Anal. Chem. 2003, 75, 1325-1330
Conformations of Gas-Phase Lysozyme Ions Produced from Two Different Solution Conformations Dunmin Mao, Kodali Ravindra Babu,† Yu-Luan Chen,‡ and D. J. Douglas*
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada
With the development of matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), the structures and chemistry of gas-phase ions of large biomolecules have become of interest.1 An interesting question is whether the gasphase ions retain memory of their solution conformations. Proteins in solution can adopt folded native conformations or be unfolded by denaturing reagents, such as acids or organic solvents. A direct
comparison of the properties of the gas-phase ions produced from the two solution conformations is of interest. However, as discussed by Wang et al.,2 gas-phase ions produced by different solution conformations often have very different charge states. A protein unfolded in solution often produces much higher charge states in ESI than the same protein does in its native conformation in solution.3 The different numbers of charges can substantially affect the properties of the ions, making direct comparisons of ions formed from two different solution conformations difficult. For example, in an early study of the collision cross sections of cytochrome c, ions with charges +7 to +10 were produced from the native conformation in solution, and ions with charges +9 to +20 were produced from the denatured conformation.4 Cross sections could be directly compared for only the +9 and +10 ions produced from the two solutions; ions produced from the native conformation showed slightly smaller cross sections.4 In some cases, proteins denatured in solution produce charge states in ESI similar to native state proteins. In a study of bovine pancreatic trypsin inhibitor, which contains three disulfide bonds, similar charge-state ions were produced by the native and disulfide-reduced proteins,5 and a direct comparison of collision cross sections of these two forms of the protein was possible. For a given charge state, the reduced protein showed cross sections ∼9-17% greater than the oxidized protein. Wang et al. showed that the N terminal domain of cardiac muscle troponin produced the same charge states, +6 to +8, when sprayed from native and denatured conformations in solution.2 The +6 and +7 ions formed from the different solution conformations showed differences in their hydrogen/deuterium (H/D) exchange behavior, suggesting the ions retain some “memory” of the solution structure. Hen egg white lysozyme, mw 14 304, has 129 amino acid residues, with four disulfide bonds between cysteine residues at positions 6-127, 30-115, 64-80, and 76-94.6 It has been a model protein for solution folding/unfolding studies with various denaturing reagents, such as guanidine hydrochloride,7,8 2,2,2-trifluoroethanol,9 acetic acid,7 and methanol,10 investigated by various
* To whom correspondence should be addressed. Phone: (604) 822-3057. Fax: (604) 822-2847. E-mail:
[email protected]. † Present address: LPVD, Rocky Mountain Laboratory, 903 S 4th St., Hamilton, MT 59840. ‡ Present address: Fujisawa Research Institute of America, 1801 Maple Ave., Evanston, IL 60201. (1) (a) Jarrold, M. F. Ann Rev. Phys. Chem. 2000, 51, 179-207. (b) HoaglundHyzer, C. S.; Counterman A. E.; Clemmer, D. E. Chem. Rev. 1999, 99, 30373079.
(2) Wang, F.; Freitas, M. A.; Marshall, A. G.; Sykes, B. D. Int. J. Mass Spectrom. 1999, 192, 319-325. (3) (a) Chowdhury, S. K.; Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1990, 112, 9012-9013. (b) Mirza, U. A.; Cohen, S. L.; Chait, B. T. Anal. Chem. 1993, 65, 1-6. (c) Feng, R.; Konishi, Y. J. Am. Soc. Mass Spectrom. 1993, 4, 638645. (4) Covey, T.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1993, 4, 616-623. (5) Nesatiy, V.; Chen, Y.-L.; Collings, B. A.; Douglas, D. J. Rapid Commun. Mass Spectrom. 1998, 12, 40-44. (6) Canfield, R. E.; Liu, A. K. J. Biol. Chem. 1965, 240, 1997-2002.
