γ-Ray-Mediated Oxidative Labeling for Detecting Protein

Anal. Chem. 2008, 80, 2222-2231

γ-Ray-Mediated Oxidative Labeling for Detecting Protein Conformational Changes by Electrospray Mass Spectrometry Xin Tong, J. Clara Wren, and Lars Konermann*

Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada

Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of γ-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent firstorder rate constant for oxidative labeling can be approximated as kapp ) kRAD/([P]tot + C/ku), where kRAD is the primary rate of •OH formation, [P]tot is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model N rikch by proposing that ku ) ∑i)1 i , where N is the number of amino acids, ri is a measure for the solvent exposure of residue i, and kch i is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H3PO4 increases kapp by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such “secondary” effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall •OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known to be minimal and where [P]tot is constant. Under physiological conditions, most proteins adopt a tightly folded structure, representing the lowest possible free energy of the polypeptide chain and the surrounding solvent.1-3 Exposure * Corresponding author. Phone: (519) 661-2111 ext. 86313. Fax: (519) 6613022. E-mail: [email protected]. Web: http://publish.uwo.ca/∼konerman. (1) Anfinsen, C. B. Science 1973, 181, 223-230. (2) Kresge, N.; Simoni, R. D.; Hill, R. L. J. Biol. Chem. 2006, 281, e11-e13.

2222 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

to denaturants such as acid, urea, and heat or the removal of strongly bound cofactors can result in protein unfolding.1 A number of spectroscopic and calorimetric techniques are available for monitoring these structural changes.4 Nonetheless, many aspects of the protein folding problem remain poorly understood, in part because most existing structure determination methods cannot easily be applied to denatured and partially folded polypeptide chains.5-7 Mass spectrometry (MS)-based techniques have become an indispensable tool for monitoring protein folding, conformational dynamics, and interactions with biomolecular binding partners.8 Many of these approaches employ labeling steps in bulk solution, making use of the fact that tightly folded protein regions afford a certain degree of protection, whereas amino acids located in solvent-exposed and unfolded areas are usually more reactive. The readout of these experiments is based on mass shifts caused by the binding of the labeling agent to the protein. Spatially resolved information can be obtained by incorporating proteolytic peptide mapping, often in combination with tandem mass spectrometry. Hydrogen/deuterium exchange (HDX) represents the most widely used labeling technique.9-20 Hydrogens in O-H, N-H, and S-H groups can be replaced with deuterium upon exposure to a (3) Dill, K. A.; Chan, H. S. Nat. Struct. Biol. 1997, 4, 10-19. (4) Pain, R. H. Mechanisms of Protein Folding, 2nd ed.; Oxford University Press: New York, 2000. (5) Mittermaier, A.; Kay, L. E. Science 2006, 312, 224-228. (6) Han, J.-H.; Batey, S.; Nickson, A. A.; Teichmann, S. A.; Clarke, J. Nat. Rev. Mol. Cell. Biol. 2007, 8, 319-330. (7) Naganathan, A. N.; Doshi, U.; Fung, A.; Sadqi, M.; Munoz, V. Biochemistry 2006, 45, 8466-8475. (8) Kaltashov, I. A.; Eyles, S. J. Mass Spectrometry in Biophysics; John Wiley and Sons, Inc.: Hoboken, NJ, 2005. (9) Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R.; Dobson, C. M. Science 1993, 262, 896-900. (10) Mandell, J. G.; Falick, A. M.; Komives, E. A. Anal. Chem. 1998, 70, 39873995. (11) Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296-8301. (12) Englander, S. W. J. Am. Soc. Mass Spectrom. 2006, 17, 1481-1489. (13) Eyles, S. J.; Kaltashov, I. A. Methods 2004, 34, 88-99. (14) Liang, Z.-X.; Lee, T.; Resing, K. A.; Ahn, N. G.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9556-9561. (15) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158-170. (16) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135-146. (17) Pan, J. X.; Rintala-Dempsey, A.; Li, Y.; Shaw, G. S.; Konermann, L. Biochemistry 2006, 45, 3005-3013. (18) Woodward, C. J. Am. Soc. Mass Spectrom. 1999, 10, 672-674. (19) Chik, J. K.; Schriemer, D. C. J. Mol. Biol. 2003, 334, 373-385. (20) Lanman, J.; Lam, T. T.; Barnes, S.; Sakalian, M.; Emmett, M. R.; Marshall, A. G.; Previlege, P. E. J. Mol. Biol. 2003, 325, 759-772. 10.1021/ac702321r CCC: $40.75

© 2008 American Chemical Society Published on Web 02/09/2008

D2O-containing solvent. Most HDX studies are concerned with amide backbone exchange. HDX at sites that are located in the protein interior and involved in hydrogen bonds is mediated by short-lived unfolding (opening/closing) events that induce the transient disruption of H-bonds and permit solvent access.21-24 The exchange mechanism is given by22 kop

kch

X - Hclosed {\ } X - Hopen 9 8 exchanged k DO cl

2

(1)

where kop and kcl are the rate constants for the opening and closing, respectively, of a particular exchangeable site. The “chemical” rate constant kch describes the exchange kinetics for a fully unprotected site.21,25 In the commonly encountered EX2 regime,26 characterized by kcl . kch, the overall HDX rate constant is given by

kex )

kop k kcl ch

(2)

