P DDD PROBD 1 PROBD 2 PROBD 3 0:07
7h
The probabilities of all molecular species with the same number of deuterons are now summed to generate the overall probability at each deuteration level (Figure 4(a), middle).
1270
Ensembles from NH Exchange
Figure 5. Comparison of experimental mass spectra (pH 9.79, 30 C) with simulations in which four or ®ve NHs exchange cooperatively. The model shown in the top three panels is called 4C/9NC and assumes that a group of four NHs exchange cooperatively and nine NHs exchange non-cooperatively. The model shown in the bottom panels is referred to as 5C/8NC. For both models, kobs for the cooperative NHs was assumed to be 0.008 secondÿ1. The remaining NHs were assumed to exchange noncooperatively with residue-speci®c values of kobs.
M0 P HHH 0:14
8a
M1 P DHH P HDH P HHD 0:45
8b
M2 P DDH P DHD P HDD 0:34 M3 P DDD 0:07
8c 8d
In these expressions M represents mass and the subscripts are the number of deuterons or the deuteration level. A full mass spectrum is obtained by multiplying the probability at each mass (Figure 4(a), middle) by the natural mass distribution of a molecule and then summing the resultant peaks (Figure 4(a), bottom). The algorithm can also account for completely cooperative exchange, de®ned as two or more NHs that always exchange together. For the example illustrated in Figure 4(b), the molecule contains six NHs, four of which exchange cooperatively with a single value of kobs (Figure 4(b), top). Thus, only three values of kobs are required in this example. As before, one minute of exchange will be simulated using the same values of kobs, except that kobs(3) pertains to four cooperative NHs instead of a single NH. With a group of four NHs that is either all protonated or all deuterated, the mass distribution (Figure 4(b), middle) is as follows: M0 P HHH4 0:14
9a
M1 P DHH4 P HDH4 0:40 M2 P DDH4 0:20
9b 9c
M3 0 M4 P HHD4 0:05
9d 9e
M5 P DHD4 P HDD4 0:14 M6 P DDD4 0:07
9f 9g
where the subscript in the probability expressions represents the four cooperative NHs. The cooperativity leads to a bimodal histogram and the spectrum at the bottom of Figure 4(b) is broadened relative to that shown in Figure 4(a). In general,
uncorrelated exchange results in a single peak while any degree of correlated or cooperative exchange leads to peak broadening. The broadening observed in the MS data for OMTKY3 (Figure 3; lower panels) thus suggests the presence of a subset of cooperatively exchanging NHs. Partial cooperativity of NH exchange in OMTKY3 The MS peak simulation algorithm has been used to investigate the extent of cooperativity exhibited by the 13 slowly exchanging NHs of OMTKY3. In these simulations, each NH was assigned to either a cooperative or non-cooperative group. The assignments were made based on structural proximity and similarity of kop values (Figure 2, Table 1). Two groups of NHs seem likely candidates for cooperative structural units. The ®rst group is located within the a-helix: Phe37, Cys38, Asn39, Ala40, and Val41. The other group forms hydrogen bonds across the ®rst and second strands of the b-sheet: Leu23, Gly25, Lys29, and Tyr31. NHs located in the b-turn and those forming hydrogen bonds with the third strand of the b-sheet (Cys24, Ser26, Asn28, and Ser51) are probably not responsible for the cooperative exchange evident in the mass spectra. Exchange at these NHs is, at most, midway between EX2 and EX1 type exchange, which will lead to signi®cant uncorrelated exchange. Moreover, the rapid and continuous 4 amu shift in the MS peak maximum at early time points (Figure 3) is most consistent with the relatively rapid kobs values for these four NHs (Figure 1, Table 1). For all MS simulations of partially cooperative exchange, the cooperative structural unit was arbitrarily assigned to NHs in the a-helix. Similar results were obtained by assigning residues in the b-sheet to be the cooperative structural unit. Results of initial simulations quickly narrowed the size of the putative cooperative unit down to
Ensembles from NH Exchange
1271
Figure 6. The full time course of NH exchange as monitored by ESI-MS at (a) pH 9.79, 30 C and (b) pH 9.56, 30 C. Superimposed on the experimental spectra are simulations from the 4C/9NC model. The values of kobs used to generate the simulated spectra were derived from the pH dependence of NH exchange (Figure 1).
about four NHs. Figure 5 presents simulations in which either four or ®ve NHs exchange cooperatively; the models are referred to as 4C/9NC and 5C/8NC, respectively. The simulations are superimposed upon experimental data. In both models the cooperative NH group was assumed to exchange with a kobs of 0.008 secondÿ1, the mean value for the NHs in the a-helix. The remaining NHs were assumed to exchange independently with their residue-speci®c values of kobs. Although simulated spectra at t ®ve seconds and t 12 minutes are nearly identical for both partially cooperative models, signi®cant differences are evident at the intermediate exchange time
(1.5 minutes). The 1.5 minute 4C/9NC spectrum simulates the experimental data well while the analogous 5C/8NC simulation is too broad. The broadness of the simulated peak is a direct result of the larger cooperative structural unit. Further increasing the size of the cooperative structural unit to six NHs leads the single peak to split into two peaks. Of all the exchange models simulated thus far, the 4C/9NC model shows the best qualitative agreement with the experimental data. This is illustrated in Figure 6, where 4C/9NC simulations are superimposed on the full time course of MS exchange results for OMTKY3 at 30 C, pH 9.79
1272 (Figure 6(a)) and pH 9.56 (Figure 6(b)). In both cases, values of kobs were derived from the NMR experiments (Figure 1). The agreement between simulation and experiment at different pH values suggests that the 4C/9NC model is reasonable. More generally, these results focus attention on the wealth of structural information to be found within MS peak shapes. Minor discrepancies exist between the simulated and experimental MS results (Figure 6). Residual EX2 exchange serves as one source of error and results in more narrow spectra than expected for pure EX1 exchange. Consequently, the 4C/9NC simulations may underestimate the size of the cooperative structural unit. The degree of error is probably small, since exchange at the nine NHs hypothesized to exchange cooperatively retain only 20 % residual EX2 character. This degree of EX2 exchange only has a modest effect on spectral peak shape (Miranker et al., 1996) and is probably not enough to explain differences between the experimental and simulated spectra (Figure 3, upper panels). The simulation algorithm does a good job of capturing the rich information found in subtle differences in MS peak shapes, but further re®nements are likely to provide a more complete interpretation of experimental MS exchange results. Currently, the algorithm only accounts for the extremes of exchange, fully cooperative and completely uncorrelated exchange. In the future we would like to include partially correlated exchange, which would take into account the conditional probability or likelihood of different sites unfolding and exchanging together. Other possible re®nements include: (1) accounting for the effect of residual EX2 exchange on spectral peak shapes; (2) globally ®tting NMR- and MS-derived native-state NH exchange data; and (3) exploring the exchange behavior and degree of correlation for additional NHs beyond the most slowly exchanging NHs. Further experiments will also facilitate a more complete interpretation of the current exchange results. High-resolution MS analysis of monoisotopic peak heights as a function of exchange time should provide more precise evidence regarding the size of the cooperative structural unit. Also, NH exchange and protein fragmentation followed by MS analysis of the peptide fragments should permit identi®cation of residues involved in cooperative exchange (Zhang & Smith, 1993; Zhang et al., 1996; Smith et al., 1997). The simulation routine contains several important features required to accurately model NH exchange spectra. First, it reproduces the inherent binomial distribution of ESI-MS peak shapes. Second, it utilizes atomic resolution NH exchange rate constants determined by NMR spectroscopy. Third, it accounts for the different rate-limiting processes (i.e., EX2 and EX1 exchange) and can model varying sizes and combinations of cooperative structural units. According to the simulations, the simplest explanation of the experimental results
Ensembles from NH Exchange