Near pH 2.0, lysozyme in water is in its native conformation, and in water/methanol (2/8) it adopts a helical denatured conformation (Kamatari et al. Protein Sci. 1998, 7, 681-688). Hydrogen/deuterium (H/D) exchange of lysozyme in solution confirms that it is partially unfolded at pH 2.0 in water/methanol (v/v ) 2/8). With electrospray ionization (ESI) mass spectrometry (MS), lysozyme in water produces ions with charges +7 to +12, with the greatest intensity at +10, whereas lysozyme in water/methanol (2/8) produces ions with charges +6 to +12 with the greatest intensity at +7. Thus, lysozyme is an exception to the rule that a protein denatured in solution forms higher charge states than the same protein in its folded native conformations in solution. Because the same charge states are produced from these two solution conformations, a direct comparison of the properties of the gas-phase ions produced from two very different solution conformations is possible. The conformations of lysozyme ions in the gas phase were studied using cross section measurements and gas-phase H/D exchange. Similar cross sections and H/D exchange levels were observed for same-charge states of lysozyme ions formed from the native and helical denatured conformations in solution. Cross sections show that the ions have compact structures. Thus, disulfide-intact gaseous lysozyme ions generated from the denatured state in water/methanol (2/ 8) refold into compact structures in the gas phase on a time scale of milliseconds or less.
10.1021/ac020647x CCC: $25.00 Published on Web 02/21/2003
© 2003 American Chemical Society
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techniques.7-10 However, there is limited information correlating solution- and gas-phase conformations. To date, the effects of removing the four disulfide bonds have been studied. Removing the disulfide bonds causes substantial changes to the solution structure of lysozyme. The protein unfolds and produces higher charge states in ESI.11-16 Surface imprinting experiments provide direct evidence for elongated structures, particularly for disulfidereduced lysozyme.14 Measurements of the rate constants of charge-transfer reactions with various bases to give apparent gasphase basicities of lysozyme ions provided evidence for multiple conformations of the +7 through +11 ions from disulfide-reduced lysozyme and +8 through +10, +12, and +13 ions from disulfideintact lysozyme.15 An ion mobility study16 showed that ions from the disulfide-reduced protein have much larger cross sections than those from the disulfide-intact protein. When protons were removed through proton-transfer reactions, ions from the disulfideintact lysozyme favored compact structures, whereas those from the disulfide-reduced protein refolded in less than 10-30 ms to conformations with cross sections similar to the disulfide-intact ions.16 Without removing the disulfide bonds, substantial changes in the aqueous solution conformation of lysozyme can be caused by the addition of methanol. Kamatari et al. have studied in detail the methanol-induced conformation changes of disulfide-intact lysozyme in solution by far- and near-UV CD and NMR spectroscopies, ANS binding, and small-angle X-ray scattering.10 At pH 2.0 in water, lysozyme is in its native state (the “N” state). At pH 2.0 and high methanol concentrations, the protein undergoes a cooperative transition into a helical denatured state (the “H” state) in which the tertiary structure is lost, but the R-helix content increases. The H state is expanded considerably and has a flexible broken rodlike chain conformation. Small-angle X-ray scattering shows that the radius of gyration (rg) of the protein in the H state is 24.9 Å, much larger than that of the protein in the native state, 15.7 Å, but somewhat smaller than that of the fully urea-denatured protein (28.7 Å). A phase diagram correlating the N and H states with pH and methanol concentration was constructed.10 In this paper, we report a study of conformations of gas-phase lysozyme ions produced from the native and helical denatured state proteins in solution. Disulfide-intact gas-phase lysozyme ions were generated from water and water/methanol (v/v ) 2/8) solutions at pH near 2.0. Native and helical denatured lysozyme in solution are shown to produce similar charge states in ESI. This allows a direct comparison of conformations of the gas phase (7) Kato, S.; Okamura, M.; Shimamoto, N.; Utiyama, H. Biochemistry 1981, 20, 1080-1085. (8) Yang, H. H.; Li, X. C.; Amft, M.; Grotemeyer, J. Anal. Biochem. 1998, 258, 118-126. (9) Buck, M.; Radford, S. E.; Dobson, C. M. Biochemistry 1993, 32, 669-678. (10) Kamatari, Y. O.; Konno, T.; Kataoka, M.; Akasaka, K. Protein Sci. 1998, 7, 681-688. (11) Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990, 62, 693-698. (12) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1993, 115, 6317-6321. (13) Przybylski, M.; Glocker, M. O. Angew. Chem., Int. Ed. Engl. 1996, 35, 806826. (14) Reimann, C. T.; Sullivan, P. A.; Axelsson, J.; Quist, A. P.; Altmann, S.; Roepstorff, P.; Vela´zquez I.; Tapia, O. J. Am. Chem. Soc. 1998, 120, 76087616. (15) Gross, D. S.; Schnier, P. D.; Rodriguez-Cruz, S. E.; Fagerquist, C. K.; Williams, E. R. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 3143-3148. (16) Valentine, S. J.; Anderson, J. G.; Ellington, A. D.; Clemmer, D. E. J. Phys. Chem. B 1997, 101, 3891-3900.
