D Exchange- and Mass Spectrometry-Based Strategy for the

Ying Xu , Sebastian Schmitt , Liangjie Tang , Ursula Jakob and Michael C. Fitzgerald ... Erin D. Hopper , Adrianne M. C. Pittman , Chandra L. Tucker ,...
1 downloads 0 Views 158KB Size
Download PDF
Anal. Chem. 2007, 79, 5869-5877

H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein-Ligand Binding Liangjie Tang,† Erin D. Hopper,† Yan Tong,†,‡ Jack D. Sadowsky,§ Kimberly J. Peterson,§ Samuel H. Gellman,§ and Michael C. Fitzgerald*,†

Department of Chemistry, Duke University, Durham, North Carolina 27708, and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706

The equilibrium unfolding properties of four model protein systems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometrybased technique for measuring the free energy (∆Gf) and m-value (δ∆Gf/δ[denaturant]) associated with the folding/ unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (>∼15 kDa) or multidomain proteins. In the absence of ligand, ∆Gf and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligandbound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein-ligand dissociation constants (Kd values) could also be ascertained. The SUPREX-derived binding free energies and Kd values evaluated here were in good agreement with those reported on the same complexes using other techniques. The combination of mass spectrometry and amide H/D exchange has become an increasingly useful tool for studying the structural and biophysical properties of protein-ligand binding interactions.1-8 Both matrix-assisted laser desorption/ionization * Corresponding author. Tel: 919-660-1547. Fax: 919-660-1605. E-mail: [email protected]. † Duke University. ‡ Present address: McKinsey & Co., Shanghai 200020, China. § University of Wisconsin. (1) Englander, J. J.; Del Mar, C.; Li, W.; Englander, S. W.; Kim, J. S.; Stranz, D. D.; Hamuro, Y.; Woods, V. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (12), 7057-7062. (2) Mandell, J. G.; Falick, A. M.; Komives, E. A. Anal. Chem. 1998, 70, 39873995. (3) Lam, T. T.; Lanman, J. K.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G.; Prevelige, P. E. J. Chromatogr., A 2002, 982 (1), 85-95. (4) Neubert, T. A.; Walsh, K. A.; Hurley, J. B.; Johnson, R. S. Protein Sci. 1997, 6 (4), 843-850. (5) Powell, K. D.; Fitzgerald, M. C. Biochemistry 2003, 42, 4962-4970. (6) Zhu, M. M.; Rempel, D. L.; Du, Z. H.; Gross, M. L. J. Am. Chem. Soc. 2003, 125 (18), 5252-5253. (7) Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (15), 8296-8301. 10.1021/ac0700777 CCC: $37.00 Published on Web 06/21/2007

© 2007 American Chemical Society

(MALDI)- and electrospray ionization-based approaches have been developed to examine the amide H/D exchange properties of proteins and protein-ligand complexes. Such mass spectrometryand amide H/D exchange-based approaches can provide important information about protein-ligand binding interactions such as the location of ligand binding sites,2,3 the conformational changes that can result from ligand binding events,4 and the strength of ligand binding interactions.5,6 SUPREX (stability of unpurified proteins from rates of H/D exchange) is one such mass spectrometry-based approach that we have developed for quantifying the strength of protein-ligand binding interactions.5,9-12 Like other amide H/D exchange- and mass spectrometry-based approaches, SUPREX is amenable to the analysis of proteins and protein-ligand complexes in both purified and unpurified samples as well as in samples with small amounts of protein or with low protein concentrations. This is in contrast to spectroscopy-based methods (e.g., circular dichroism, fluorescence, UV absorption, or nuclear magnetic resonance) that require relatively large amounts of highly purified protein for analysis. One additional advantage of SUPREX over other amide H/D exchange- and mass spectrometry-based strategies for making similar measurements of protein-ligand binding affinities is the capacity of the technique for high-throughput analysis.13 To date, the majority of protein and protein-ligand systems investigated by SUPREX have involved relatively small (∼15 kDa) or multidomain proteins do not display such two-state folding behavior. Thus, SUPREX cannot be used to obtain absolute folding (8) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1-25. (9) Ma, L. Y.; Fitzgerald, M. C. Chem. Biol. 2003, 10 (12), 1205-1213. (10) Powell, K. D.; Ghaemmaghami, S.; Wang, M. Z.; Ma, L.; Oas, T. G.; Fitzgerald, M. C. J. Am. Chem. Soc. 2002, 124, 10256-10257. (11) Roulhac, P. L.; Powell, K. D.; Dhungana, S.; Weaver, K. D.; Mietzner, T. A.; Crumbliss, A. L.; Fitzgerald, M. C. Biochemistry 2004, 43 (50), 1576715774. (12) Tong, Y.; Wuebbens, M. M.; Rajagopalan, K. V.; Fitzgerald, M. C. Biochemistry 2005, 44 (7), 2595-601. (13) Powell, K. D.; Fitzgerald, M. C. J. Comb. Chem. 2004, 6, 262-269.