Advantages of HDX techniques include their noninvasive nature and conceptual simplicity. Regardless of the amino acid side chains adjacent to an amide group, the chemical change is always the same (N-H f N-D). This is in contrast to some other labeling methods where competing reaction pathways and more than a single product may be encountered. In addition, HDX rates can be conveniently modulated through pD changes, taking advantage of the dependence of kch on [D+] and [OD-].15,16,25,27 On the other hand, isotope back exchange is a potentially complicating factor that has to be accounted for through suitable control experiments.28 Also, H/D scrambling represents an impediment in studies using gas-phase fragmentation for spatially resolved isotope labeling experiments.29,30 Covalent labeling methods represent an interesting alternative for mapping solvent-accessible areas of proteins. For example, acetylation of Lys side chains is a widely used strategy,31 but a host of reagents targeting other residues (e.g., Cys, Arg, Tyr, Asp, Glu) are available as well.8,32,33 For all of these approaches, the permanent nature of the chemical modifications greatly facilitates the localization of labeled sites, when compared to HDX experiments. Hydroxyl radicals (•OH) represent a relatively nonspecific (21) Hvidt, A.; Nielsen, S. O. Adv. Protein Chem. 1966, 21, 287-386. (22) Krishna, M. M. G.; Hoang, L.; Lin, Y.; Englander, S. W. Methods 2004, 34, 51-64. (23) Maity, H.; Lim, W. K.; Rumbley, J. N.; Englander, S. W. Protein Sci. 2003, 12, 153-160. (24) Li, R.; Woodward, C. Protein Sci. 1999, 8, 1571-1590. (25) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins: Struct., Funct., Genet. 1993, 17, 75-86. (26) Miranker, A.; Robinson, C. V.; Radford, S. E.; Dobson, C. M. FASEB J. 1996, 10, 93-101. (27) Konermann, L.; Simmons, D. A. Mass Spectrom. Rev. 2003, 22, 1-26. (28) Wu, Y.; Kaveti, S.; Engen, J. R. Anal. Chem. 2006, 78, 1719-1723. (29) Jorgensen, T. J. D.; Gardsvoll, H.; Ploug, M.; Roepstorff, P. J. Am. Chem. Soc. 2005, 127, 2785-2793. (30) Ferguson, P. L.; Pan, J.; Wilson, D. J.; Dempsey, B.; Lajoie, G.; Shilton, B.; Konermann, L. Anal. Chem. 2007, 79, 153-160. (31) Wang, X.; Kim, S.-H.; Ablonczy, Z.; Crouch, R. K.; Knapp, D. R. Biochemistry 2004, 43, 11153-11162. (32) Abramczyk, O.; Rainey, M. A.; Barnes, R.; Martin, L.; Dalby, K. N. Biochemistry 2007, 46, 9174-9186. (33) Hager-Braun, C.; Tomer, K. B. Biochemistry 2002, 41, 1759-1766.

covalent modifier.34-40 The reactivity of individual side chains with •OH is determined by their solvent accessibility and by their intrinsic reactivity.35 The sulfur-containing residues Cys and Met are most reactive, followed by the aromatic side chains of Trp, Tyr, and Phe. Also His, Leu, Ile, Arg, Lys, Val, Pro, Gln, and Glu represent potential modification sites, whereas the remaining residues are less reactive.41-45 Most hydroxl radical-induced side chain modifications result in the incorporation of oxygen, corresponding to mass shifts of +16 Da or multiples thereof.35,45 While the use of •OH for nucleic acid footprinting is well established,46,47 the application of this labeling agent to proteins is still at a relatively early stage of development. For example, the dramatic dependence of the oxidation kinetics on the protein concentration has not been systematically investigated until recently.48 The chemical modifications resulting from •OH exposure are thought to be largely independent of the procedure employed for radical production.35,41,49 One of the best characterized approaches is the radiolysis of water by synchrotron X-ray pulses, offering the possibility to carry out footprinting studies with a time resolution in the millisecond range.50 Water radiolysis by γ-rays (usually from 60Co or 137Ce) requires longer exposure times; however, γ-ray sources have the advantage that they are more widely accessible.34,40,51,52 Another elegant method is the homolytic cleavage of hydrogen peroxide by UV light.53 When used in combination with a pulsed laser, this approach permits labeling times as short as a few microseconds.54-56 Alternatively, reactive oxygen species may be generated by Fenton chemistry,38,57 in the vicinity of endogenous transition metal ions,39,58,59 or in the ion source of electrospray ionization (ESI) mass spectrometers under (34) Nukuna, B. N.; Sun, G.; Anderson, V. E. Free Radical Biol. Med. 2004, 37, 1203-1213. (35) Takamono, K.; Chance, M. R. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 251-276. (36) Maleknia, S. D.; Downard, K. Mass Spectrom. Rev. 2001, 20, 388-401. (37) Hambly, D. M.; Gross, M. J. Int. J. Mass Spectrom. 2007, 259, 124-129. (38) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Anal. Biochem. 2003, 313, 216225. (39) Lim, J.; Vachet, R. W. Anal. Chem. 2003, 75, 1164-1172. (40) Sharp, J. S.; Sullivan, D. M.; Cavanagh, J.; Tomer, K. B. Biochemistry 2006, 45, 6260-6266. (41) Xu, G.; Chance, M. R. Anal. Chem. 2004, 76, 1213-1221. (42) Xu, G.; Kiselar, J.; He, Q.; Chance, M. R. Anal. Chem. 2005, 77, 30293037. (43) Xu, G.; Chance, M. R. Anal. Chem. 2005, 77, 4549-4555. (44) Xu, G.; Chance, M. R. Anal. Chem. 2005, 77, 2437-2449. (45) Xu, G.; Chance, M. R. Chem. Rev. 2007, 107, 3514-3543. (46) Sclavi, B.; Sullivan, M.; Chance, M. R.; Brenowitz, M.; Woodson, S. A. Science 1998, 279, 1940-1943. (47) Lipfert, J.; Das, R.; Chu, V. B.; Kudaravalli, M.; Boyd, N.; Herschlag, D.; Doniach, S. J. Mol. Biol. 2007, 365, 1393-1406. (48) Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382. (49) Xu, G.; Takamoto, K.; Chance, M. R. Anal. Chem. 2003, 75, 6995-7007. (50) Maleknia, S. D.; Brenowitz, M.; Chance, M. R. Anal. Chem. 1999, 71, 39653973. (51) Sharp, J. S.; Tomer, K. B. Biophys. J. 2007, 92, 1682-1692. (52) Mousseau, G.; Thomas, O. P.; Oppilliart, S.; Coirier, A.; Salcedo-Serna, A.; Thai, R.; Beau, F.; Renault, J.-P.; Pin, S.; Cintrat, J.-C.; Rousseau, B. Anal. Chem. 2007, 79, 5444-5448. (53) Sharp, J. S.; Becker, J. M.; Hettich, R. L. Anal. Chem. 2004, 76, 672-683. (54) Hambly, D. M.; Gross, M. J. J. Am. Soc. Mass Spectrom. 2005, 16, 20572063. (55) Hambly, D. M.; Gross, M. Internat. J. Mass Spectrom. 2007, 259, 124129. (56) Aye, T. T.; Low, T. Y.; Sze, S. K. Anal. Chem. 2005, 77, 5814-5822. (57) Heyduk, E.; Heyduk, T. Biochemistry 1994, 33, 9643-9650. (58) Bridgewater, J. D.; Lim, J.; Vachet, R. W. Anal. Chem. 2006, 78, 24322438. (59) Lim, J.; Vachet, R. W. Anal. Chem. 2004, 76, 3498-3504.

