Thermodynamic Analysis of Protein Stability and Ligand Binding

Graham M. West, Liangjie Tang, and Michael C. Fitzgerald*. Department of Chemistry, Duke ..... constant for the oxidation of an unprotected site in th...
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Anal. Chem. 2008, 80, 4175–4185

Thermodynamic Analysis of Protein Stability and Ligand Binding Using a Chemical Modification- and Mass Spectrometry-Based Strategy Graham M. West, Liangjie Tang, and Michael C. Fitzgerald* Department of Chemistry, Duke University, Durham, North Carolina 27708 Described here is a new technique, termed SPROX (stability of proteins from rates of oxidation), that can be used to measure the thermodynamic stability of proteins and protein-ligand complexes. SPROX utilizes hydrogen peroxide in the presence of increasing concentrations of a chemical denaturant to oxidize proteins. The extent of oxidation at a given oxidation time is determined as a function of the denaturant concentration using either electrospray or matrix-assisted laser desorption/ionization mass spectrometry. Ultimately, the denaturant concentration dependence of the oxidation reaction rate is used to evaluate a folding free energy (∆Gf) and m value (δ∆Gf/ δ[Den]) for the protein’s folding/unfolding reaction. Measurements of such SPROX-derived ∆Gf and m values on proteins in the presence and absence of ligands can also be used to evaluate protein-ligand affinities (e.g., ∆∆Gf and Kd values). Presented here are SPROX results obtained on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). SPROX-derived ∆Gf and m values on these proteins are compared to values obtained using more established techniques (e.g., CD spectroscopy and SUPREX). The dissociation constants of several known protein-ligand complexes involving these proteins were also determined using SPROX and compared to previously reported values. The complexes included the CypA-cyclosporin A complex and the BCAII-4-carboxybenzenesulfonamide complex. The accuracy and precision of SPROX-derived thermodynamic parameters for the model proteins and protein-ligand complexes in this study are discussed as well as the caveats of the technique. Conventional techniques for measuring the thermodynamic stability of proteins and protein-ligand complexes typically utilize calorimetric or spectroscopic methods to evaluate the thermodynamic parameters associated with protein folding and ligand binding reactions.1–6 A disadvantage to such methods is that they typically require relatively high concentrations of highly purified * Corresponding author. Mailing address: Department of Chemistry, Box 90346, Duke University, Durham, NC 27708-0346. Tel: 919-660-1547. Fax: 919660-1605. E-mail: [email protected]. (1) Brandts, J. F.; Lin, L. N. Biochemistry 1990, 29, 6927–6940. (2) Hill, J. J.; Royer, C. A. Methods Enzymol. 1997, 278, 390–416. (3) Sigurskjold, B. W. Anal. Biochem. 2000, 277, 260–266. (4) Straume, M.; Freire, E. Anal. Biochem. 1992, 203, 259–268. 10.1021/ac702610a CCC: $40.75  2008 American Chemical Society Published on Web 05/06/2008