for OMTKY3 is that only a subset of the most protected NHs in native OMTKY3 exchange during a single cooperative unfolding event. Overall, a number of conformational equilibria are responsible for exchange from these most slowly exchanging NHs. Conformational ensembles from native-state NH exchange In the present study residue-speci®c unfolding rate constants from NMR have been used to extract structural and dynamic information from MS peak shapes. From the NMR data kop varies by two orders of magnitude for the most slowly exchanging NHs in OMTKY3 (Figure 2, Table 1). Interestingly, the variation in kop is not correlated with GHX (Figure 7, Table 1; Clarke & Fersht, 1996). Excluding the three residues with the largest values of GHX (dubbed ``superprotected'' because their values of GHX are greater than Gu), GHX for the other NHs are equivalent to Gu within experimental error. These results indicate that residues with similar thermodynamics of exchange are not necessarily involved in the same conformational equilibria. From the MS results it appears that only a subset of the most slowly exchanging NHs in native OMTKY3 exchange cooperatively. MSderived NH exchange data thus argue that conformational motion at residues with similar thermodynamics and kinetics of unfolding is not necessarily cooperative. Overall, the NMR and MS results paint a self-consistent picture that multiple conformational equilibria are responsible for exchange at the slowest exchanging NHs in native OMTKY3. Combined analysis of native-state NH exchange by NMR and ESI-MS is likely to be one of the most informative experimental methods for exploring the energy landscape of proteins (Bryngelson et al., 1995; Dill & Chan, 1997). Currently, the majority of
Figure 7. Comparison of residue-speci®c values of kop with residue-speci®c values of GHX. Free energies were determined using the following expression: GHX ÿ RT ln(kop/kcl). Lower limits of kop (denoted as open triangles) are shown for Cys24, Ser26, Asn28, Asn33, and Ser51. The error bars are ®tting errors at two standard deviations.
1273
Ensembles from NH Exchange
information regarding energy landscapes is derived from simulations. However, questions remain concerning the extent to which simulated results re¯ect physical reality (Brooks, 1998). What is the nature of the conformational ¯uctuations that lead to exchange at the most protected NHs in OMTKY3? The major conformational ¯uctuation leading to exchange at Asn33 is nearly two orders of magnitude more frequent than for the residues in the (a-helix and across the ®rst two strands of the b-sheet (Figure 2 (red ball), Table 1). The NH of Asn33 has no obvious hydrogen-bond partner in the crystal structure of OMTKY3 (Bode et al., 1985); it points directly into the center of the (a-helix where, presumably, solvent exclusion results in slowed NH exchange. Perhaps the elevated kop value for Asn33 results from an energetically costly ¯uctuation followed by solvent penetration. Disruption of the third strand of the b-sheet and the b-turn between strands one and two also seems to occur more frequently than for the remainder of the sheet and helix (Figure 2 (yellow balls), Table 1). Similar kinetic patterns have been observed in molecular dynamics simulations of unfolding for other proteins. For example, in barnase it was noted that hydrogen-bond disruption occurred at both ends of the b-sheet early in the unfolding process while the center of the sheet remained mostly intact (Li & Daggett, 1998). This behavior coincides with NH-exchange results for residues in b-strands one and two of OMTKY3. NH exchange at residues 26 and 28 reports on the more rapid conformational motion at the end of strands one and two, while exchange at Leu23, Gly25, Lys29 and Tyr31 reports on the persistent structural integrity of the center of these b-strands. For the nine residues with the smallest values of kop, it seems reasonable to suppose that global unfolding is required for exchange (Figure 2 (bluegreen balls), Table 1). However, since these residues do not appear to unfold together, they probably re¯ect ensemble averages rather than a simple unfolding trajectory. A more complete understanding of the thermodynamic and kinetic properties obtained from these NH-exchange studies must come from statistical mechanical models (Hilser & Freire, 1996; MunÄoz et al., 1998).
Materials and Methods Materials OMTKY3 was puri®ed from turkey egg whites as described (Swint & Robertson, 1993). Deuterium oxide (99.9 atom %, 2H2O) was purchased from Cambridge Isotope Laboratories (Cambridge, MA) and deuterium chloride (2HCl) was obtained from Stohler Isotope Chemicals (Rutherford, NJ). pH measurements were made using an Orion Research model 611 pH meter equipped with a 3 mm glass electrode (Wilmad Glass Company, Buena, NJ). Two-point calibration of the pH meter was performed using standards from VWR Scienti®c (West Chester, PA) and Fisher Scienti®c (Pittsburgh,
PA). pH values reported herein are for 2H2O solutions and have not been corrected for isotope effects. NH exchange The following experimental protocol was designed to monitor exchange at the most protected NHs in OMTKY3 using ESI-MS. Lyophilized OMTKY3 was dissolved in ice-cold, acidic 2H2O containing 15 mM each of glycine and glycylglycine (pH 3.5). At this pH and temperature, the half-life for exchange at solvent-exposed NHs is on the order of tens of minutes to hours. Protein solutions (5 mM, 200 ml) were then heated in a water bath for 30 seconds to the desired reaction temperature (30 C). Rapid NH exchange was initiated by manually mixing protein solutions with 100 ml of prewarmed (30 C) alkaline 2H2O solutions (®nal pH 9-10). At various times thereafter, 10 ml aliquots were removed from the reaction solution and rapidly mixed with 990 ml of an ice-cold, acidic 2H2O quench solution (®nal pH 3). Quenched samples were stored at ÿ20 C until ESI-MS analysis. Formic acid and ammonium hydroxide were used to make acidic and alkaline pH adjustments, respectively. For each reaction, aliquots were collected at 10-12 time points ranging from ®ve seconds to 50 minutes. Reaction pH was measured in the solution remaining after the ®nal time point. The ®nal concentration of OMTKY3 in the quenched samples was 200 mg/ml. A similar protocol was used to prepare OMTKY3 samples for NMR analysis. Modi®cations to the ESI-MS protocol include: (1) 2HCl was used to make acidic pH adjustments; (2) the volumes of the protein solution and alkaline 2H2O solution were scaled up by a factor of 4; (3) the quench solution contained 10 mM each of glycine and glycylglycine; (4) 150 ml aliquots of the reaction solution were quenched with 650 ml of quench solution (®nal pH 3.5); (5) seven samples were collected at reaction times ranging from ®ve seconds to ®ve minutes; and (6) the concentration of OMTKY3 in the quenched samples was 3.8 mg/ml. ESI-MS measurements Mass spectra were obtained using an Autospec mass spectrometer equipped with an electrospray interface (Micromass Inc., UK). Samples were introduced into the electrospray interface by direct infusion with a Harvard syringe pump 22. Flow rates were between 5 and 10 ml/ minute. To prevent isotopic back-exchange during mass analysis, the system was ¯ushed with 2H2O between samples. Nitrogen gas was used both as bath and nebulizer gas. The electrospray ion source was held at 80 C and the spray needle was held at 8000 V. Tetraethyl ammonium iodide (Aldrich) in acetonitrile was used as the calibrant (Hop, 1996). The manufacturer's software, OPUSTM (Micromass Inc., UK), was used to acquire and transform the ESI-MS data. Approximately 30 scans, with a mass range of 700 to 2200 Da were collected for each sample. The time required to collect 30 scans was ®ve minutes and 80-100 ml of quenched protein solution was consumed. NMR measurements The NMR spectrometer speci®cations have been described (Swint-Kruse & Robertson, 1996). Either 256 or 512 transients consisting of 8000 time-domain data
1274 points were summed to generate free induction delays (FIDs) for each sample. The spectral width was 6000 Hz, the recycle delay was 1.5 seconds and, depending on the number of transients, the total data acquisition time was either nine or 18 minutes. The values of kobs were determined as described (Arrington & Robertson, 1997). MS peak simulation algorithm Using kobs data derived from NMR experiments, the computer algorithm (provided as Supplementary Material) was used to calculate the probability of all molecular combinations of protons and deuterons. Also, the algorithm summed the probabilities of all molecular species with the same mass. Using a spreadsheet program, two additional steps were taken to simulate mass spectra. First, the probability at each mass was multiplied by the natural mass distribution of OMTKY3 to generate a daughter peak. Second, all daughter peaks were summed to generate the complete mass spectrum. The natural mass distribution corresponding to the molecular formula of OMTKY3 was calculated using the OPUSTM software.