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corona discharge conditions.36 Many previous protein •OH footprinting applications have focused on the mapping of proteinprotein interfaces.31,33,35,41,49,60-62 These experiments make use of the fact that the formation of intermolecular noncovalent contacts reduces the solvent accessibility of residues at the interface, thereby suppressing their oxidative labeling. Despite the potential of oxidative modifications to cause structural damage, the magnitude of conformational changes caused by •OH labeling has been found to be surprisingly small.36,48,56,63 Similar to intermolecular binding events, protein folding and unfolding transitions are associated with dramatic changes in the solvent exposure of amino acid side chains, particularly those that form the hydrophobic core of globular proteins.4,64 This core typically encompasses many aromatic residues that exhibit an intrinsically high reactivity toward hydroxyl radicals.41-45 Thus, folded and unfolded protein conformations would be expected to be easily distinguishable based on their oxidation level,62 in a manner that is comparable to the isotope exchange behavior in HDX experiments.9,45 Surprisingly, there are only few reported attempts to use •OH labeling for studies of this kind.55,65-67 This work addresses the question under what conditions the overall labeling level represents a viable probe of large scale protein structural changes. Previous studies provide a somewhat inconclusive picture, where alterations of the protein conformation either result in quite pronounced changes in overall labeling65,66 or in hardly any change at all.55 Because the ongoing development of oxidative labeling methods is at least partly inspired by advancements in HDX technology,45 it is interesting to highlight some of the conceptual analogies and differences of the two methods. The current study demonstrates that •OH labeling is complicated by the fact that commonly used denaturants and solvent additives act as highly effective radical scavengers, often masking effects caused by conformational transitions. Only under carefully controlled conditions is it possible to distinguish different protein conformers based on their overall oxidation level. EXPERIMENTAL PROCEDURES Materials. Ferri-holo-myoglobin (horse skeletal muscle) and cytochrome c (horse heart, average mass 12 360 Da) were purchased from Sigma, St. Louis, MO. Bradykinin (RPPGFSPFR, monoisotopic mass 1059.6 Da) was from Bachem Bioscience, King of Prussia, PA. Apo-myoglobin (aMb) was produced from the holoprotein (hMb) by butanone extraction at pH 2, followed by extensive dialysis against water (pH 7) using Slide-A-Lyzer cassettes (Pierce, Rockford, IL) with a nominal 7 kDa molecular weight cutoff.68 Small amounts of insoluble debris were removed by centrifugation. Protein concentrations were adjusted based on UV-vis measurements, using 408 ) 188 000 M-1 cm-1 for hMb (60) Gerega, S. K.; Downard, K. M. Bioinformatics 2006, 22, 1702-1709. (61) Guan, J. Q.; Takamono, K.; Almo, S. C.; Reisler, E.; Chance, M. R. Biochemistry 2005, 44, 3166-3175. (62) Maleknia, S. D.; Wong, J. W. H.; Downard, K. M. Photochem. Photobiol. Sci. 2004, 3, 741-748. (63) Wong, J. W. H.; Maleknia, S. D.; Downard, K. M. Anal. Chem. 2003, 75, 1557-1563. (64) Myers, J. K.; Pace, C. N.; Schotz, J. M. Protein Sci. 1995, 4, 2138-2148. (65) Chance, M. R. Biochem. Biophys. Res. Commun. 2001, 287, 614-621. (66) Maleknia, S. D.; Downard, K. M. Eur. J. Biochem. 2001, 268, 5578-5588. (67) Kiselar, J. G.; Janmey, P. A.; Almo, S. C.; Chance, M. R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3942-3947. (68) Teale, F. W. J. Biochim. Biophys. Acta 1959, 35, 543.