protein. Several amide H/D exchange-based techniques using NMR7 and mass spectrometry-based readouts8–16 have been established for measuring the thermodynamic properties of proteins and protein-ligand complexes. H/D exchange and mass spectrometry-based techniques, such as SUPREX8,10–12,14–16 and PLIMSTEX,13 have the advantage over spectroscopy- and calorimetry-based methods that they can handle small quantities (e.g., picomoles) of proteins. In theory, they also have the ability to analyze proteins in complex mixtures such as cell lysates. In practice, the ability to analyze proteins in complex mixtures by mass spectrometry is often limited by signal suppression issues and the resolving power of modern mass spectrometers. The most common approach for overcoming such signal suppression and resolution issues involves the use of fractionation strategies. Unfortunately, many fractionation strategies are not compatible with amide H/D exchange (i.e., they cannot be performed under conditions that preserve the protein’s deuterated state). It would be especially useful to have a mass spectrometrybased method for measuring the thermodynamic properties of proteins that would be compatible with the fractionation strategies (e.g., chromatographic techniques such as size exclusion chromatography, gel electrophoresis, isoelectric focusing, and ionexchange chromatography) commonly used to facilitate mass spectral analyses of proteins in complex mixtures. Such a method would create a unique opportunity to perform thermodynamic measurements on proteins in complex biological mixtures, such (5) Xie, D.; Gulnik, S.; Erickson, J. W. J. Am. Chem. Soc. 2000, 122, 11533– 11534. (6) Robertson, A. D.; Murphy, K. P. Chem. Rev. 1997, 97, 1251–1268. (7) Englander, S. W.; Mayne, L. Annu. Rev. Biophys. Biomol. Struct. 1992, 21, 243–265. (8) Dai, S. Y.; Fitzgerald, M. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1535– 1542. (9) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1–25. (10) Roulhac, P. L.; Powell, K. D.; Dhungana, S.; Weaver, K. D.; Mietzner, T. A.; Crumbliss, A. L.; Fitzgerald, M. C. Biochemistry 2004, 43, 15767–15774. (11) Tang, L.; Hopper, E. D.; Tong, Y.; Sadowsky, J. D.; Peterson, K. J.; Gellman, S. H.; Fitzgerald, M. C. Anal. Chem. 2007, 79, 5869–5877. (12) Wang, M. Z.; Shetty, J. T.; Howard, B. A.; Campa, M. J.; Patz, E. F., Jr.; Fitzgerald, M. C. Anal. Chem. 2004, 76, 4343–4348. (13) Zhu, M. M.; Rempel, D. L.; Du, Z.; Gross, M. L. J. Am. Chem. Soc. 2003, 125, 5252–5253. (14) Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8296–8301. (15) Powell, K. D.; Ghaemmaghami, S.; Wang, M. Z.; Ma, L.; Oas, T. G.; Fitzgerald, M. C. J. Am. Chem. Soc. 2002, 124, 10256–10257. (16) Powell, K. D.; Fitzgerald, M. C. Biochemistry 2003, 42, 4962–4970.

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as cell lysates. In theory, the dynamic range and complexity of protein samples that can be successfully analyzed by the technique described here will be defined by the LC-MS and/or gel-based proteomics technologies to which the methodology is interfaced. From a practical standpoint the ability to analyze proteins in complex mixtures eliminates the need to perform tedious and time-consuming protein purification steps prior to analysis; and from a fundamental standpoint it allows for the study of proteins in their true biological context. Described here is a new technique termed SPROX (stability of proteins from rates of oxidation) for measuring the thermodynamic properties of proteins and protein-ligand complexes. SPROX is fundamentally compatible with the fractionation strategies used in mass spectrometry-based proteomic analyses. The technique is closely related to the SUPREX technique on which we have previously reported.8,10–12,14–16 Like SUPREX, the SPROX technique utilizes a mass spectrometry readout to generate thermodynamic information about the chemical denaturantinduced equilibrium unfolding/refolding properties of proteins in solution. The primary difference between SPROX and SUPREX is that SPROX utilizes the oxidation rates of globally protected sites in a protein to generate such thermodynamic information; whereas SUPREX utilizes the H/D exchange rates of globally protected amide groups in proteins to determine the thermodynamic properties of a protein’s folding/unfolding reaction. A major advantage of SPROX over SUPREX is the irreversible nature of the oxidation reaction. The inherent chemical stability of the oxidized protein product(s) allows for greater manipulation of the protein following modification. For example, the stability of the covalent modification provides the opportunity to define the thermodynamic properties of a protein or protein-ligand complex while it is in a complex biological mixture; but after the reaction is quenched, it is possible to incorporate standard fractionation techniques (e.g., gel electrophoresis, ion exchange chromatography, etc.) to maximize the number of proteins detected in the mass spectrometry readout. In this work a protocol for SPROX is developed and the technique is tested on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new method’s ability to accurately and precisely evaluate protein folding free energies, protein folding m values, and dissociation constants of proteinligand complexes is discussed. EXPERIMENTAL SECTION Materials. Ubiquitin, RNaseA, BCAII from bovine erythrocytes (88 wt %), 4-carboxybenzenesulfonamide (CBS), sinapinic acid (SA), sodium hydrogenphosphate and acetonitrile were purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA) was from Halocarbon. Cyclosporin A (CsA) was purchased from LKT Laboratories, Inc. Hydrogen peroxide (H2O2) (30% w/w) was purchased from ScienceLab.com. Guanidine hydrochloride (GdmCl), omni pure, was from VWR. L-Methionine (98+% pure) was from Acros Organics. Sodium phosphate was purchased from Mallinckrodt. The Zip-Tips were purchased from Millipore. The cyclophilin A (human) used in this work was a gift from Prof. Michael Campa (Duke University Medical Center); and it was obtained by recombinant DNA methods that involved overexpressing the CypA protein as a glutathione S-transferase (GST) fusion protein in 4176