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ions produced from these two solution conformations. The conformations of lysozyme in water and in water/methanol (2/8) were reinvestigated by solution phase H/D exchange, whereas conformations of ions in the gas phase were studied by cross section measurements and gas-phase H/D exchange. Solution H/D exchange confirms that lysozyme in the helical denatured state has a more open structure than that in the native state. Ions generated by ESI from the helical denatured state are found to have cross sections and hydrogen exchange levels similar to ions produced from native lysozyme. The cross sections also show that the gas-phase ions are similar in size to the native conformation in solution. Thus, lysozyme ions produced from the helical denatured state refold substantially during ESI to form compact structures in the gas phase. EXPERIMENTAL SECTION Reagents. Hen egg white lysozyme from Sigma (L6876, St. Louis, MO) was used without further purification. HPLC grade methanol and hydrochloric acid were from Fisher Scientific (Nepean, ON, Canada). Deuterium oxide (99.9%) and methanold4 (99.8%) were from Cambridge Isotope Laboratories, Inc. (Andover, MA). Deuterium chloride was from Aldrich Chemical Co. (Milwaukee, WI). Sample Preparation and pH Measurements. The concentration of lysozyme in water and water/methanol (v/v ) 2/8) was 20 µM. Solution pH, measured with an Accumet pH meter (model 15, Fisher Scientific), was controlled between 2.0 and 2.1 by adding HCl. The pH in D2O/CD3OD (v/v ) 2/8) was adjusted by adding DCl. The corresponding pD of the solution was corrected for D2O by adding 0.4 to the pH meter reading.17 The correction to the pH measurement for methanol is very small (pH ) meter reading + 0.06)18 and is not included in the reported values. Solution H/D Exchange. The solution H/D exchange of lysozyme in water and water/methanol (2/8) at room temperature was monitored by an ESI quadrupole mass spectrometer system built in house.19 The protein was first dissolved in deionized water and diluted from a 1 mM stock solution by a factor of 50 in D2O or D2O/CD3OD (2/8) at the desired pD to initiate the H/D exchange reaction. The sample solution was then immediately infused to the ESI source at a flow rate of 1.5 µL/min, and mass spectra of the +10 charge state were recorded at various exchange times. The voltage difference between the sampling orifice and radio frequency (RF)-only quadrupole rod was 85 V. The scanning step size was 0.2 Da with a 20 ms dwell time to scan a mass range of 15 Da around the +10 ion. The number of hydrogens incorporated at the various exchange times was calculated from the product of the mass shift and the charge. Cross Section Measurements. The cross sections of lysozyme ions were measured through energy loss experiments on a triple quadrupole mass spectrometer, as described previously.4,20,21 Protonated lysozyme ions, generated by ESI, passed through an (17) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190. (18) (a) DeLigny, C. L.; Rehbach, M. Rec. Trav. Chim. 1960, 79, 727-730. (b) Bates, R. G. Determination of pH, Theory and Practice; John Wiley and Sons Inc.: New York, 1964; p 224. (19) Konermann, L.; Collings, B. A.; Douglas D. J. Biochemistry 1997, 36, 55545559. (20) Douglas, D. J. J. Am. Soc. Mass Spectrom. 1994, 5, 17-18. (21) Chen, Y.-L.; Collings, B. A.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 1997, 8, 681-687.