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and 280 ) 13 980 M-1 cm-1 for aMb.69 D2O was obtained from Cambridge Isotope Laboratories. Ammonium acetate and formic acid were bought from Fluka, Buchs, Switzerland. The AB15 pH meter and all other chemicals were from Fisher Scientific (Nepean, ON, Canada). Measured pD values for D2O-containing solutions were corrected for isotope effects based on the relationship pD ) pHread + 0.4. UV-vis absorption spectra were recorded on a Cary 100 spectrophotometer (Varian, Mississauga, ON, Canada). On-Line Continuous Hydrogen/Deuterium Exchange. HDX was initiated by exposing 5 µM protein to 85:15 (v/v) D2O/ H2O at pD 7. The mixture was infused on-line into the ion source of the mass spectrometer at a flow rate of 5 µL min-1 using a syringe pump (Harvard Apparatus, South Natick, MA). Mass shifts ∆M were determined from the most intense peak in the spectra by converting the mass-to-charge ratio values of the original data according to ∆M ) (rz) - mcharge - M0, where r is the mass-tocharge ratio, z is the charge state of the ion, mcharge is the combined mass of the z charge carriers (protons and deuterons, their numbers being determined by the D2O to H2O ratio in solution), and M0 is the mass of the unlabeled protein. The M0 values and number of exchangeable hydrogens for hMb and aMb are 17 568/ 264 and 16 952/262, respectively.70 The reported relative HDX levels were normalized to the number of exchangeable hydrogens and corrected for the 85% D2O content of the solution. γ-Ray Exposure. Oxidative labeling was conducted under aerobic conditions at room temperature. All experiments were performed using an MDS Nordion 220 Excel Gammacell which has 60Co pencils as the radiation source, providing a uniform radiation field in the sample compartment. The absorbed dose in water, measured by Fricke dosimetry, was 2.4 Gy s-1, corresponding to a primary hydroxyl radical production rate of kRAD ) 0.67 µM s-1. An amount of 0.25 mL of aqueous protein solution was placed in a closed Eppendorf cup (head space volume 1.75 mL) in the center of the sample compartment. The sample was then lowered by an automated mechanism into the lead-shielded radiation zone and retrieved after a predetermined time. All samples were flash-frozen in liquid nitrogen immediately after irradiation and stored at -80 °C in order to quench any secondary oxidation reactions.48 The pH of the solutions was stable before and after irradiation to within 0.2 units, even in unbuffered solutions. Mass Spectrometry. ESI mass spectra were acquired on a Q-TOF Ultima API mass spectrometer (Waters/Micromass) equipped with a Z-spray ESI source, using the positive ion mode at a sprayer voltage of 3 kV and a desolvation temperature of 150 °C. For experiments on bradykinin and for comparative studies on native and acid-unfolded proteins, the mass spectrometer was coupled to a Waters 1525µ HPLC system employing a C4 (Symmetry 300) 2.1 mm × 100 mm reverse phase column. The proteins were eluted using a water/acetonitrile gradient in the presence of 0.05% trifluoroacetic acid at a flow rate of 135 µL min-1. Under these denaturing LC conditions, myoglobin is detected as apoprotein, regardless of the solution conditions used during irradiation. For HDX experiments, as well as for comparative studies on native hMb/aMb, all samples were analyzed by direct (69) Antonini, E.; Brunori, M. Hemoglobin and Myoglobin in Their Reactions With Ligands; North-Holland Publishing Company: Amsterdam, The Netherlands, 1971. (70) Wang, F.; Tang, X. Biochemistry 1996, 35, 4069-4078.

infusion. In the latter case, 1% acetic acid was added prior to injection. Deconvoluted mass distributions were obtained using MaxEnt software supplied by the instrument manufacturer. The extent of oxidative labeling is reported as the fraction of unmodified protein, Fu, determined from the deconvoluted mass distributions according to

Fu )

Aunmod Atot

(3)

where Aunmod is the integrated area of the single peak corresponding to the unmodified protein and Atot represents the area of the entire mass distribution. Fu can range from unity for nonirradiated controls down to almost zero for extensively oxidized samples. Peak tailing of the nonirradiated controls necessitates a slight correction to the denominator of eq 3, which also forms the basis of the reported errors. Details of this procedure are provided in ref 48. Fu values below approximately 0.04 could not be reliably determined and hence were omitted from the analysis (e.g., in Figures 6, 7, and 9). The oxidative labeling data discussed below were found to be highly reproducible when testing multiple (three or more) independently prepared protein samples. RESULTS AND DISCUSSION HDX and Covalent Labeling of Native hMb/aMb. For studying the effects of protein conformation on the extent of γ-raymediated oxidative labeling, we initially focus on myoglobin (Mb), a protein that has previously served as model system in several •OH labeling studies and numerous folding experiments.38,54,55,65,66 Native holo-myoglobin (hMb) possesses eight R-helices that form a hydrophobic pocket into which a heme group is bound. Unfolding under acidic conditions leads to disruption of the heme-protein interactions, thereby forming apo-myoglobin (aMb).71-73 At neutral pH, the heme-free protein adopts an overall fold resembling that of hMb. However, this “native” aMb is more structurally dynamic, and the heme binding region (including helix F) exhibits a certain degree of disorder.71,72 In neutral solution, the HDX properties of hMb and aMb are dramatically different. The deconvoluted mass distributions obtained after 50 min of deuterium in-exchange reveal two baselineseparated peaks (Figure 1A). The apoprotein approaches complete exchange after a few tens of minutes, whereas hMb retains about 20% of unexchanged hydrogens after several hours (Figure 1B). NMR experiments suggest that most of these highly protected sites are located in the A, G, and H helices,72 a result that is in agreement with chemical shift analyses.71 HDX data very similar to those depicted in Figure 1 have been reported previously;74 they are shown here to allow a comparison with the behavior of the two Mb forms in •OH labeling experiments. Deconvoluted mass distributions of aMb and hMb obtained after 80 s of γ-ray exposure at pH 7 are shown in Figure 2A. As a result of oxygen incorporation, the spectra reveal series of +16 Da adducts. The (71) Eliezer, D.; Yao, J.; Dyson, H. J.; Wright, P. E. Nat. Struct. Biol. 1998, 5, 148-155. (72) Hughson, F. M.; Wright, P. E.; Baldwin, R. L. Science 1990, 249, 15441548. (73) Sogbein, O. O.; Simmons, D. A.; Konermann, L. J. Am. Soc. Mass Spectrom. 2000, 11, 312-319. (74) Johnson, R. S.; Walsh, K. A. Protein Sci. 1994, 3, 2411-2418.