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Escherichia coli (BL21-DE3); purifying the fusion protein using a GST-binding resin; removing the GST-tag in an overnight incubation with 5 units of thrombin per mg of fusion protein; and then removing the GST and thrombin with GST-binding resin and HiTrap benzamidine FF, respectively. The oxidized ubiquitin was generated by reacting 0.28 µmol of ubiquitin with 44 mmol of hydrogen peroxide for 90 s in 1.8 mL of buffer A (water containing 0.1% TFA). The oxidized RNaseA was generated by reacting 0.37 µmol of RNaseA with 3.5 mmol of hydrogen peroxide for 60 s in 1.5 mL of buffer A. Both oxidation reactions were performed at room temperature under ambient light. In these experiments to obtain oxidized ubiquitin and RNaseA, the oxidation reactions were quenched upon freezing the sample for lyophilization. Both of the ubiquitin and RNaseA oxidation reaction mixtures were frozen and lyophilized to obtain powders that were redissolved in buffer A and purified using reversed-phase HPLC using a C4 column. The pure fractions (as determined by ESI mass spectrometry) of each oxidized protein (i.e., ubiquitin with one oxygen and RNaseA with 4 oxygens) were pooled, lyophilized, and dissolved in buffer A to generate concentrated stock solutions of each oxidized protein. The oxidized CypA sample (+5 oxygens) was prepared by combining a 16 µL aliquot of a 100 µM CypA solution with 80 µL of a 20 mM sodium phosphate buffer (pH 7.4) containing 2 M GdmCl. After a 30 min equilibration, 16 µL of a 30% (w/w) hydrogen peroxide solution was added to initiate the oxidation reaction, which was allowed to proceed at room temperature under ambient light conditions. After 2 min, 16 µL of a 50 unit/µL solution of catalase was added to the solution to terminate the oxidation reaction by decomposing the hydrogen peroxide. The quenched sample was centrifuged for 2 min, and the resulting solution was used in the SUPREX experiments. The oxidized BCAII/Zn2+ sample (+2 oxygens) was prepared by combining 16 µL of a 150 µM BCAII solution with 80 µL of a 20 mM sodium phosphate buffer (pH 7.4) containing 3 M GdmCl and 225 µM Zn2+. After a 2 h equilibration time, 16 µL of a 30% (w/w) hydrogen peroxide solution was added to initiate the oxidation reaction, which was allowed to proceed at room temperature under ambient light. After 3.5 min, a 16 µL aliquot of a 50 unit/µL catalase aqueous solution was introduced to quench the oxidation reaction. The quenched samples were centrifuged for 2 min, and the resulting solution was used for the SUPREX experiments. Instrumentation. MALDI mass spectra were acquired on an UltraFlex II TOF/TOF (Bruker Daltonics, Inc., Billerica, MA) mass spectrometer in the linear and positive ion mode using a smartbeam Nd:YAG laser (355 nm). Spectra were collected using the following instrument parameters: 25 kV ion source 1 voltage, 23.4 kV ion source 2 voltage, 6 kV lense voltage, 100-130 ns pulsed ion extraction, and matrix gating to 3000 Da. ESI mass spectra were acquired on a PE Sciex 150EX electrospray mass spectrometer using a 0.1 m/z step size. Solution pH measurements were made using a Jenco 6072 pH meter equipped with a Futura calomel pH electrode from Beckman Instruments (Fullerton, CA). Refractive index measurements were made using a Bausch & Lomb (Rochester, NY) refractometer.17 Reversed-phase high-performance liquid chromatography (RP-HPLC) analyses (17) Nozaki, Y. Methods Enzymol. 1972, 26, 43–50.