orifice skimmer interface (orifice-skimmer voltage difference 80V) and entered an RF-only quadrupole ion guide (Q0). The pressure in the ion guide was 6.5 mTorr, sufficient to cool the ion translational energies and energy spreads to 1 eV or less.22 Ions then passed through a quadrupole (Q1) operated in RF-only mode and were injected into the quadrupole collision cell (Q2) with an initial kinetic energy, E0, of 10 eV per charge. The collision gas in Q2 was argon at pressures of 0-1 mTorr, measured with a precision capacitance manometer (model 120AA, MKS Instruments, Andover, MA). The kinetic energies, E, of ions leaving Q2 were determined from stopping curves generated with the rod offset of the mass analyzing quadrupole (Q3).4 Cross sections (σ) were estimated by fitting E to
(
)
-Cdnm2lσ E ) exp E0 m1
(1)
where Cd is a drag coefficient for diffuse scattering; n, the argon number density in the cell; l, the collision cell length (20.6 cm); and m1 and m2, the masses of the lysozyme ions and argon, respectively. H/D Exchange of Gas-Phase Ions. The H/D exchange of gas-phase lysozyme ions was studied with a linear quadrupole ion trap reflectron time-of-flight mass spectrometer (LIT-rTOFMS) system, described previously.23 Ions generated by ESI passed through a curtain gas (N2), an orifice in a curtain plate, and an orifice-skimmer assembly to enter a chamber containing two quadrupoles operated in RF-only mode. Nitrogen from the source region flowed into the chamber to give a background pressure of 2.4 mTorr. The first quadrupole (Q0) guided the ions to the second quadrupole (Q1), where ions were trapped radially by the RF voltage between the rods and axially by timed stopping potentials applied to aperture plates at the entrance and exit. The orifice skimmer voltage difference was +175 V. The RF voltage on Q1 (LIT) was 411 Vp-p (pole-to-ground), giving Mathieu parameters (q) between 0.085 and 0.170 for Lys + nH+ ions (n ) 6-12). For H/D exchange, D2O was introduced continuously into the trap through a 0.9-mm-diameter nozzle, 4 mm radially out from the center of the trap.24,25 The D2O vapor formed a free jet to produce a locally high density of reagent over a length of ∼40 mm in the ion trap. The background D2O pressure in the trap chamber was 2.6 mTorr measured by a precision capacitance manometer (model 120AA, MKS Instruments, Andover, MA). Integration of the number density of the free jet of D2O over the length of the trapping quadrupole (20 cm) gives an equivalent background pressure from the free jet of 0.4 mTorr.24,25 Therefore, the effective D2O number density in the trap was equivalent to a pressure of 3.0 mTorr, the sum of the average number densities in the trap from the free jet and from the background D2O. Ions accumulated in Q0 in the previous trapping cycle were emptied from Q0 and Q1 for 100 ms by keeping both the entrance and the exit aperture plates at low potentials. The exit aperture plate (22) Douglas, D. J.; French, J. B. J. Am. Soc. Mass Spectrom. 1992, 3, 398-408 (23) (a) Campbell, J. M.; Collings, B. A.; Douglas, D. J. Rapid Commun. Mass Spectrom. 1998, 12, 1463-1474. (b) Collings, B. A.; Campbell, J. M.; Mao, D.; Douglas, D. J. Rapid Commun. Mass Spectrom. 2001, 15, 1777-1795. (24) Mao, D.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 2003, 14, 85-94. (25) Mao, D.; Ding, C.; Douglas, D. J. Rapid Commun. Mass Spectrom. 2002, 16, 1941-1945.