Figure 1. (A) Deconvoluted ESI mass distributions obtained after isotope exchange of 5 µM hMb and aMb in 85% D2O/15% H2O containing 5 mM ammonium acetate (pD 7) for 50 min. (B) Plot of the relative HDX level as a function of time.

extent of aMb labeling slightly exceeds that of hMb. Timedependent dose-response curves are depicted in Figure 2B. Kinetic Model. The approximately linear appearance of the semilogarithmic dose-response curves (Figure 2B) is consistent with the results of a kinetic model,48 which predicts that the time dependence of labeling is given by

Fu ) exp(-kappt)

(4)

with

kapp )

kRAD [P]tot + C/ku

(5)

where [P]tot is the total protein concentration and kRAD is the M primary hydroxyl radical production rate. C ) ∑j)1 rj[Cj] reflects the presence of M competing species Cj in solution that react with •OH at rate constants r . These other species include solvent j additives, as well as primary and secondary water radiolysis products.75 Equations 4 and 5 are based on the simplifying assumption that all these Cj instantly adopt steady-state concentrations. Slight deviations from linearity in the semilogarithmic plots of Figure 2B for t < 40 s suggest that the latter supposition may not be completely justified for early reaction times. The rate constant ku in eq 5 describes the reaction of •OH with the protein. (75) Wren, J. C.; Ball, J. M. Radiat. Phys. Chem. 2001, 60, 577-596.

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a conformational factor, i.e., steric protection (eq 6) vs an equilibrium constant that reflects how frequently a site resides in an open conformation (eq 2).21,76 For quantifying the extent to which protein denaturation affects the degree of oxidative labeling we introduce the parameter Q, defined as

Q)

kapp(non-native) kapp(native)

(7)

where kapp(non-native) and kapp(native) are the apparent rate constants measured under the corresponding solvent conditions (eq 4). When comparing results for a single time point, Q can be estimated according to

Q)

Figure 2. (A) Deconvoluted ESI mass distributions obtained after 80 s of γ-ray exposure in 5 mM aqueous ammonium acetate (pH 7) for 13 µM hMb (blue) and aMb (red). Some peaks are labeled according to the number of incorporated oxygen atoms. The data have been normalized, such that the peaks corresponding to unmodified protein (“0”) have the same intensity. (B) Fraction of unmodified protein, Fu, plotted vs γ-ray exposure time for hMb and aMb.

N In an earlier study,48 we used the notation ku ) ∑i)1 ki, where N is the number of amino acid residues and ki represents the oxidation rate constant of residue i. In order to explicitly consider protein conformational effects, it is useful to formulate the parameter ku in a slightly different way. The reactivity of side chains with •OH is determined by their solvent accessibility and their chemical nature,35,45,53 suggesting that it should be possible to express each of the ki values as ki ) ch Rikch i , where ki represents the “chemical” oxidation rate constant that would apply in the case of a completely unprotected side chain. The set of dimensionless parameters Ri (with 0 e Ri e 1) depends on the protein conformation. With the possible exception of Met,53 the R value of a given residue is expected to increase with its degree of solvent exposure. Within this model, ku in eq 5 becomes

N

ku )

∑ Rk

ch i i

(6)

i)1

Protein unfolding will increase the Ri values, thereby enhancing ku, and resulting in an elevated level of oxidative labeling due to a larger kapp (eq 5). This proposed framework bears conceptual analogies to the HDX formalism under EX2 conditions. In both cases, the chemical reactivity of individual sites is modulated by 2226

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ln(Fu(non-native)) ln(Fu(native))

(8)

This expression, which follows from eq 4, also allows the quantitation of labeling differences in cases where dose-response curves exhibit deviations from exponential behavior. Q > 1 represents the case where denaturation enhances the degree of oxidation, whereas in the absence of any labeling differences Q ) 1. The comparative measurements depicted in Figure 2, with Q ) 1.2 ( 0.1, were carried out at the same protein concentration and under identical solvent conditions, such that [P]tot and C in eq 4 are constant. Hence, the slightly elevated oxidation levels for native aMb are consistent with a certain degree of unfolding compared to native hMb,71,74,77 resulting in small changes in the overall Ri pattern (eq 6). The reduced steric shielding in the heme binding region upon cofactor removal may also play a role. An earlier •OH footprinting study reported the overall hMb/aMb labeling levels to be virtually identical.55 Figure 2 suggests that the two forms are distinguishable when using very carefully controlled conditions. The magnitude of the differences in oxidative labeling, however, is exceedingly small. Only for the t ) 160 s time point do the error bars of comparative measurements not overlap. This is in striking contrast to the dramatic differences seen in the HDX data of Figure 1. Oxidative Labeling of Native hMb and Acid-Unfolded aMb. Our focus will now shift to a comparison of native hMb and acidunfolded aMb. Lowering the pH represents a very common method to induce protein denaturation.8 At pH 2, Mb exists as an extensively unfolded species.71,77 This structural change is much more pronounced than the conformational transition monitored for Figures 1 and 2. Comparative HDX studies at different pH are not straightforward due to the pronounced dependence of kch in eq 2 on the acidity of the solvent.25 Hence, it was not attempted to complement the following oxidative labeling measurements by isotope exchange studies. For the experiments of Figure 3, the protein was irradiated in aqueous solution containing 5 mM ammonium acetate. This solvent was chosen based on its widespread use in ESI-MS experiments.78 Oxidative labeling of (76) Bai, Y.; Sosnick, T. R.; Mayne, L.; Englander, S. W. Science 1995, 269, 192-197. (77) Dobo, A.; Kaltashov, I. A. Anal. Chem. 2001, 73, 4763-4773.