Acknowledgements This work was supported by the National Institutes of Health (GM46849) and a National Science Foundation Shared Instrument Grant (9510004).
References Arrington, C. B. & Robertson, A. D. (1997). Microsecond protein folding kinetics from native-state hydrogen exchange. Biochemistry, 36, 8686-8691. Bahar, I., Wallqvist, A., Covell, D. G. & Jernigan, R. L. (1998). Correlation between native-state hydrogen exchange and cooperative residue ¯uctuations from a simple model. Biochemistry, 37, 1067-1075. Bai, Y., Milne, J. S., Mayne, L. & Englander, S. W. (1993). Primary structure effects on peptide group hydrogen exchange. Proteins: Struct. Funct. Genet. 17, 75-86. Bevington, P. R. & Robinson, D. K. (1992). Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill Inc., New York. Bhuyan, A. K. & Udgaonkar, J. B. (1998). Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state. Biochemistry, 37, 9147-9155. Bode, W., Epp, O., Huber, R., Laskowski, M. & Ardelt, W. (1985). The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3). Eur. J. Biochem. 147, 387-395. Brandts, J. F. (1969). Conformational transitions of proteins in water and in aqueous mixtures. In Structure and Stability of Biological Molecules (Timasheff, S. N. & Fasman, G. D., eds), pp. 213-290, Marcel Dekker Inc., New York. Brooks, C. L., III (1998). Simulations of protein folding and unfolding. Curr. Opin. Struct. Biol. 8, 222-226. Bryngelson, J. D., Onuchic, J. N., Socci, N. D. & Wolynes, P. G. (1995). Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins: Struct. Funct. Genet. 21, 167-195.
Ensembles from NH Exchange Chung, E. W., Nettleton, E. J., Morgan, C. J., Groû, M., Miranker, A., Radford, S. E., Dobson, C. M. & Robinson, C. A. (1997). Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci. 6, 1316-1324. Clarke, J. & Fersht, A. R. (1996). An evaluation of the use of hydrogen exchange at equilibrium to probe intermediates on the protein folding pathway. Folding and Design, 1, 243-254. Dill, K. A. & Chan, H. S. (1997). From Levinthal to pathways to funnels. Nature Struct. Biol. 4, 10-19. Hilser, V. J. & Freire, E. (1996). Structure-based calculation of the equilibrium folding pathway of proteins. Correlation with hydrogen exchange protection factors. J. Mol. Biol. 262, 756-772. Hop, C. E. C. A. (1996). Generation of high molecular weight cluster ions by electrospray ionization; implications for mass calibration. J. Mass Spect. 31, 1314-1316. Hvidt, A. (1964). Discussion of the pH dependence of the hydrogen-deuterium exchange of proteins. C. R. Trav. Lab. Carlsberg, 34, 299-317. Hvidt, A. & Nielsen, S. O. (1966). Hydrogen exchange in proteins. Advan. Protein Chem. 21, 287-386. Johnson, M. L. & Faunt, L. M. (1992). Parameter estimation by least-squares methods. Methods Enzymol. 210, 1-37. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946-950. Lazaridis, T. & Karplus, M. (1997). ``New view'' of protein folding reconciled with the old through multiple unfolding simulations. Science, 278, 19281931. Li, A. & Daggett, V. (1996). Identi®cation and characterization of the unfolding transition state of chymotrypsin inhibitor 2 by molecular dynamics simulations. J. Mol. Biol. 257, 412-429. Li, A. & Daggett, V. (1998). Molecular dynamics simulation of the unfolding of barnase: characterization of the major intermediate. J. Mol. Biol. 275, 677694. Miranker, A., Robinson, C. V., Radford, S. E., Aplin, R. T. & Dobson, C. M. (1993). Detection of transient protein folding populations by mass spectrometry. Science, 262, 896-900. Miranker, A., Robinson, C. V., Radford, S. E. & Dobson, C. M. (1996). Investigation of protein folding by mass spectrometry. FASEB J. 10, 93-101. MunÄoz, V., Henry, E. R., Hofrichter, J. & Eaton, W. A. (1998). A statistical mechanical model for b-hairpin kinetics. Proc. Natl Acad. Sci. USA, 95, 5872-5879. Smith, D. L., Deng, Y. & Zhang, Z. (1997). Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry. J. Mass Spect. 32, 135-146. Swint, L. & Robertson, A. D. (1993). Thermodynamics of unfolding for turkey ovomucoid third domain: thermal and chemical denaturation. Protein Sci. 2, 20372049. Swint-Kruse, L. & Robertson, A. D. (1996). Temperature and pH dependences of hydrogen exchange and global stability for ovomucoid third domain. Biochemistry, 35, 171-180. Yi, Q. & Baker, D. (1996). Direct evidence for a twostate protein unfolding transition from hydrogendeuterium exchange, mass spectrometry, and NMR. Protein Sci. 5, 1060-1066.