Figure 1. Mass spectra of lysozyme in (a) water and (b) water/ methanol (v/v ) 2/8) near pH ) 2.0. Numbers denote the charge states of ions.
voltage was then increased, and ions were accumulated in Q1 for 50 ms. Following this, ions were confined in Q1 for up to 10 s by raising the entrance aperture plate potential. H/D exchange mainly occurred in this period. Ions leaving the ion trap for 50 ms passed through a stack of lenses and entered the acceleration region of an orthogonally coupled rTOFMS for mass analysis. RESULTS Charge State Distributions. Mass spectra of lysozyme near pH 2.0 in water and in water/methanol (v/v ) 2/8), recorded with the rTOFMS, are shown in Figure 1. Spectra obtained with the single and triple quadrupole mass spectrometers are similar. In water (0% methanol) where the protein is in its native conformation in solution, a charge state distribution ranging from +7 through +12 with the maximum intensity at +10 is produced (Figure 1a). In 80% methanol, where the protein is in the helical denatured state, similar charges are produced, but the maximum intensity shifts to the +7 peak. In contrast to other proteins in methanol,26 lysozyme did not show a bimodal charge state distribution at any methanol concentration. Solution H/D Exchange. Neutral disulfide-intact lysozyme has 255 exchangeable hydrogens. The hydrogen exchange levels of lysozyme in D2O and D2O/CD3OD (2/8) at different exchange times at pD ) 2.0 are shown in Figure 2. The initial exchange rate of the protein in D2O is greater than that in D2O/CD3OD (2/8) when the exchange time is less than ∼0.2 h. However, lysozyme in D2O exchanges more slowly than that in D2O/CD3OD (2/8) at longer exchange times (>0.2 h). After ∼50 h of exchange, on average from two repeated experiments, lysozyme in the native state exchanges 172 ( 3 hydrogens (exchange percent, 67%), but lysozyme in the helical denatured state exchanges 209 ( 4 hydrogens (exchange percent, 82%). (26) (a) Babu, K. R.; Moradian, A.; Douglas, D. J. J. Am. Soc. Mass Spectrom. 2001, 12, 317-328. (b) Babu, K. R.; Douglas, D. J. Biochemistry 2000, 39, 14702-14710.
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Figure 2. Hydrogen exchange levels of lysozyme in water (D2O, O) and water/methanol (D2O/CD3OD, v/v ) 2/8, 4) at pD ) 2.0 at various exchange time intervals, determined from the +10 ion in the mass spectrum. Figure 4. Mass spectra of lysozyme +10 ions produced from 100% water at pH ) 2.1 at (a, b) 0, (c) 1, (d) 5, (e) 7, and (f) 10 s of trapping. The pressures in the trap chamber were (a) 5.0 mTorr of nitrogen, and (b-f) 2.4 mTorr of nitrogen and 2.6 mTorr of D2O.
Figure 3. Cross sections of lysozyme ions produced from water (b) and water/methanol (v/v ) 2/8) (4) near pH ) 2.0.
Cross Section Measurements. The collision cross sections of lysozyme ions in charge states +6 to +12 produced from the N and H states are shown in Figure 3. The error bars in the figure are the standard deviations of four repeat measurements. The cross sections vary from 990 Å2 for the +6 ions to 1500 Å2 for the +12 ions. The cross sections increase with charge and the +6 and +7 ions have significantly smaller cross sections than the higher charge state ions. For a given charge state, and within the experimental uncertainty, the cross sections of the lysozyme ions produced from the N state are the same as those produced from the H state. Gas-Phase H/D Exchange. Mass spectra of +10 ions of lysozyme produced from water at pH 2.1, trapped for various times in the presence of D2O vapor, are shown in Figure 4. The mass spectrum of lysozyme with no trapping and with 5.0 mTorr of N2 is also shown (Figure 4a). As the lysozyme ions exchange hydrogen for deuterium, the width of the isotopic distribution increases, and the peak increases in mass. The levels of hydrogen exchange with D2O of lysozyme +10 ions produced from the N and H states, at various trapping times, are shown in Figure 5a. Even without trapping, the ions exchange up to 26 hydrogens, and the peak broadens somewhat, because ions accumulate in the quadrupole for 50 ms before the trapping begins. During this period, ions can undergo H/D exchange, as 1328 Analytical Chemistry, Vol. 75, No. 6, March 15, 2003
Figure 5. (a) Hydrogen exchange levels of lysozyme +10 ions generated from water (b) and water/methanol (v/v ) 2/8) (4) vs trapping time. (b) Hydrogen exchange levels of gas-phase lysozyme ions produced from water (b) and water/methanol (4) solutions at pH ) 2.1.