Figure 3. (A) Mass distributions obtained after 80 s of γ-ray exposure for 20 µM Mb in the presence of 5 mM aqueous ammonium acetate. Blue, native hMb at pH 7; red, acid-unfolded aMb in the presence of phosphoric acid at pH 2.0. The spectra are normalized as in Figure 2. (B) Dose-response curve for the native and the acidunfolded protein.

native hMb and acid-unfolded aMb for 80 s results in very different mass distributions and dose-response curves with Q ) 1.5 ( 0.2 (Figure 3). One important issue for oxidative labeling experiments is the tendency of many compounds to react with •OH, thereby acting as radical scavengers that can suppress protein oxidation.45,54,55,79 Protein unfolding studies normally employ increasing denaturant concentrations. By design, these experiments involve solvent conditions that are not constant.64,80 This introduces an obvious risk to cause artifactual differences in labeling, originating from factors other than protein conformational changes. In the following, we will refer to undesirable phenomena of this kind as “secondary” effects. For the experiments of Figure 3, phosphoric acid was used as the denaturant because phosphates have been reported to cause minimal interferences.45,79 A casual observer might thus ascribe the different labeling behavior in Figure 3 exclusively to the large-scale conformational changes occurring under the conditions of this experiment. Unfortunately, the control experiments discussed in the following section strongly suggest that this simplistic interpretation is not correct. Bradykinin Control Experiments. Bradykinin was used for probing the extent of artifactual changes in the oxidation level. (78) Peschke, M.; Kebarle, P. J. Am. Chem. Soc. 2002, 124, 11519-11530. (79) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref. Data 1988, 17, 513-886. (80) Schellman, J. A. Biophys. Chem. 2002, 96, 91-101.

This peptide remains unstructured regardless of solution pH, and therefore any solvent-induced differences in its •OH labeling behavior must be ascribed to secondary effects.81,82 Figure 4A,B shows the results of comparative bradykinin measurements at pH 7 and pH 2 in 5 mM ammonium acetate, mimicking the conditions used for the Mb experiments of Figure 3. Notably, labeling of the peptide for 80 s at pH 7 results in a much lower degree of oxidation than at pH 2 (Q ) 3.1 ( 0.3). The observed behavior reveals that secondary effects are highly prevalent under these conditions. Differences in labeling for Figure 4A,B are attributed to the presence of ammonium acetate. Organic acids such as HCOOH, CH3-COOH, and their conjugate bases act as highly effective radical scavengers.83 Considering the pKa of acetic acid (4.7), acetate will remain deprotonated at pH 7, whereas it is protonated at pH 2. The primary product for the reaction of acetate with •OH is •CH2-COO-, formed with a rate constant of r ) 9 × 107 M-1 s-1. The formation of •CH2-COOH from acetic acid proceeds more slowly (r ) 2 × 107 M-1 s-1).79 The fact that racetate > raceticacid results in CpH7 > CpH2 (eq 5), such that the extent of bradykinin oxidation is lower at neutral pH than under acidic conditions (Figure 4A,B). This finding implies that the differential Mb labeling in Figure 3 is not exclusively caused by an increased exposure of reactive sites upon unfolding. In an effort to reduce the magnitude of secondary effects, bradykinin labeling experiments were repeated without ammonium acetate (Figure 4C,D). Exclusion of the radical scavenger from the solutions results in a higher degree of oxidation. More importantly, the labeling level is less dependent on pH. Interestingly, the observed trend is now opposite to that in the presence of acetate, i.e., bradykinin is somewhat less oxidized at pH 2 than at pH 7 (Q ) 0.82 ( 0.08). The reasons underlying this behavior are not completely clear. The primary rate of •OH production, kRAD, is virtually constant over the pH range studied here.84 However, even in pure water, hydroxyl radicals cause a host of reactions and coupled equilibria involving radiolysis products such as eaq, •H, HO •, H , H O , H+, etc. Since many of these processes are 2 2 2 2 somewhat pH dependent, a slight variation in bradykinin oxidation level upon acidification is not entirely surprising.75,79,84 The experiment was also repeated in the presence of 5 mM acetamide as the scavenger. This compound was chosen because, other than acetate, acetamide does not undergo protonation upon acidification, and therefore its scavenging activity should be less dependent on pH. This expectation is confirmed by the results in Figure 4E,F, with Q ) 0.95 ( 0.09. The absence of pronounced pH effects for bradykinin in acetamide-containing solutions was confirmed for a range of irradiation times (40-160 s) and for different bradykinin concentrations (10 and 20 µM, data not shown). The control experiments of Figure 4 illustrate that factors other than alterations in protein structure can lead to dramatic changes in the extent of oxidative labeling. These secondary effects are highly prevalent in solutions containing substances that exhibit pH-dependent scavenging activity, such as acetate. The parameter (81) Katta, V.; Chait, B. T. J. Am. Chem. Soc. 1993, 115, 6317-6321. (82) Hossain, B. M.; Simmons, D. A.; Konermann, L. Can. J. Chem. 2005, 83, 1953-1960. (83) LaVerne, J. A. Radiat. Res. 2000, 153, 196-200. (84) Spinks, J. W. T.; Woods, R. J. An Introduction to Radiation Chemistry, 2nd ed.; John Wiley & Sons: New York, 1990.

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Figure 4. [M + 2H]2+ region of bradykinin ESI mass spectra obtained after 80 s of γ-ray exposure at a peptide concentration of 20 µM in aqueous solution. (A) 5 mM ammonium acetate, pH 7; (B) 5 mM ammonium acetate with phosphoric acid, pH 2.0; (C) water without additives, pH 7.0; (D) water with phosphoric acid, pH 2.0. (E) 5 mM acetamide, pH 7.0; (F) 5 mM acetamide with phosphoric acid, pH 2.0.