Figure 1. Schematic representation of the SPROX protocol.

were performed using a Rainin instrument consisting of a Dynamax variable wavelength UV/visible absorbance detector. An analytical reversed-phase C4 column (4.6 × 150 mm, 300 Å) was used with 214 nm UV detection, and a semipreparative reversed-phase C4 column (10 × 250 mm, 300 Å) was used with 230 nm UV detection. All columns were obtained from Vydac. Chromatographic separations were achieved using linear gradients of buffer B in A (buffer A was 0.1% TFA in water, and buffer B was 90% acetonitrile in water containing 0.09% TFA). Circular dichroism (CD) spectra were acquired on an Applied Photophysics PiStar 180 CDF spectrometer fitted with a temperature-controlled cell holder and equipped with a Microlab 500 automated titrator. SPROX Protocol. The basic SPROX protocol developed and used in this work is outlined in Figure 1. In this protocol, an aliquot of each protein or protein-ligand stock solution is added to a series of buffers. Each buffer in the series contains a constant amount of H2O2 and an increasing amount of chemical denaturant. For proteins or protein-ligand complexes that are slow to reach their folding/unfolding equilibrium in denaturant-containing buffers, the protein or protein-ligand complexes can be equilibrated for the necessary amount of time before the oxidation reaction is initiated by adding aliquots of a 30% (w/w) hydrogen peroxide solution. After a specified oxidation time (a time that is the same for each denaturant-containing buffer) the oxidation reaction in each denaturant-containing SPROX buffer is quenched. This can be accomplished upon initiation of a desalting protocol using ZipTips, upon addition of catalase, or upon addition of excess methionine to each SPROX buffer. Ultimately, a mass spectrum of the protein sample in each SPROX buffer is recorded using either a MALDI or ESI mass spectrometer in order to determine the extent of oxidation (see SPROX Data Analysis below). The Zip-Tip quenching method is essential for experiments utilizing ESI. The Zip-Tip quenching method used in this work involved wetting the tips with 2 × 10 µL volumes of acetonitrile followed by 2 × 10 µL volumes of buffer A. The protein from each oxidation reaction is loaded onto a Zip-Tip, each Zip-Tip is washed with 2 × 10 µL volumes of buffer A, and protein is eluted from the Zip-Tip for mass spectral analysis. Protein was eluted for ESIMS analyses using 6 aspirations of a 10 µL aliquot of an acetonitrile/water solution (75/25 v/v) containing 0.1% TFA. Protein was eluted for MALDI-MS analysis using 6 aspirations of a 10 µL aliquot of a matrix solution containing 50% buffer B in A and saturated with SA.