discussed previously.24,25 Lysozyme ion intensities from water were only 20% of those from water/methanol (2/8). Ion signals became weak, and signal-to-noise ratios were poor at trapping times >10 s. Therefore, only results up to 10 s are discussed here. Figure 5a shows that after 10 s of trapping with D2O, the +10 ion produced from the native state has 71 ( 7 hydrogens replaced,
whereas that from the H state has 63 ( 3 hydrogens incorporated. The quoted uncertainties are the standard deviations of five repeat measurements. The H/D exchange levels of the +8 to +11 ions from water and the +6 to +12 ions from water/methanol (2/8) were measured, and the exchange levels after 10 s of trapping are shown in Figure 5b. (The ion signals from the +6, +7, and +12 ions from water were too weak to allow gas-phase exchange experiments.) On average, the +8 to +11 ions from the native state have nine more hydrogens exchanged than ions of the same charge produced from the H state. However this difference is close to the combined experimental errors of the data. At all trapping times, and for each charge state, only one peak was observed in the mass spectra. DISCUSSION Charge States. Figure 1 shows that native lysozyme in water produces a charge state distribution with a maximum intensity at the +10 ion. When the four disulfide bonds of lysozyme are reduced, it unfolds in solution and produces a charge state distribution in ESI that is centered near the +14 or +15 ion.11-16 In contrast to this, the helical denatured state produces the same charge states as the native conformation, although with different relative intensities. This also contrasts to the helical denatured states of cytochrome c, β-lactoglobulin, ubiquitin,26a and myoglobin,26b which produce higher charges than the native states. At intermediate methanol concentrations, proteins can show a bimodal charge state distribution, suggesting an equilibrium between folded and unfolded conformations in solution.26 Lysozyme, however, does not exhibit a bimodal distribution at any methanol concentration near pH 2.0, presumably because the N and H states have similar charge state distributions. Evidently, the four disulfide bonds of lysozyme allow the H state to remain sufficiently folded that a substantial shift in the charge state distribution does not occur. Because the same charge states are produced by the N and H states, lysozyme provides an opportunity to study the properties of the gas-phase protein ions produced directly from two very different solution conformations. H/D Exchange (Solution). In solution, unfolded denatured proteins exchange more hydrogens than tightly folded native proteins.12,26-28 Figure 2 shows that after 50 h, native lysozyme at pH 2.0 has exchanged 177, or 69%, of its exchangeable hydrogens. This is somewhat higher than observed by Katta and Chait, who observed exchange of 159 (62%) of the labile hydrogens in 30 min in 1% CH3COOD/D2O12 and Przybylski and Glocker who reported exchange of 132 hydrogens (52%) in 1% CH3COOD/D2O with an exchange time of 12 h.13 King et al., in a study of the effects of substrate binding on lysozyme H/D exchange, found the free protein exchanged 196 hydrogens in D2O/H2O (1/1).29 The differences between these experiments may reflect different solvents, different pH, and different exchange times. Under the conditions used here, lysozyme in water/methanol (2/8) has 37 more hydrogens incorporated (for a total of 209 or 82%) than lysozyme in water. Therefore, H/D exchange also shows that (27) Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1991, 5, 214-217. (28) (a) Wagner, D. S.; Anderegg, R. J. Anal. Chem. 1994, 66, 706-711. (b) Miranker, A.; Robinson, C. V.; Radford, S. E.; Dobson, C. M. FASEB J. 1996, 10, 93-101. (c) Eyles S. J.; Dresch, T.; Gierasch, L. M.; Kaltashov, I. A. J. Mass Spectrom. 1999, 34, 1289-1295. (29) King, D.; Lumpkin, M.; Bergmann, C.; Orlando, R. Rapid Commun. Mass Spectrom. 2002, 16, 1569-1574.