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5869

free energies on such systems. However, in the case of some nontwo-state folding proteins, we have shown that SUPREX can still be used to extract relative thermodynamic information about a protein’s folding reaction. For example, we have shown that relative protein-ligand binding affinities (i.e., ∆∆Gf values) could be determined by SUPREX, even though the method did not permit the absolute measurement of a ∆Gf value.9,11 One potential problem with the SUPREX analysis of larger or multidomain proteins is that the fraction of amide protons undergoing exchange when the protein globally unfolds can be small compared to the fraction of amide protons undergoing exchange when the protein locally unfolds. In such cases, the measurement of an accurate ∆Gf value by SUPREX is precluded using the SUPREX data acquisition and analysis methods developed to date.5,7,14 This is because these methods assume that there is a significant fraction of amide protons that undergoes H/D exchange only when the protein globally unfolds. As part of the work described here, we document the SUPREX behavior of several model systems that are large or multidomain proteins with folding reactions that do not appear to be dominated by denaturant-dependent global folding/unfolding events, but rather include a large number of denaturant-independent local unfolding/refolding events. The model protein systems in this work include bovine calmodulin (CaM), bovine carbonic anhydrase II (BCAII), d-TDP-D-glucose 4,6-dehydratase (RmlB), and Bcl-xL. These protein systems were also used to validate a new experimental approach that facilitates the SUPREX analysis of ligand binding interactions in protein systems with protein folding/ unfolding reactions that are not dominated by a global unfolding event. The approach uses stabilizing ligands of a protein to reduce the number of denaturant-independent local unfolding/refolding events. This facilitates detection of the protein’s denaturantdependent global unfolding reaction using SUPREX. We show that quantitative thermodynamic SUPREX analyses of ligand binding in these systems can be performed in one of two different ways: (1) by using a known ligand to stabilize the protein so that the binding affinity of a second ligand can be quantified, or (2) by using one ligand at a time to stabilize the protein so that relative binding affinities of different ligands can be measured. EXPERIMENTAL SECTION Materials. The following materials were purchased from Sigma-Aldrich (St. Louis, MO): CaM from bovine brain (46 wt %), BCAII from bovine erythrocytes (88 wt %), melittin from honeybee venom (70 wt %), trypsin inhibitor from soybean, aldolase from rabbit muscle, cytochrome c from horse heart, deuterium oxide (D2O; 99.9 atom % D), potassium deuteroxide (35 wt % in D2O, 99.9 atom % D), deuterium chloride (20 wt % in D2O, 99.5 atom % D), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), dipicolinic acid, 1,4-piperazinediethanesulfonic acid (PIPES), sulfanilamide (SULFA), 4-carboxybenzenesulfonamide (CBS), nicotinamide adenine dinucleotide (NAD+), thymidine monophosphate (TMP), and sinapinic acid (SA). Ethylenediaminetetraacetic acid disodium salt (EDTA) and urea were purchased from Mallinckrodt (Hazelwood, MO). Guanidine hydrochloride (GdmCl) was from EMD Chemicals Inc. (Gibb(14) Powell, K. D.; Wales, T. E.; Fitzgerald, M. C. Protein Sci. 2002, 11 (4), 841-851.