1275
Ensembles from NH Exchange Zaidi, F. N., Nath, U. & Udgaonkar, J. B. (1997). Multiple intermediates and transition states during protein unfolding. Nature Struct. Biol. 4, 1016-1024. Zhang, Z. & Smith, D. L. (1993). Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci. 2, 522-531. Zhang, Z., Post, C. B. & Smith, D. L. (1996). Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase. Biochemistry, 35, 779-791.
http://www.academicpress.com/jmb
Edited by P. E. Wright (Received 9 September 1998; received in revised form 26 October 1998; accepted 26 October 1998)
Supplementary material for this paper, comprising a computer algorithm, is available from JMB Online.
J. Mol. Biol. (1999) 285, 1265±1275
Defining Protein Ensembles with Native-state NH Exchange: Kinetics of Interconversion and Cooperative Units from Combined NMR and MS Analysis Cammon B. Arrington1,2, Lynn M. Teesch3 and Andrew D. Robertson1* 1
Department of Biochemistry
2
Medical Scientist Training Program and 3The Highresolution Mass Spectrometry Facility University of Iowa, Iowa City IA 52242, USA
Previous studies of native-state peptide hydrogen atom (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded the thermodynamics and kinetics of unfolding and folding for the 14 slowest-exchanging peptide hydrogen atoms (NHs). Unfolding rate constants and free energies for nine of the NHs are very similar, suggesting that these NHs exchange during a single cooperative unfolding event. Electrospray ionization mass spectrometry (ESI-MS) has been used to test this hypothesis. ESI-MS data and MS peak simulations suggest that this hypothesis is incorrect: in spite of the similarity in their unfolding rate constants, only three to ®ve of the nine residues exchange in a cooperative manner. Thus, residues with similar thermodynamics and kinetics of exchange are probably involved in multiple conformational equilibria. Overall, combined NMR and MS analysis of NH exchange provides a rich and complex picture of the ensemble properties of native proteins. # 1999 Academic Press
*Corresponding author
Keywords: ensembles; protein unfolding; hydrogen atom exchange; electrospray mass spectrometry; ovomucoid third domain
Introduction Recent results from experimental and computational studies suggest the possibility of multiple unfolding pathways for native proteins (Zaidi et al., 1997; Bhuyan & Udgaonkar, 1998; Li & Daggett, 1996; Lazaridis & Karplus, 1997). This view of the kinetics of unfolding is consistent with the emerging picture of proteins as conformational ensembles (Brandts, 1969; Dill & Chan, 1997). Native-state peptide hydrogen atom (NH) exchange is proving to be one of the most informative experimental probes of equilibrium ensemble properties (Hilser & Freire, 1996; Bahar et al., 1998). Recent NMR studies suggest that kinetic ensemble properties can also be examined by native-state NH exchange (Arrington & Robertson, 1997). Here, combined NMR and mass spectral (MS) analysis of Abbreviations used: NH(s), peptide hydrogen atom(s); OMTKY3, turkey ovomucoid third domain; ESI-MS, electrospray ionization mass spectrometry; MS, mass spectral; amu, atomic mass unit. E-mail address of the corresponding author: [email protected] 0022-2836/99/031265±11 $30.00/0
native-state NH exchange demonstrates that the most slowly exchanging NHs in turkey ovomucoid third domain (OMTKY3) report on multiple conformational equilibria. Previous NMR studies of NH exchange were used to determine the thermodynamics (SwintKruse & Robertson, 1996) and kinetics (Arrington & Robertson, 1997) of unfolding at multiple sites throughout native OMTKY3. For the 14 most slowly exchanging NHs, exchange-derived free energies, GHX, were similar to the overall free energy of unfolding, Gu, suggesting that global unfolding precedes exchange at these sites. For ten of these NHs, unfolding rate constants were also similar and overlapped at two standard deviations, with a mean rate constant for unfolding of 0.008 (0.004) secondÿ1 at 30 C. The simplest hypothesis to explain these results is that exchange at these ten NHs results from the cooperative unfolding of OMTKY3. The present study uses electrospray ionization mass spectrometry (ESI-MS) to address this hypothesis. The rationale for using ESI-MS to assess the cooperativity of NH exchange at multiple NHs is based on the well-established kinetic mechanism # 1999 Academic Press
1266
Ensembles from NH Exchange
for slow NH exchange in native proteins (Hvidt, 1964): kop
kch
NH closed NH open * exchanged kcl
1
Each slowly exchanging NH participates in an equilibrium between a closed conformation, NH (closed), from which exchange cannot occur, and an open solvent-exposed state, NH(open). The rate constants for interconversion between closed and open conformations are kcl and kop, respectively. From the open state, NH exchange can occur at the intrinsic chemical rate constant, kch, known from model compound studies (Bai et al., 1993). The exchange reaction is effectively irreversible in the presence of a vast excess of 2H2O. The resulting substitution of peptide hydrogen atoms for deuterium can be followed by ESI-MS. Under conditions favoring the native state, i.e. kcl4kop, the following relation describes the observed rate constant of exchange, kobs, for an individual NH: kobs kop kch = kcl kch
2
Depending on the relative magnitudes of kcl and kch, two limiting cases can be derived from equation (2) (Hvidt & Nielsen, 1966). When kch4kcl, equation (2) simpli®es to: kobs kop
3
This rate-limiting case is referred to as EX1 exchange. In this case exchange occurs with every opening event. In the case of cooperative unfolding, exchange occurs at different NHs simultaneously. Cooperativity of exchange at multiple NHs can be determined by ESI-MS (Miranker et al., 1993; Yi & Baker, 1996; Chung et al., 1997). Under EX1 conditions, cooperative unfolding and subsequent correlated exchange is detected as a bimodal mass spectrum: the lower mass peak represents a protonated molecular population that has not yet opened to exchange, while the higher mass peak represents deuterated molecules that have undergone the exchange reaction. The mass separation of the two peaks indicates the number of cooperatively exchanging NHs. If exchange is purely cooperative, the mass of each peak remains constant with time while the lower and higher mass peaks decrease and increase in intensity, respectively. The second limiting case, known as EX2 exchange, occurs when kcl4kch. In this case, equation (2) simpli®es as follows: kobs kch kop =kcl
4
This rate-limiting case is characterized by numerous visits to the open state prior to exchange. Under EX2 conditions, exchange happens in a random or uncorrelated manner at different NHs, regardless of whether cooperative unfolding occurs. Resulting mass spectra contain a single
peak that shifts to higher mass as exchange proceeds. The same spectral pattern will also exist under EX1 conditions when molecular ¯uctuations are uncorrelated or non-cooperative. Native-state NH exchange in OMTKY3 has been monitored by ESI-MS under the same solution conditions as used in the NMR studies, where G0u is about 7 kcal/mol. To discern the molecular cooperativity of exchange, the experiments have been conducted under EX1 conditions, 30 C and pH > 9.5 (Arrington & Robertson, 1997). Experimental results have been interpreted using a new program for the simulation of MS peak shapes resulting from NH exchange.