SPROX analyses on ubiquitin and RNaseA were performed using a set of buffers composed of 50 mM phosphate (pH 7.4) and concentrations of GdmCl that varied between 0 and 5 M. In these analyses the protein concentration was between 1 and 117 µM, the equilibration time was 15 min, the H2O2 concentration in each buffer was 2.3 M, the oxidation times ranged from 2 to 120 min (depending on the experiment), and either C18 Zip-Tips (for ESI-MS analyses) or a 5-fold molar excess of methionine (for MALDI-MS analyses) was employed to quench the oxidation reaction. SPROX analyses on CypA were performed using a set of buffers composed of 20 mM sodium phosphate (pH 7.4) and concentrations of GdmCl that varied between 0 and 4 M. In these analyses, the protein concentration was 100 µM, no equilibration time was used, the H2O2 concentration in each buffer was 1.6 M, the oxidation time was 60 s, and catalase was employed to quench the oxidation reaction. SPROX analyses on the CypA-CsA complex were performed using a set of buffers composed of 20 mM sodium phosphate (pH 7.4), DMSO (20%), and concentrations of GdmCl that varied between 0 and 4 M. In these analyses on CypA-CsA, the protein concentration was 100 µM, the CsA concentration was 2 mM, a 5 min equilibration time was employed for protein-ligand complex formation prior to dilution into the SPROX buffers, the H2O2 concentration in each buffer was 1.6 M, the oxidation time was 60 s, and catalase was used to quench the oxidation reaction. SPROX analyses on the BCAII-Zn2+ samples were performed using a set of buffers composed of 20 mM phosphate (pH 7.4) and concentrations of GdmCl that varied between 0 and 4 M. In these analyses the BCAII and Zn2+ concentrations were 150 and 225 µM (respectively), no equilibration time was employed, the H2O2 concentration in each buffer was 1.6 M, the oxidation time was 60 s, and catalase was employed to quench the oxidation reaction. The SPROX analyses of the BCAII-Zn2+-CBS complexes were identical to those described above for the BCAII-Zn2+ sample except that solutions of the BCAII-Zn2+ bound with the CBS ligand were equilibrated for 4 h prior to dilution into the denaturant-containing buffers. The final concentration of CBS in the denaturant containing buffers was 450 µM. SPROX Data Analysis. The ion signals generated in each mass spectral analysis of the wild-type protein and the oxidation products were used to derive a weighted average m/z value, m/zwt,av, according to eq 1. m ⁄ zwt,av )

∑ m I /∑ I i i

i

(1)

In eq 1, Ii is the intensity detected at m/z value mi. The m/zwt,av values calculated in this work were calculated using a window of the mass spectrum that included the m/z values expected for the wild-type ion signal and all the expected oxidation products (see below). The m/zwt,av was converted to a weighted average mass for the protein, Masswt,av, and a weighted average change in mass, ∆Masswt,av, was calculated by subtracting the molecular weight of the unoxidized protein from the Masswt,av value. Ultimately, a ∆Masswt,av versus [Denaturant] plot was constructed. One of two methods was used to extract a ∆Gf and m value from SPROX data. In method 1, the ∆Masswt,av versus [Denaturant] plots were fit to eq 2. Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

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∆Masswt,av ) ∆M∞ + (∆M0 - ∆M∞)e[-(kOX⁄(1+Kfold))t]

(2)

In eq 2, Kfold ) e-(∆Gf+m[Den])/RT, ∆M0 is ∆Masswt,av before global oxidation, ∆M∞ is ∆Masswt,av after global oxidation, t is the time of oxidation in seconds, kOX is the average pseudo-first-order rate constant for the oxidation of an unprotected site in the protein, ∆Gf is the free energy of folding in the absence of denaturant, [Den] is the denaturant concentration, m is δ∆Gf/δ[GdmCl], R is the gas constant, and T is the temperature in kelvins. In fitting the ∆Masswt,av versus [Denaturant] plots to eq 2, ∆M0, ∆M∞, ∆Gf, and m were allowed to float and kOX was assigned a value based on the exact H2O2 concentration used in the experiment and an experimentally determined second order rate constant for the oxidation of the unprotected sites in each protein (see below). In method 2, a C1/2SPROX value (i.e., the [Den] at which the oxidation of the globally protected sites proceeded halfway to completion) was obtained for each ∆Masswt,av versus [Denaturant] plot by fitting the data to a 4-parameter sigmoidal function in SigmaPlot. The C1/2SPROX values obtained using different oxidation times were fit to eq 3 using a linear least-squares analysis. RT ln(koxt/0.693-1) ) -mC1⁄2SPROX - ∆Gf

(3)

In eq 3, R, T, t , m, C1/2SPROX and kOX are as defined above. The y-intercept and the slope of the best-fit line were taken as the ∆Gf value and the m value, respectively. Kd Value Determinations. The Kd values for the CypA-CsA complex were determined using eq 4. Kd )

[L] e-∆∆Gf⁄NRT - 1

(4)