5870

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

stown, NJ). Acetonitrile (MeCN) and zinc chloride were from Fisher (Fairlawn, NJ). Trifluoroacetic acid (TFA) was from Halocarbon (River Edge, NJ). Phosphoric acid-d3 (99 atom % D) was purchased from Cambridge Isotope Laboratories (Andover, MA). The Microcon centrifugal filter device (YM-3, membrane NMWL 3000) and C4 ZipTips were from Millipore (Billerica, MA). Deuterated urea, GdmCl, and HEPES were prepared by repeated (four times) dissolution in D2O and lyophilization. Rm1B was kindly provided by Professor Eric J. Toone (Duke University). Protein Expression and Peptide Synthesis. Bcl-xL was obtained as follows. A pET-30a(+) vector (Novagen, Madison, WI) containing human Bcl-xL (residues 1-196), which lacks the C-terminal hydrophobic region, with an N-terminal His tag was transformed into Escherichia coli BL21(DE3) cells. Gene sequencing indicated the following sequence of the protein: MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMSMSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYG. Cells were grown at 37 °C in LB medium with 30 µg/mL kanamycin to an OD600 of 0.6. Protein expression was induced with 1 mM IPTG, and cells were grown for an additional 3 h. The cell pellet was resuspended in lysis buffer (50 mM Tris, 200 mM NaCl, 1 mM β-mercaptoethanol; pH 7.5) with 40 mg of lysozyme and incubated on ice for 1 h. Cells were disrupted by pulse sonication, and the soluble fraction was loaded onto a column of nickel-nitrilotriacetic acid agarose (Qiagen, Valencia, CA). The column was washed with 5 mM imidazole, and the protein was eluted with 500 mM imidazole. The protein was dialyzed to ion exchange buffer A (20 mM Tris, 1 mM β-mercaptoethanol; pH 7.5) and purified using a HiTrapQ FF 1 mL column (GE Healthcare, Piscataway, NJ) with a gradient of 15-45% ion exchange buffer B (20 mM Tris, 1 M NaCl, 1 mM β-mercaptoethanol; pH 7.5) over 34 min. The final purified Bcl-xL sample had a concentration of 7 µM and was stored at 4 °C in a PBS buffer containing 2 mM DTT and 2 mM EDTA. The solution was concentrated with a Microcon centrifugal filter device with a 3000 Da cutoff membrane by centrifuging for 99 min at 14000g. The final concentration of BclxL was determined by measuring the A280 of Bcl-xL in an 8 M GdmCl solution using a molar extinction coefficient of 41940 M-1cm-1.15 The concentration was found to be 130 µM. The Bcl-xL ligands are Bak-derived peptides that will be referred to hereafter as Bak1 (H2N-GQLGRQLAIIGDDINRCONH2) and Bak2 (H2N-GQLGRQLAI-norleucine-GDDFNRCONH2). These peptides were obtained by standard solid-phase peptide synthesis as previously described.16 Bak1 and Bak2 peptides were stored as a lyophilized powder and were reconstituted in water to a final concentration of 1.3 mM. After reconstitution, the peptides were stored at 4 °C. Preparation of Protein Samples and SUPREX Buffers. The apo-CaM sample was prepared according to a previously (15) Manion, M. K.; O’Neill, J. W.; Giedt, C. D.; Kim, K. M.; Zhang, K. Y. Z.; Hockenbery, D. M. J. Biol. Chem. 2004, 279 (3), 2159-2165. (16) Sadowsky, J. D.; Fairlie, W. D.; Hadley, E. B.; Lee, H.-S.; Umezawa, N.; Nikolovska-Coleska, Z.; Wang, S.; Huang, D. C. S.; Tomita, Y.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129 (1), 139-154.