lysozyme in the H state has a less protected structure than in water, consistent with the data of Kamatari et al.10 Katta and Chait found that disulfide-reduced lysozyme exchanged 252, or 96%, of 263 possible hydrogens.12 Pryzybylski and Glocker found reduced lysozyme exchanged 227 hydrogens (86%) under the conditions that native lysozyme exchanges 132.13 Thus, in the H state, the number of hydrogens exchanged is not as great as that in the fully reduced and denatured protein, possibly because some hydrogens are protected in non-native helices.26 Helical denatured states of cytochrome c, β-lactoglobulin and ubiquitin,26a and myoglobin26b in 80% methanol show somewhat higher exchange levels than the native conformation. However, at still higher methanol concentrations, the helix content can increase, and at 90% methanol, the exchange level can decrease to be similar to that of the native protein.26 Solution-phase H/D exchange of proteins has been classified into two mechanisms.28 If there are two protein conformations that exchange at different rates and interconvert more slowly than the exchange rate, two peaks can be expected in a mass spectrum (EX1 mechanism). If the conformations interconvert more rapidly than the exchange rate, only one peak is observed (EX2 mechanism). A single peak was obtained from the protein in both D2O and D2O/CD3OD during the entire exchange time. This indicates either that both native and denatured lysozyme have only one conformation or that if multiple conformers exist, they interconvert rapidly compared to the exchange rate. Cross Sections. Ion mobilities of native and reduced lysozyme ions have been measured and converted to cross sections using a formula that assumes hard sphere collisions.16 Both a consideration of diffuse and specular scattering models21 and direct scattering calculations30 show that the hard sphere approximation can overestimate cross sections by 20-25%. The diffuse scattering model used here gives cross sections similar to the direct scattering model used to interpret mobility experiments.21 The cross sections of the +8 to +10 ions determined here are very close to those determined by ion mobility experiments that assume hard sphere scattering. This agreement may be partly fortuitous, because different scattering dynamics are assumed in interpreting the energy loss experiments. The cross sections here are close to the cross sections for the most compact conformers seen in the mobility experiments (as shown in Figure 7 of ref 16). However, the cross sections here show a greater increase with charge, with the +10 ion having a cross section 54% greater than that of the +6 ion. The difference may result from differences in the ion activation in the two instruments, differences in the scattering behavior at the different ion energies in mobility and energy loss experiments, different solution conditions, and the different time scales of the experiments. As well, the mobility experiments showed unresolved conformers that have greater cross sections than the mobility cross sections used for the comparison here.16 These conformers would not be resolved in our energy loss experiments, and if similar conformers are present in this experiment, the cross sections here would contain contributions from several conformations to give larger cross sections than the most compact conformations of the mobility experiments. (30) Shelimov, K. B.; Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2987-2994.