In eq 4, [L] is the concentration of free ligand, N is the number of independent equivalent binding sites, ∆∆Gf is the binding free energy, and R and T are as defined above. In these experiments the concentration of free ligand was estimated as the total ligand concentration, as the CypA ligand was present in 10-fold excess over the protein. The ∆∆Gf value was determined using eq 5. ∆∆Gf ) m∆C1⁄2SPROX

(5)

In eq 5, ∆C1/2SPROX is the difference in a protein’s C1/2SPROX value recorded in the presence and in the absence of ligand, and m is as defined above. In our studies on the BCAII-CBS complex, the CBS ligand was present at less than a 10-fold excess over the protein concentration. Therefore, eq 6 was needed to evaluate the Kd.

Kd )

4Ltotale-∆∆Gf ⁄ NRT - 4Ptotal(e-∆∆Gf ⁄ NRT - 1) (2e-∆∆Gf ⁄ NRT - 1)2 - 1

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∆Masswt,av ) ∆Masswt,av,0 + A(1 - e-kt)

(6)

(7)

In eq 7, ∆Masswt,av,0 is the ∆Masswt,av value measured immediately after the protein was contacted with H2O2 and quenched (i.e., the ∆Masswt,av value at t ) 0), A is the amplitude of the curve, and k is the pseudo-first-order oxidation rate constant at 100 mM H2O2. Ultimately, the k value determined for each protein was divided by the [H2O2] (i.e., 100 mM) to generate a second-order rate constant for the oxidation of unprotected sites in each protein. Conventional Equilibrium Unfolding Experiments. CD denaturation curves for the oxidized ubiquitin (+1 oxygen) and the oxidized RNaseA (+4 oxygens) samples were monitored at 222 nm; RNaseA (unoxidized) was monitored at 225 nm. In all cases the CD denaturation curves were recorded using the automated titration system on the CD instrument. In each, titration 0 and 6 M GdmCl solutions containing the protein in 20 mM Tris (pH 7.4) buffer were mixed in different ratios. CD signals were collected in 0.1 M increments. The mixing time was 20 s, there was a delay of 5 s, ∼5000 CD signals were collected over the course of 30 s, and the signals were averaged. The averaged CD signals were used to generate ∆Gf and m values according to the linear extrapolation method.18 The thermodynamic stabilities of the oxidized CypA and BCAII/Zn2+ samples were determined by SUPREX using previously established protocols for data acquisition and analysis.16 The deuterated SUPREX buffers used to characterize these samples were composed of 20 mM sodium phosphate buffer (pD 7.4) and increasing concentrations of GdmCl. SUPREX curves were recorded for the oxidized BCAII/Zn2+ sample at room temperature using H/D exchange times that ranged from 10 to 280 min. SUPREX transition midpoints (i.e., C1/2SUPREX values) were determined for each curve, and these values were plotted against exchange time according to eq 8 as previously described.16

RTln(〈kint 〉tEX ⁄ 0.693 - 1))-mC1⁄2SUPREX - ∆Gf

In eq 6, the derivation of which has been previously reported,11 Ltotal is the concentration of ligand, and Ptotal is the concentration of protein. N, R, T and ∆∆Gf are as defined above. Oxidation Rate Determinations. Second-order rate constants for the H2O2 oxidation of unprotected sites in each protein were experimentally determined. In these experiments, a 1 mg/mL 4178

solution of each protein was prepared in a GdmCl-containing buffer solution. The GdmCl concentration was 5.0, 3.7, 2.5, and 4.0 for ubiquitin, RNaseA, CypA, and BCAII (respectively). The GdmCl concentrations used for each protein were high enough to ensure that each protein was >99% unfolded. The oxidation reactions were carried out in the presence of 100 mM H2O2. The extent of oxidation (calculated using eq 1) was determined from ESI mass spectral data in the case of ubiquitin, RNaseA, and BCA II and from MALDI mass spectral data in the case of CypA. Ultimately, a plot of ∆Masswt,av vs time was constructed and the data were fit to the single exponential function given by eq 7.