Results and Discussion Residue-specific unfolding rate constants in native OMTKY3 NH exchange has been monitored at the 14 slowest-exchanging NHs in OMTKY3 by NMR spectroscopy. In the context of this study, the rationale for the NMR measurements is twofold: (1) to obtain residue-speci®c values of kobs for interpretation of the MS exchange data and (2) to test the extent to which MS exchange samples were prepared under EX1 conditions. The pH dependence of kobs for 13 of the 14 slowly exchanging NHs has been reported previously (Arrington & Robertson, 1997). For nine of these NHs, values of kobs were determined above pH 9.5 and a switch from EX2 to EX1 exchange was detectable. The remaining ®ve NHs (Cys24, Ser26, Asn28, Asn33, and Ser51) exchanged too rapidly to measure above pH 9.5 and a switch from EX2 to EX1 exchange was not detected. To obtain estimates of kobs for these residues at high pH, a quench protocol has been employed to measure more rapid exchange. Updated plots of log(kobs) versus pH are shown in Figure 1 for 12 of the slowly exchanging NHs (data not shown for Ser26 and Asn33). Values of kobs obtained from the quench experiment are displayed as open circles. The data for Ser26 are similar to those for Cys24, whereas NH exchange for Asn33 is still too rapid to detect using the quench protocol; lower limits of kobs have been estimated for Asn33 at high pH. Fits for nine of the NHs in Figure 1 are very similar to those shown previously (Arrington & Robertson, 1997). With the additional data point at high pH, more accurate simulations of log(kobs) versus pH have been performed for Cys24, Ser26, Asn28, Asn33 and Ser51. From the ®tted or simulated curves, residuespeci®c values of kop and kcl as well as exchangederived free energies, GHX, have been estimated (Table 1). In the absence of the high pH data point, Asn28 was previously grouped with the nine NHs with relatively low kop values. Present results clearly demonstrate that Asn28 belongs to the more rapidly exchanging group.
1267
Ensembles from NH Exchange
ent with a switch from EX2 to EX1 exchange, where the plateau value of kobs asymptotically approaches kop. For the majority of residues, the results in Figure 1 indicate that EX1 exchange predominates above pH 9.5. In Figure 2, residue-speci®c values of kop (Table 1) are mapped onto the three-dimensional structure of OMTKY3. The values of kop are derived from the plateaux described by the data in Figure 1. The nine NHs hypothesized to exchange during a single cooperative unfolding event are colored blue-green, with kop 0.008 secondÿ1: Leu23, Gly25, Lys29, Tyr31, Phe37, Cys38, Asn39, Ala40, and Val41. Figure 1. The pH dependence of kobs for 12 of the 14 NHs monitored in this study. The data point at pH 9.65 (open circle) is derived from the manual mixing experiment described in this study while all other data points are from previous work (Arrington & Robertson, 1997). The observed rate constants for all residues except Cys24, Ser26 (not shown), Asn28, Asn33 (not shown) and Ser51 were subjected to non-linear least-squares analysis using equation (2) (Johnson & Faunt, 1992). Plateaux in the data for Cys24, Ser26, Asn28, Asn33 and Ser51 were insuf®ciently de®ned for ®tting, so simulations of equation (2) were performed to obtain lower limits for kop and kcl. Continuous and broken lines represent the ®tted or simulated curves for each NH. The exchange data for Ser26 are similar to those for Cys24, while the data for Asn33 show little deviation from pure EX2 exchange.
Plots of log(kobs) versus pH can be used to distinguish between EX2 and EX1 exchange. For example, the exchange behavior of Cys24 is characteristic of EX2 exchange wherein log(kobs) increases linearly with a slope of 1. However, systematic deviation from a unit slope is evident in the data for Cys38 above pH 8.5. This deviation is consist-
ESI-MS test for cooperative exchange ESI-MS has been used to investigate the cooperativity of NH exchange for the most slowly exchanging NHs in native OMTKY3. ESI-MS exchange samples were prepared under EX1 conditions (pH > 9.5). Under these conditions, 87 of the 100 exchangeable protons in OMTKY3 are deuterated in the time required to initiate exchange and quench the ®rst sample (®ve seconds). The remaining 13 slowly exchanging NHs complete the exchange process in approximately 12-25 minutes. At high pH Asn33 exchanges too rapidly to detect in these experiments. The MS results in Figure 3 (thin line) re¯ect the time-course of NH exchange for OMTKY3 at 30 C, pH 9.79. To determine whether cooperative exchange of nine residues can account for these results, simulated spectra are superimposed upon the experimental spectra (Figure 3, upper panels). For the simulations, nine residues were assumed to exchange cooperatively with an average rate constant of 0.008 secondÿ1. The remaining four NHs were assumed to exchange in an uncorrelated manner with residue-speci®c values of kobs ranging
Table 1. Values of kop and kcl for native OMTKY3 at 30 C Residue Leu23 Cys24b Gly25 Ser26b Asn28b Lys29 Tyr31 Asn33b Phe37 Cys38 Asn39 Ala40 Val41 Ser51b
kop 102 (secondÿ1) 0.3 (0.2, 58 0.8 (0.6, 54 55 1.0 (0.7, 0.7 (0.5, 530 1.0 (0.5, 0.6 (0.4, 2.0 (1.0, 0.8 (0.5, 0.6 (0.4, 57
0.6) 1.2) 1.6) 1.2) 2.5) 0.9) 6.2) 2.3) 1.0)
kcl (secondÿ1)
GHXa (kcal/mol)
1.6 (0.5, 4.3)102 52104 2.0 (1.0, 3.7)103 55103 54103 1.6 (0.8, 3.0)103 0.9(0.4, 1.8)103 53104 1.0 (0.3, 3.3)103 4.3 (2.0, 8.3)103 1.6 (0.5, 5.8)104 6.0 (2.2, 20)103 7.5 (3.8, 15)102 55103
6.6 7.4 7.5 7.1 6.8 7.2 7.1 6.9 7.0 8.2 8.2 8.1 7.1 6.7
Values for all residues except Cys24, Ser26, Asn28, Asn33 and Ser51 were determined from non-linear regression analysis using equation (2). Numbers in parentheses denote lower and upper limits corresponding to a 95 % con®dence level. The ®tting error was determined using F analysis (Bevington & Robinson, 1992). a Estimated error for GHX is 0.5 kcal/mol (Swint-Kruse & Robertson, 1996). b Simulations of equation (2) were used to obtain lower limits for Cys24, Ser26, Asn28, Asn33 and Ser51.