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From Figure 3, it is evident that gas-phase lysozyme ions produced from the N and H states in solution have very similar cross sections. These cross sections correspond to compact structures. The crystal structure has been estimated16 to give a cross section of 1180 Å2, and the cross section calculated from the radius of gyration of the native conformation is 1290 Å2 (σ ) 5/ πr2 ).31 In contrast, the cross section of the H state, calculated 3 g from the radius of gyration, is 3246 Å2 and that of lysozyme stretched into a string16 is 3750 Å2. Thus, the lysozyme ions formed from these two solution conformations have gas-phase conformations similar in “size” to the solution or crystal structures. Ions formed from the H state must refold from solution structures with cross sections of ∼3246 Å2 to structures with cross sections of 1000-1500 Å2 on the time scale of the cross section measurement (∼1 ms) or less. H/D Exchange (Gas Phase).Figure 5b shows that ions produced from the native and H states in solution have very similar hydrogen exchange levels. The cross sections of Figure 3 show that the +6 and +7 ions are ∼250 Å2 smaller than the +8 to +12 ions (∼20% smaller). The +6 and +7 ions exchange ∼20 fewer hydrogens than the +8 to +12 ions (∼28% less). Thus, both the cross sections and H/D exchange suggest that the +6 and +7 ions are significantly more compact than the +8 - +12 ions. Green and Lebrilla also found an increase in exchange level of ions of disulfide-intact lysozyme with charge state.32 This was partially attributed to the lower reactivity of arginine residues relative to other basic residues. However, the exchange level in that experiment was much lower than in this work because of the much lower pressure of deuterating reagent (1 × 10-7 Torr of CH3OD). Although multiple conformers were seen in ion mobility experiments, only a single peak is observed in all of the exchange experiments here. Thus, if multiple conformers are present, they have similar exchange levels or interconvert rapidly relative to the seconds time scale of the exchange experiment (similar to the EX2 mechanism in solution). The widths of the peaks in Figure 4 provide some indirect evidence for multiple conformations. As a protein ion exchanges hydrogens, the width of the isotopic peak broadens, but then narrows again as the exchange approaches completion (e.g., myoglobin +12 ions25). (31) Glatter, O.; Kratzky, O. Small Angle X-ray Scattering; Academic Press: New York, 1982; p 156. (32) Green, M. K.; Lebrilla, C. B. Int. J. Mass Spectrom. Ion Proc. 1998, 175, 15-26. (33) Campbell, S.; Rodgers, M. T.; Marzluff, E. M.; Beauchamp, J. L. J. Am. Chem. Soc. 1995, 117, 12840-12854. (34) Goldberg, M. E.; Guillou, Y. Protein Sci. 1994, 3, 883-887. (35) Arteca, G. A.; Reimann, C. T.; Tapia, O. Chem. Phys. Lett. 2001, 350, 277285 and ref 18.
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The peaks of Figure 4f remain quite broad relative to the unexchanged protein (Figure 4a), even though the data of Figure 5a suggest that the exchange is approaching completion between 5 and 10 s. The peak-broadening might be due to unresolved conformations that exchange slightly different numbers of hydrogens. The mechanism of H/D exchange of protein ions in the gas phase is less well understood than the mechanism of solution exchange. For peptides, Campbell et al. have proposed several mechanisms of gas-phase H/D exchange.33 The H/D exchange of proteins may be more complex. Nevertheless, by analogy to solution-phase exchange, it is plausible that more tightly folded states will exchange lower numbers of hydrogens than more open unfolded states. Notably, in this work, the higher charge states, which have greater cross sections, also exchange somewhat greater numbers of hydrogens. Although the relation between folding and exchange level is uncertain, different exchange levels and exchange rates likely indicate different conformations and similar exchange rates and levels suggest similar conformations. Thus, as probed by H/D exchange, lysozyme ions produced from the N and H states have similar gas-phase conformations. The results here show that disulfide-intact lysozyme unfolded in solution produces compact ions in the gas phase. This contrasts with disulfide-reduced lysozyme that produces more highly charged ions with substantially greater cross sections. The greater cross sections of the more highly charged ions are usually attributed to greater Coulomb repulsion in the ions. It is possible the disulfide bonds allow the protein to retain sufficient structure that it refolds more quickly in ESI34 or has access to additional folding pathways that allow it to refold.35 The net effect is that ions produced from lysozyme unfolded in solution largely lose memory of the solution structure. ACKNOWLEDGMENT This work was supported through a Natural Sciences and Engineering Research Council Discovery Grant and a Natural Sciences and Engineering Research Council-SCIEX Industrial Research Chair. NOTE ADDED IN PROOF Evans et al. have recently reported studies of H/D exchange of protein ions in a 3-D ion trap (Int. J. Mass Spectrom. 2003, 222, 175-187). Received for review October 17, 2002. Accepted January 10, 2003. AC020647X