(8)

In eq 8, C1/2SUPREX is the transition midpoint of a SUPREX curve, 〈kint〉 is the average intrinsic H/D exchange rate of an unprotected amide proton, tEX is the H/D exchange time, and the values R, T, m, and ∆Gf are as defined above. Ultimately, the y-intercept and the slope of the best-fit line were taken as the -∆Gf value and the -m value, respectively. The 〈kint〉 value used in our calculations (18) Pace, C. N. Methods Enzymol. 1986, 131, 266–280.

was 29.4 h-1. This value was determined using the SPHERE program19,20 and the entire primary amino acid sequence of BCAII. Attempts were made to acquire SUPREX curves for the oxidized CypA sample using H/D exchange times of 5 and 10 min at room temperature and 0 °C, respectively. However, a SUPREX transition was not detected in either case. RESULTS SPROX Analysis of Two-State Folding Proteins. Ubiquitin and RNaseA, two well-studied protein systems with documented two-state folding behavior in chemical denaturant-induced equilibrium unfolding experiments,21–25 were each subjected to SPROX analyses according to the new protocol outlined in Figure 1. Shown in Figures 2 and 3 are typical SPROX data acquired for ubiquitin and RNaseA using a 2 min oxidation time in the SPROX protocol. Both MALDI and ESI mass spectrometry were utilized to readout the extent of oxidation in the different denaturant-containing SPROX buffers. The MALDI and ESI mass spectra in Figures 2 and 3 show that the ion signal intensity for the unoxidized protein is decreased at higher GdmCl concentration, whereas the ion signal intensities for the oxidized species are increased. The MALDI- and ESI-generated SPROX curves for ubiquitin and RNaseA (see Figures 2C and 3C) show a single cooperative transition that is reasonably well-fit to eq 2. The average ∆Gf and m values extracted from three replicate SPROX curves using eq 2 according to method 1 are summarized in Table 1. The ubiquitin and RNaseA systems were also subject to additional SPROX analyses using a range of different oxidation times in the SPROX protocol. Shown in Figure 4A and B are SPROX curves obtained for ubiquitin and RNaseA using oxidation times ranging from 25 s to 120 min. A C1/2SPROX value was determined for each SPROX curve; and these values were plotted against the exchange time according to eq 3. A linear least-squares analysis of the data obtained for each protein (see Figure 4C) was used to generate y-intercept and slope values, which were taken as measures of each protein’s -∆Gf and -m value, respectively, according to method 2 (see Table 1). Only the data obtained at the four shortest exchange times for each protein were included in the linear least-squares analysis, as a visual inspection of the data revealed that the data points at the longer exchange times were not well-fit to eq 3 (see Figure 4C). This was likely due to increased oxidation at low denaturant concentrations (see Discussion below). SPROX Analysis of Non-Two-State Folding Proteins. Cyclophilin A (CypA) and BCAII were also analyzed by SPROX. BCAII is a known non-two-state folding protein,26,27 and it has been (19) Bai, Y.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins 1993, 17, 75– 86. (20) Zhang, Y.-Z. University of Pennsylvania, 1995. (21) Dai, S. Y.; Gardner, M. W.; Fitzgerald, M. C. Anal. Chem. 2005, 77, 693– 697. (22) Gladwin, S. T.; Evans, P. A. Fold. Des. 1996, 1, 407–417. (23) Ibarra-Molero, B.; Loladze, V. V.; Makhatadze, G. I.; Sanchez-Ruiz, J. M. Biochemistry 1999, 38, 8138–8149. (24) Pace, C. N.; Laurents, D. V.; Thomson, J. A. Biochemistry 1990, 29, 2564– 2572. (25) Sivaraman, T.; Arrington, C. B.; Robertson, A. D. Nat. Struct. Biol. 2001, 8, 331–333. (26) Henkens, R. W.; Kitchell, B. B.; Lottich, S. C.; Stein, P. J.; Williams, T. J. Biochemistry 1982, 21, 5918–5923. (27) Andersson, D.; Hammarstrom, P.; Carlsson, U. Biochemistry 2001, 40, 2653–2661.