1268
Ensembles from NH Exchange
Figure 2. Structural distribution of kop for the most slowly exchanging NHs in native OMTKY3. The magnitude of the rate constants has been color-coded onto balls representing individual NHs. The ribbon diagram was generated in part with MOLSCRIPT (Kraulis, 1991) and PDB ®le 2OVO (Bode et al., 1985).
from 0.03 to 0.05 secondÿ1. Due to the more rapid uncorrelated exchange at four NHs, a four atomic mass unit (amu) shift in the peak maximum occurs early in the simulation. This mass shift is complete by approximately 30 seconds and two peaks, separated by 9 amu, are observed subsequently as a result of cooperative exchange at nine NHs. The comparison of simulated and experimental spectra demonstrates that cooperative unfolding at nine NHs cannot explain the experimental results. To assess whether completely uncorrelated exchange can account for the experimental results, simulated spectra have been generated in which the 13 NHs were assumed to exchange independently of each other (Figure 3, lower panels). Residue-speci®c values of kobs were obtained from NMR experiments and ranged from 0.003 to 0.05
secondÿ1. Although the mass shifts of the simulated and experimental spectra are similar, differences in peak shape are apparent. For example, the ®rst experimental spectrum (®ve seconds) is broadened (mostly at the base) and skewed towards higher mass. In addition, the experimental peak shape at 1.5 minutes is much broader than the simulated peak. These results and the simulations discussed below suggest that a subset of the most slowly exchanging NHs in OMTKY3 may exchange in a cooperative manner. Cooperative protein fluctuations from deconvolution of NH exchange mass spectra In MS studies of NH exchange, two distinct mass distributions contribute to spectral peak
Figure 3. Time dependence of NH exchange at 30 C and pH 9.79 as monitored by mass spectrometry. Time of exchange is denoted in the upper right corner of each panel. The experimental data (®ne lines) in the top three panels are superimposed on simulated spectra generated by assuming that nine NHs exchange cooperatively and four NHs exchange in an uncorrelated manner. In the bottom three panels, data are superimposed on simulations of completely uncorrelated exchange at 13 NHs.
1269
Ensembles from NH Exchange
tations) are summed to generate the overall probability at a given deuteration level or mass outcome. Lastly, the probability at each deuteration level is multiplied by the natural mass distribution of a molecule and the resulting peaks are summed to generate a full mass spectrum. The algorithm will be illustrated in more detail by simulating spectra for a hypothetical molecule with three exchangeable NHs (Figure 4(a)). Using NMR-derived values of kobs, the program calculates the probability of an amide being protonated, PROBH, or deuterated, PROBD:
Figure 4. Diagrammatic representation of the approach used to simulate MS exchange results. (a) Simulated results for a hypothetical molecule with three exchangeable NHs. All possible combinations of protons (H) and deuterons (D) are listed and separated by color into groups with the same number of deuterons. The histogram represents the molecular probability at each deuteration level after one minute of exchange and assuming the values of kobs listed in the text. The probability at each deuteration level is multiplied by the natural mass distribution of OMTKY3 to generate the colored peaks. The continuous black line is the full mass spectrum obtained by summing the colored peaks. (b) Simulated results for a hypothetical molecule with six exchangeable NHs, four of which exchange cooperatively. The number 4 in parentheses represents the group of NHs that exchange cooperatively.
shapes. First, mass spectra of pure proteins exhibit ®nite line widths characteristic of the protein's molecular formula and the presence of naturally occurring isotopes (e.g., 13C, 15N, etc.). Second, the molecular population has a distribution of protons and deuterons as a result of NH exchange. A computer algorithm has been developed to simulate mass spectra by accounting for the two mass distributions. In outline, the program ®rst de®nes all possible molecular species in terms of proton/deuteron occupancy and then calculates the probability of each species using NMR-derived NH exchange rate constants. Next, the probabilities of all species with the same number of deuterons (i.e., indistinguishable proton/deuteron permu-
PROBH i exp ÿkobs i t
5
PROBD i 1-PROBH i
6
where i represents an exchangeable NH and t is the exchange time. Let us assume that, kobs(l) 0.5 minuteÿ1, kobs(2) 1.2 minuteÿ1, and kobs(3) 0.3 minuteÿ1. After one minute of exchange, the site-speci®c probabilities are as follows: PROBH(1) 0.61, PROBD(1) 0.39; PROBH(2) 0.30, PROBD(2) 0.70; PROBH(3) 0.74, PROBD(3) 0.26. The total number of molecular species distinguished by different patterns of protonation and deuteration equals 2N, where N is the number of exchanging NHs. For the sample molecule with three NHs there are eight possible molecular species (Figure 4(a), top). If exchange at the three NHs is uncorrelated, then the probability of each molecular species is the product of the site-speci®c probabilities for the three sites. Probabilities for each molecular species in the present example are as follows: P HHH PROBH 1 PROBH 2 PROBH 3 0:14
7a
P DHH PROBD 1 PROBH 2 PROBH 3 0:09
7b
P HDH PROBH 1 PROBD 2 PROBH 3 0:31
7c
P HHD PROBH 1 PROBH 2 PROBD 3 0:05 P DDH PROBD 1 PROBD 2
7d
PROBH 3 0:20 P DHD PROBD 1 PROBH 2
7e
PROBD 3 0:03
7f
P HDD PROBH 1 PROBD 2 PROBD 3 0:11
7g
P DDD PROBD 1 PROBD 2 PROBD 3 0:07
7h
The probabilities of all molecular species with the same number of deuterons are now summed to generate the overall probability at each deuteration level (Figure 4(a), middle).
1270
Ensembles from NH Exchange
Figure 5. Comparison of experimental mass spectra (pH 9.79, 30 C) with simulations in which four or ®ve NHs exchange cooperatively. The model shown in the top three panels is called 4C/9NC and assumes that a group of four NHs exchange cooperatively and nine NHs exchange non-cooperatively. The model shown in the bottom panels is referred to as 5C/8NC. For both models, kobs for the cooperative NHs was assumed to be 0.008 secondÿ1. The remaining NHs were assumed to exchange noncooperatively with residue-speci®c values of kobs.