C in eq 5 cannot be considered to be a constant under such conditions. Exclusion of titratable scavengers greatly reduces artifactual modulations of the oxidation level. In principle, it should be possible to eliminate undesired secondary effects through suitable adjustments of the radiation dose. However, such a procedure would require a calibrant that exactly mimics the parameters C and ku of the protein, a requirement that is difficult to fulfill in practice (obviously, [P]tot would have to be the same as well). Attempts to use such a calibration procedure in the absence of matching C and ku values are not meaningful. For example, using the bradykinin data in Figure 4A,B (with Q ) 3.1 ( 0.3) to calibrate the oxidative labeling of Mb under the conditions of Figure 3 suggests that the γ-ray exposure time should be ∼3-fold lower for acid-unfolded aMb than for the native protein. The dose-response curves of Figure 3B show that this would result in a much lower labeling level for the unfolded protein than for the native state, which is inconsistent with the changes in solvent exposure. The use of peptide models as calibrants for oxidative labeling, therefore, does not appear to be promising. Nonetheless, control experiments on these compounds are very useful for identifying conditions under which undesired secondary effects are small (Figure 4C,D) or almost absent (Figure 4E,F). Native hMb and Acid-Unfolded aMb in Acetate-Free Solutions. Figure 5A shows mass distributions obtained for native hMb in water (pH 7) and acid-unfolded aMb in dilute H3PO4 (pH 2). Unfolding enhances oxidative labeling, with Q ) 1.2 ( 0.1. The corresponding dose-response curves are depicted in Figure 6 (solid lines). Very similar results were obtained in 2.5 mM phosphate buffer and when employing HCl or H2SO4 as denaturants (data not shown). The bradykinin control experiments carried out in purely aqueous solution indicated a small but noticeable suppression of oxidation at pH 2 (Figure 4C,D). This phenomenon is expected to diminish the extent of conformationally induced labeling changes in the Mb experiments of Figure 5A. In contrast, studies in solutions containing 5 mM acetamide should be virtually free of undesired secondary effects, as demonstrated in Figure 2228 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

Figure 5. Mb mass distributions obtained after 80 s of γ-ray exposure in aqueous solution at a protein concentration of 13 µM. (A) Comparison of native hMb at pH 7 (blue) without solvent additives and acid-unfolded aMb in phosphoric acid, pH 2.0 (red). The spectra are normalized as in Figure 2. (B) Acid-unfolded aMb in formic acid, pH 2.0; (C) unfolded protein in 8 M urea, pH 7.0.

4E,F. Comparative Mb measurements conducted under these conditions result in Q ) 1.3 ( 0.1 (Figure 7). As expected, the differences in labeling are somewhat more pronounced than without acetamide (Figure 5A). Taken together, these data confirm that the presence of a more “open” protein conformation enhances

Figure 6. Dose-response curves for the various conditions of Figure 5.

the extent of labeling, as long as the measurements are carried out under conditions where secondary effects are small. However, considering the vast conformational differences between native and acid-unfolded Mb, the changes in labeling remain surprisingly small. To emphasize the importance of secondary effects further, we note that unfolding in formic acid (Figures 5B and 6) or acetic acid (not shown) at pH 2 results in a dramatically lower degree of oxidation than for the native protein at pH 7 (Figure 5A). This effect is attributed to the high radical scavenging activity of these organic acids.83 Protein unfolding can also be achieved by denaturants other than acid, such as urea.85 Earlier reports have suggested that urea does not scavenge radicals to any significant extent.65,66,86 •OH reacts with urea more slowly (r ≈ 106 M-1 s-1) than, for example, with formic acid (r ≈ 108 M-1 s-1).79 However, the urea concentrations typically used (up to 8 M)65,66,85 are much larger than the acid concentrations required to achieve unfolding, e.g., roughly 0.5 M formic acid at pH 2. Thus, significant scavenging activity can also occur in urea-containing solutions. Figures 5C and 6 demonstrate that unfolded Mb in 8 M urea shows a much lower extent of oxidation than the native protein at pH 7. Similar observations were made for unfolded Mb in 6 M guanidinium hydrochloride (data not shown). Clearly, any attempts to use scavenger-active denaturants for studies of this kind are futile, since these compounds mask conformationally induced changes in oxidation level by modifying the parameter C in eq 5. Protein Concentration. According to eq 5, an increase in [P]tot will reduce the rate constant kapp.48 This concentration dependence is another factor that has to be considered in studies that aim to use the extent of oxidation as a probe for protein conformational changes. Comparative experiments will be mean(85) Barrick, D.; Baldwin, R. L. Biochemistry 1993, 32, 3790-3796. (86) Maleknia, S. D.; Ralston, C. Y.; Brenowitz, M. D.; Downard, K. M.; Chance, M. R. Anal. Biochem. 2001, 289, 103-115.

ingful only when they are conducted at exactly the same [P]tot. Figure 8 shows the dose-response curves of native hMb in water and acid unfolded aMb in dilute H3PO4. In contrast to the data of Figure 5, this experiment was carried out at a 10-fold lower protein concentration ([P]tot ) 1.3 µM). The much faster oxidation kinetics allow the measurement to be conducted in a fraction of the time (note the x-axis in Figure 8), while maintaining a significant labeling difference for the two conformers, e.g., Q ) 1.3 ( 0.1 for t ) 6 s. The relatively pronounced deviations from linearity in Figure 8 likely originate from the fact that water radiolysis products require a certain time period to reach their steady-state concentrations, as discussed previously.48 Because reactions with these radiolysis products represent a dominant •OH deactivation pathway at low protein concentration,48 deviations from linearity in Figure 8 are more prevalent than for higher values of [P]tot. Oxidative Labeling of Native and Acid-Unfolded Cytochrome c. The effects of protein conformational changes on the extent of γ-ray induced oxidative labeling were also examined for another system. Like Mb, cytochrome c (cyt c) is a protein that has been thoroughly studied previously.87-89 Native cyt c at neutral pH adopts a largely R-helical conformation that is tightly folded around a covalently attached heme prosthetic group. Exposure to pH 2 causes the protein to unfold.90,91 Comparative labeling experiments on 13 µM cyt c in 5 mM acetamide result in very pronounced differences. The mass distribution of the acid-unfolded protein for t ) 80 s reveals a much higher oxidation level than (87) Maity, H.; Maity, M.; Krishna, M. M. G.; Mayne, L.; Englander, S. W. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 4741-4746. (88) Scott, R. A.; Mauk, A. G. Cytochrome c: A Multidisciplinary Approach; University Science Books: Sausalito, CA, 1996. (89) Akiyama, S.; Takashi, S.; Ishimori, K.; Morishima, I. Nat. Struct. Biol. 2000, 6, 514-520. (90) Moore, G. R.; Pettigrew, G. W. Cytochromes c: Evolutionary, Structural and Physicochemical Aspects; Springer-Verlag: New York, 1990. (91) Konermann, L.; Douglas, D. J. Biochemistry 1997, 36, 12296-12302.