Figure 2. SPROX analysis of ubiquitin. Representative MALDI and ESI mass spectra obtained at selected denaturant concentrations are shown in (A) and (B) (respectively). The +6 charge state is highlighted in (B). “Ox” represents oxygen. (C) SPROX curves generated using a 2 min oxidation time and MALDI and ESI readouts. The filled circles and solid line represent the MALDI-generated data and fit to eq 2. The open circles and dashed line represent the ESI data and fit to eq 2.

suggested that CypA is also a non-two-state folding protein based on the low cooperativity of its folding transition.12 Shown in Figure 5 are typical SPROX curves generated using a 1 min oxidation time for each protein alone and in the presence of known ligands. The average ∆Gf and m values extracted using eq 2 (i.e., method 1) to analyze the replicate SPROX curves obtained for each protein and protein-ligand complex are summarized in Table 2, as are the ∆C1/2SPROX, ∆∆Gf, and Kd values calculated from the data on each protein-ligand complex. Analytical Chemistry, Vol. 80, No. 11, June 1, 2008

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Table 1. Thermodynamic Parameters Obtained for the Folding/Unfolding Reactions of Ubiqutin and RNaseA protein ubiquitin

technique

SPROX-ESI (method SPROX-MALDI (method 1) SPROX-ESI (method CD oxidized ubiquitin CD RNaseA SPROX-ESI (method SPROX-MALDI (method 1) SPROX-ESI (method CD oxidized RNaseA CD

m (kcal/ (mol M))

∆Gf (kcal/mol)

1) 2.1 ± 0.1a 1.6 ± 0.3a

-8.0 ± 0.3a -6.3 ± 1.0a

2) 1.9 ± 0.2b 2.1 ± 0.2b,c 1.9 ± 0.1 1) 2.0 ± 0.2a 2.7 ± 0.4a

-8.4 ± 0.6b -8.5 ± 0.3b,c -5.1 ± 0.1 -5.3 ± 0.5a -6.8 ± 0.8a

2) 2.2 ± 0.4b 2.1 ± 0.1b 1.3 ± 0.1b

-6.4 ± 0.2b -6.8 ± 0.3b -1.8 ± 0.1b

a Values are the average and standard deviation of three replicate measurements. b Error given is the fitting error obtained in a linear least-squares analysis. c Value is from ref 8.

Figure 3. SPROX analysis of RNaseA. Representative MALDI and ESI mass spectra obtained at selected denaturant concentrations are shown in (A) and (B) (respectively). The +8 charge state is highlighted in (B). (C) SPROX curves generated using a 2 min oxidation time and MALDI and ESI readouts. The filled circles and solid line represent the MALDI-generated data and fit to eq 2. The open circles and dashed line represent the ESI data and fit to eq 2.

Conventional Equilibrium Unfolding Experiments. The GdmCl-induced equilibrium unfolding behaviors of RNaseA, oxidized RNaseA, and oxidized ubiquitin were examined using CD spectroscopy. The raw data collected in these experiments are shown in the Supporting Information (Figures SI-1A, SI-2A, and SI-3A), as are the normalized unfolding curves (Figures SI1B, SI-2B, and SI-3B). The ∆Gf and m values extracted from the normalized unfolding curves are summarized in Table 1. The SUPREX technique was employed to analyze the GdmClinduced equilibrium unfolding behaviors of oxidized CypA and 4180

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oxidized BCAII. SUPREX analysis of oxidized CypA did not yield a SUPREX curve but rather ∆Mass values of ∼70 Da at the GdmCl concentrations tested (i.e., those between 0 and 2.5 M) (Figure SI-4). These ∆Mass values are consistent with those expected in the post-transition baseline of a SUPREX curve for CypA. The absence of a SUPREX transition suggests that oxidation of the CypA is highly destabilizing. We note that a CypA ∆Gf value of 3.5 kcal/mol would have yielded a SUPREX transition midpoint of 0 M when the 10 min H/D exchange time at 0 °C was used in the SUPREX protocol. Thus, we estimate that the stability of the oxidized CypA sample is