M0 P HHH 0:14
8a
M1 P DHH P HDH P HHD 0:45
8b
M2 P DDH P DHD P HDD 0:34 M3 P DDD 0:07
8c 8d
In these expressions M represents mass and the subscripts are the number of deuterons or the deuteration level. A full mass spectrum is obtained by multiplying the probability at each mass (Figure 4(a), middle) by the natural mass distribution of a molecule and then summing the resultant peaks (Figure 4(a), bottom). The algorithm can also account for completely cooperative exchange, de®ned as two or more NHs that always exchange together. For the example illustrated in Figure 4(b), the molecule contains six NHs, four of which exchange cooperatively with a single value of kobs (Figure 4(b), top). Thus, only three values of kobs are required in this example. As before, one minute of exchange will be simulated using the same values of kobs, except that kobs(3) pertains to four cooperative NHs instead of a single NH. With a group of four NHs that is either all protonated or all deuterated, the mass distribution (Figure 4(b), middle) is as follows: M0 P HHH4 0:14
9a
M1 P DHH4 P HDH4 0:40 M2 P DDH4 0:20
9b 9c
M3 0 M4 P HHD4 0:05
9d 9e
M5 P DHD4 P HDD4 0:14 M6 P DDD4 0:07
9f 9g
where the subscript in the probability expressions represents the four cooperative NHs. The cooperativity leads to a bimodal histogram and the spectrum at the bottom of Figure 4(b) is broadened relative to that shown in Figure 4(a). In general,
uncorrelated exchange results in a single peak while any degree of correlated or cooperative exchange leads to peak broadening. The broadening observed in the MS data for OMTKY3 (Figure 3; lower panels) thus suggests the presence of a subset of cooperatively exchanging NHs. Partial cooperativity of NH exchange in OMTKY3 The MS peak simulation algorithm has been used to investigate the extent of cooperativity exhibited by the 13 slowly exchanging NHs of OMTKY3. In these simulations, each NH was assigned to either a cooperative or non-cooperative group. The assignments were made based on structural proximity and similarity of kop values (Figure 2, Table 1). Two groups of NHs seem likely candidates for cooperative structural units. The ®rst group is located within the a-helix: Phe37, Cys38, Asn39, Ala40, and Val41. The other group forms hydrogen bonds across the ®rst and second strands of the b-sheet: Leu23, Gly25, Lys29, and Tyr31. NHs located in the b-turn and those forming hydrogen bonds with the third strand of the b-sheet (Cys24, Ser26, Asn28, and Ser51) are probably not responsible for the cooperative exchange evident in the mass spectra. Exchange at these NHs is, at most, midway between EX2 and EX1 type exchange, which will lead to signi®cant uncorrelated exchange. Moreover, the rapid and continuous 4 amu shift in the MS peak maximum at early time points (Figure 3) is most consistent with the relatively rapid kobs values for these four NHs (Figure 1, Table 1). For all MS simulations of partially cooperative exchange, the cooperative structural unit was arbitrarily assigned to NHs in the a-helix. Similar results were obtained by assigning residues in the b-sheet to be the cooperative structural unit. Results of initial simulations quickly narrowed the size of the putative cooperative unit down to
Ensembles from NH Exchange
1271
Figure 6. The full time course of NH exchange as monitored by ESI-MS at (a) pH 9.79, 30 C and (b) pH 9.56, 30 C. Superimposed on the experimental spectra are simulations from the 4C/9NC model. The values of kobs used to generate the simulated spectra were derived from the pH dependence of NH exchange (Figure 1).
about four NHs. Figure 5 presents simulations in which either four or ®ve NHs exchange cooperatively; the models are referred to as 4C/9NC and 5C/8NC, respectively. The simulations are superimposed upon experimental data. In both models the cooperative NH group was assumed to exchange with a kobs of 0.008 secondÿ1, the mean value for the NHs in the a-helix. The remaining NHs were assumed to exchange independently with their residue-speci®c values of kobs. Although simulated spectra at t ®ve seconds and t 12 minutes are nearly identical for both partially cooperative models, signi®cant differences are evident at the intermediate exchange time
(1.5 minutes). The 1.5 minute 4C/9NC spectrum simulates the experimental data well while the analogous 5C/8NC simulation is too broad. The broadness of the simulated peak is a direct result of the larger cooperative structural unit. Further increasing the size of the cooperative structural unit to six NHs leads the single peak to split into two peaks. Of all the exchange models simulated thus far, the 4C/9NC model shows the best qualitative agreement with the experimental data. This is illustrated in Figure 6, where 4C/9NC simulations are superimposed on the full time course of MS exchange results for OMTKY3 at 30 C, pH 9.79
1272 (Figure 6(a)) and pH 9.56 (Figure 6(b)). In both cases, values of kobs were derived from the NMR experiments (Figure 1). The agreement between simulation and experiment at different pH values suggests that the 4C/9NC model is reasonable. More generally, these results focus attention on the wealth of structural information to be found within MS peak shapes. Minor discrepancies exist between the simulated and experimental MS results (Figure 6). Residual EX2 exchange serves as one source of error and results in more narrow spectra than expected for pure EX1 exchange. Consequently, the 4C/9NC simulations may underestimate the size of the cooperative structural unit. The degree of error is probably small, since exchange at the nine NHs hypothesized to exchange cooperatively retain only 20 % residual EX2 character. This degree of EX2 exchange only has a modest effect on spectral peak shape (Miranker et al., 1996) and is probably not enough to explain differences between the experimental and simulated spectra (Figure 3, upper panels). The simulation algorithm does a good job of capturing the rich information found in subtle differences in MS peak shapes, but further re®nements are likely to provide a more complete interpretation of experimental MS exchange results. Currently, the algorithm only accounts for the extremes of exchange, fully cooperative and completely uncorrelated exchange. In the future we would like to include partially correlated exchange, which would take into account the conditional probability or likelihood of different sites unfolding and exchanging together. Other possible re®nements include: (1) accounting for the effect of residual EX2 exchange on spectral peak shapes; (2) globally ®tting NMR- and MS-derived native-state NH exchange data; and (3) exploring the exchange behavior and degree of correlation for additional NHs beyond the most slowly exchanging NHs. Further experiments will also facilitate a more complete interpretation of the current exchange results. High-resolution MS analysis of monoisotopic peak heights as a function of exchange time should provide more precise evidence regarding the size of the cooperative structural unit. Also, NH exchange and protein fragmentation followed by MS analysis of the peptide fragments should permit identi®cation of residues involved in cooperative exchange (Zhang & Smith, 1993; Zhang et al., 1996; Smith et al., 1997). The simulation routine contains several important features required to accurately model NH exchange spectra. First, it reproduces the inherent binomial distribution of ESI-MS peak shapes. Second, it utilizes atomic resolution NH exchange rate constants determined by NMR spectroscopy. Third, it accounts for the different rate-limiting processes (i.e., EX2 and EX1 exchange) and can model varying sizes and combinations of cooperative structural units. According to the simulations, the simplest explanation of the experimental results
Ensembles from NH Exchange
for OMTKY3 is that only a subset of the most protected NHs in native OMTKY3 exchange during a single cooperative unfolding event. Overall, a number of conformational equilibria are responsible for exchange from these most slowly exchanging NHs. Conformational ensembles from native-state NH exchange In the present study residue-speci®c unfolding rate constants from NMR have been used to extract structural and dynamic information from MS peak shapes. From the NMR data kop varies by two orders of magnitude for the most slowly exchanging NHs in OMTKY3 (Figure 2, Table 1). Interestingly, the variation in kop is not correlated with GHX (Figure 7, Table 1; Clarke & Fersht, 1996). Excluding the three residues with the largest values of GHX (dubbed ``superprotected'' because their values of GHX are greater than Gu), GHX for the other NHs are equivalent to Gu within experimental error. These results indicate that residues with similar thermodynamics of exchange are not necessarily involved in the same conformational equilibria. From the MS results it appears that only a subset of the most slowly exchanging NHs in native OMTKY3 exchange cooperatively. MSderived NH exchange data thus argue that conformational motion at residues with similar thermodynamics and kinetics of unfolding is not necessarily cooperative. Overall, the NMR and MS results paint a self-consistent picture that multiple conformational equilibria are responsible for exchange at the slowest exchanging NHs in native OMTKY3. Combined analysis of native-state NH exchange by NMR and ESI-MS is likely to be one of the most informative experimental methods for exploring the energy landscape of proteins (Bryngelson et al., 1995; Dill & Chan, 1997). Currently, the majority of
Figure 7. Comparison of residue-speci®c values of kop with residue-speci®c values of GHX. Free energies were determined using the following expression: GHX ÿ RT ln(kop/kcl). Lower limits of kop (denoted as open triangles) are shown for Cys24, Ser26, Asn28, Asn33, and Ser51. The error bars are ®tting errors at two standard deviations.