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Figure 7. (A) Mass distributions obtained after 80 s of γ-ray exposure for 13 µM Mb in aqueous solution containing 5 mM acetamide. Blue, native hMb at pH 7; red, acid-unfolded aMb in the presence of phosphoric acid at pH 2.0. The spectra are normalized as in Figure 2. (B) Dose-response curve for the native and the acidunfolded protein.

Figure 9. (A) Mass distributions obtained after 80 s of γ-ray exposure for 13 µM cyt c in the presence of 5 mM acetamide. Blue, native cyt c at pH 7; red, acid-unfolded cyt c in the presence of phosphoric acid at pH 2.0. The spectra are normalized as in Figure 2. (B) Dose-response curve for the native and the acid-unfolded protein.

amount of residual structure in the unfolded state92,93 are likely to play an important role. For example, the demonstrated persistence of local hydrophobic clusters in the case of aciddenatured Mb94 could restrict •OH access to potentially reactive side chains, thereby diminishing the differences in labeling for pH 7 and pH 2. In addition, the native conformations of the two proteins do not necessarily have to afford the same degree of protection from attack by hydroxyl radicals.

Figure 8. Dose-response curves obtained for native hMb at pH 7 and acid-unfolded aMb at pH 2 in aqueous solution at a protein concentration of 1.3 µM (no scavenger added).

that of native cyt c (Q ) 1.7 ( 0.1, Figure 9A), and the doseresponse curves obtained under the two conditions are very distinct (Figure 9B). These differences are considerably larger than for Mb (Q ) 1.3, Figure 7A) under otherwise identical conditions. It is an interesting question why the changes in labeling upon unfolding are different for the two proteins. Dissimilarities in the 2230 Analytical Chemistry, Vol. 80, No. 6, March 15, 2008

CONCLUSIONS This work demonstrates the feasibility of using the overall degree of γ-ray-induced oxidative labeling as a probe for monitoring large-scale protein conformational changes. Analogous to the isotope exchange behavior in HDX experiments, unfolded conformers exhibit higher labeling levels than tightly folded species. However, oxidative labeling appears to be less sensitive to changes in protein structure than the rates of deuterium incorporation. In addition, the extent to which conformers can be differentiated based on their oxidation level is protein dependent. In the current study we observed that acid unfolding enhances the rate of oxidation for cyt c by 70%, whereas only a 30% increase was seen for Mb. (92) Pletneva, E. V.; Gray, H. B.; Winkler, J. R. J. Mol. Biol. 2005, 345, 855867. (93) Yao, J.; Chung, J.; Eliezer, D.; Wright, P. E.; Dyson, H. J. Biochemistry 2001, 40, 3561-3571. (94) Schwarzinger, S.; Wright, P. E.; Dyson, H. J. Biochemistry 2002, 41, 1268112686.

Although the dose-response curves obtained upon γ irradiation do not always show ideal exponential behavior, eqs 4 and 5 provide a useful framework for interpreting the effects of various parameters in a semiquantitative fashion. Unfolding increases ku by enhancing the steric exposure of reactive amino acid side chains, thereby raising the rate at which a protein undergoes oxidation. Unfortunately, these conformational effects are easily masked if the protein concentration [P]tot is not kept constant.48 Even more importantly, changes in C can severely affect the rate of oxidation. This parameter reflects the presence of competing •OH deactivation pathways, including reactions with water radiolysis products, impurities, solvent additives, etc. Because many commonly used denaturants such as organic acids act as radical scavengers, these substances are not suitable for radical footprinting experiments. In contrast to earlier reports,65,66,86 we found that significant radical scavenging also occurs in urea-containing solutions. Control experiments on small peptides allow the identification of undesired secondary effects, i.e., factors that modify the degree of oxidative labeling without being related to protein structural changes. The results of Figure 4 suggest that the chemical rate constants kch i (eq 6) for the reaction of solvent-exposed side chains with •OH are relatively independent of pH, quite in contrast to chemical HDX rate constants.25 This implies that “nonscaveng-

ing” acids such as H3PO4 represent suitable denaturants for oxidative labeling experiments. Numerous previous studies have demonstrated the usefulness of hydroxyl radical footprinting for mapping surface areas of native proteins and protein complexes. The current work contributes to the further development of this approach as a tool for monitoring large-scale conformational changes, and for exploring the properties of semi-unfolded polypeptide chains. Investigations are currently being conducted in our laboratory to determine the exact relationship between the R values of individual residues and the corresponding solventaccessible surface areas. While R is expected to increase with the degree solvent exposure, the relationship does not necessarily have to be linear. ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and the Canada Research Chairs Program. We thank Drs. Jamie Noe¨l and Jiju M. Joseph for help with the radiolysis experiments. Received for review November 10, 2007. Accepted December 28, 2007. AC702321R

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