1273
Ensembles from NH Exchange
information regarding energy landscapes is derived from simulations. However, questions remain concerning the extent to which simulated results re¯ect physical reality (Brooks, 1998). What is the nature of the conformational ¯uctuations that lead to exchange at the most protected NHs in OMTKY3? The major conformational ¯uctuation leading to exchange at Asn33 is nearly two orders of magnitude more frequent than for the residues in the (a-helix and across the ®rst two strands of the b-sheet (Figure 2 (red ball), Table 1). The NH of Asn33 has no obvious hydrogen-bond partner in the crystal structure of OMTKY3 (Bode et al., 1985); it points directly into the center of the (a-helix where, presumably, solvent exclusion results in slowed NH exchange. Perhaps the elevated kop value for Asn33 results from an energetically costly ¯uctuation followed by solvent penetration. Disruption of the third strand of the b-sheet and the b-turn between strands one and two also seems to occur more frequently than for the remainder of the sheet and helix (Figure 2 (yellow balls), Table 1). Similar kinetic patterns have been observed in molecular dynamics simulations of unfolding for other proteins. For example, in barnase it was noted that hydrogen-bond disruption occurred at both ends of the b-sheet early in the unfolding process while the center of the sheet remained mostly intact (Li & Daggett, 1998). This behavior coincides with NH-exchange results for residues in b-strands one and two of OMTKY3. NH exchange at residues 26 and 28 reports on the more rapid conformational motion at the end of strands one and two, while exchange at Leu23, Gly25, Lys29 and Tyr31 reports on the persistent structural integrity of the center of these b-strands. For the nine residues with the smallest values of kop, it seems reasonable to suppose that global unfolding is required for exchange (Figure 2 (bluegreen balls), Table 1). However, since these residues do not appear to unfold together, they probably re¯ect ensemble averages rather than a simple unfolding trajectory. A more complete understanding of the thermodynamic and kinetic properties obtained from these NH-exchange studies must come from statistical mechanical models (Hilser & Freire, 1996; MunÄoz et al., 1998).
Materials and Methods Materials OMTKY3 was puri®ed from turkey egg whites as described (Swint & Robertson, 1993). Deuterium oxide (99.9 atom %, 2H2O) was purchased from Cambridge Isotope Laboratories (Cambridge, MA) and deuterium chloride (2HCl) was obtained from Stohler Isotope Chemicals (Rutherford, NJ). pH measurements were made using an Orion Research model 611 pH meter equipped with a 3 mm glass electrode (Wilmad Glass Company, Buena, NJ). Two-point calibration of the pH meter was performed using standards from VWR Scienti®c (West Chester, PA) and Fisher Scienti®c (Pittsburgh,
PA). pH values reported herein are for 2H2O solutions and have not been corrected for isotope effects. NH exchange The following experimental protocol was designed to monitor exchange at the most protected NHs in OMTKY3 using ESI-MS. Lyophilized OMTKY3 was dissolved in ice-cold, acidic 2H2O containing 15 mM each of glycine and glycylglycine (pH 3.5). At this pH and temperature, the half-life for exchange at solvent-exposed NHs is on the order of tens of minutes to hours. Protein solutions (5 mM, 200 ml) were then heated in a water bath for 30 seconds to the desired reaction temperature (30 C). Rapid NH exchange was initiated by manually mixing protein solutions with 100 ml of prewarmed (30 C) alkaline 2H2O solutions (®nal pH 9-10). At various times thereafter, 10 ml aliquots were removed from the reaction solution and rapidly mixed with 990 ml of an ice-cold, acidic 2H2O quench solution (®nal pH 3). Quenched samples were stored at ÿ20 C until ESI-MS analysis. Formic acid and ammonium hydroxide were used to make acidic and alkaline pH adjustments, respectively. For each reaction, aliquots were collected at 10-12 time points ranging from ®ve seconds to 50 minutes. Reaction pH was measured in the solution remaining after the ®nal time point. The ®nal concentration of OMTKY3 in the quenched samples was 200 mg/ml. A similar protocol was used to prepare OMTKY3 samples for NMR analysis. Modi®cations to the ESI-MS protocol include: (1) 2HCl was used to make acidic pH adjustments; (2) the volumes of the protein solution and alkaline 2H2O solution were scaled up by a factor of 4; (3) the quench solution contained 10 mM each of glycine and glycylglycine; (4) 150 ml aliquots of the reaction solution were quenched with 650 ml of quench solution (®nal pH 3.5); (5) seven samples were collected at reaction times ranging from ®ve seconds to ®ve minutes; and (6) the concentration of OMTKY3 in the quenched samples was 3.8 mg/ml. ESI-MS measurements Mass spectra were obtained using an Autospec mass spectrometer equipped with an electrospray interface (Micromass Inc., UK). Samples were introduced into the electrospray interface by direct infusion with a Harvard syringe pump 22. Flow rates were between 5 and 10 ml/ minute. To prevent isotopic back-exchange during mass analysis, the system was ¯ushed with 2H2O between samples. Nitrogen gas was used both as bath and nebulizer gas. The electrospray ion source was held at 80 C and the spray needle was held at 8000 V. Tetraethyl ammonium iodide (Aldrich) in acetonitrile was used as the calibrant (Hop, 1996). The manufacturer's software, OPUSTM (Micromass Inc., UK), was used to acquire and transform the ESI-MS data. Approximately 30 scans, with a mass range of 700 to 2200 Da were collected for each sample. The time required to collect 30 scans was ®ve minutes and 80-100 ml of quenched protein solution was consumed. NMR measurements The NMR spectrometer speci®cations have been described (Swint-Kruse & Robertson, 1996). Either 256 or 512 transients consisting of 8000 time-domain data
1274 points were summed to generate free induction delays (FIDs) for each sample. The spectral width was 6000 Hz, the recycle delay was 1.5 seconds and, depending on the number of transients, the total data acquisition time was either nine or 18 minutes. The values of kobs were determined as described (Arrington & Robertson, 1997). MS peak simulation algorithm Using kobs data derived from NMR experiments, the computer algorithm (provided as Supplementary Material) was used to calculate the probability of all molecular combinations of protons and deuterons. Also, the algorithm summed the probabilities of all molecular species with the same mass. Using a spreadsheet program, two additional steps were taken to simulate mass spectra. First, the probability at each mass was multiplied by the natural mass distribution of OMTKY3 to generate a daughter peak. Second, all daughter peaks were summed to generate the complete mass spectrum. The natural mass distribution corresponding to the molecular formula of OMTKY3 was calculated using the OPUSTM software.
Acknowledgements This work was supported by the National Institutes of Health (GM46849) and a National Science Foundation Shared Instrument Grant (9510004).
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http://www.academicpress.com/jmb
Edited by P. E. Wright (Received 9 September 1998; received in revised form 26 October 1998; accepted 26 October 1998)
Supplementary material for this paper, comprising a computer algorithm, is available from JMB Online.