described protocol that involved using EDTA to remove Ca2+.17 The final solution contained 80 µM CaM and 1.5 mM EDTA. The CaM-4Ca(II) complex was prepared by mixing the CaM stock solution with a CaCl2 solution and equilibrating the resulting solution at room temperature for at least 4 h. The final CaM4Ca(II) solution contained 80 µM CaM and 3.33 mM CaCl2. The CaM-4Ca(II)-melittin sample contained 80 µM CaM, 3.33 mM CaCl2, and 240 µM melittin. This sample was prepared by mixing a stock solution of melittin (prepared in HEPES buffer) with an equilibrated CaM-4Ca(II) solution. The resulting CaM-4Ca(II)melittin sample was equilibrated at room temperature for 4 h. The BCAII-Zn(II) sample was composed of 150 µM BCAII and 225 µM Zn2+. The BCAII-Zn(II)-SULFA and BCAII-Zn(II)-CBS samples contained 150 µM BCAII, 225 µM Zn2+, and either 1650 µM SULFA or 450 µM CBS. The solutions of the BCAII-Zn(II) bound with ligand (either SULFA or CBS) were equilibrated for 4 h at room temperature. Apo-BCAII was prepared by unfolding the supplied BCAII in 3.0 M GdmCl in the presence of a 20-fold excess amount of sodium dipicolinate to remove zinc. This protocol was similar to that described elsewhere.18,19 The BCAII- and dipicolinate-containing solution was incubated overnight at 4 °C, and then a 300-µL aliquot of the sample solution was transferred to the sample reservoir of a prewetted Microcon centrifugal filter device with a 3000 Da cutoff membrane. The device was centrifuged for 60 min at 14000g. A 200-µL aliquot of buffer containing 25 mM PIPES, 100 mM NaCl, and 1.5 mM dipicolinate was added to the filter device, and the sample was centrifuged again for 30 min at 14000g. The apo-BCAII solution in the filter device was subject to 16 washings (200 µL each) with the PIPES buffer to ensure complete removal of the GdmCl and zinc ions. The final zinc- and GdmCl-free BCAII sample was stored at 4 °C before use. A 665 µM RmlB protein stock solution was prepared in 150 mM NaCl and 20 mM phosphate at pH 7.6. This RmlB stock solution was diluted using phosphate buffer to prepare an apoRmlB sample with a final concentration of 100 µM. The sample of RmlB-NAD+ contained 458 µM RmlB and 4.6 mM NAD+ and was prepared by mixing the RmlB stock solution with a stock solution of NAD+ prepared in phosphate buffer. The RmlB-TMP sample contained 393 µM RmlB and 4 mM TMP, and it was prepared by mixing a stock solution of TMP prepared in phosphate buffer with the RmlB stock solution. The RmlB-NAD+TMP complex was prepared by mixing the appropriate stock solutions to yield a final solution that consisted of 511 µM RmlB, 5.1 mM NAD+, and 5.1 mM TMP. The Bcl-xL-peptide complexes were prepared directly in the SUPREX buffers. Aliquots of the 130 µM Bcl-xL stock solution were diluted 11-fold into H/D exchange buffers that already contained the peptide ligand (i.e., either the Bak1 or the Bak2 peptide). The final protein and ligand concentrations in each H/D exchange buffer were 12 and 120 µM, respectively. The H/D exchange buffers used in our SUPREX studies on CaM contained 50 mM deuterated HEPES (pD 7.8), 100 mM KCl, (17) Persechini, A.; Blumenthal, D. K.; Jarrett, H. W.; Klee, C. B.; Hardy, D. O.; Kretsinger, R. H. J. Biol. Chem. 1989, 264 (14), 8052-8058. (18) Hunt, J. B.; Rhee, M. J.; Storm, C. B. Anal. Biochem. 1977, 79 (1-2), 614617. (19) Tripp, B. C.; Bell, C. B.; Cruz, F.; Krebs, C.; Ferry, J. G. J. Biol. Chem. 2004, 279 (20), 21677-21677.

and various concentrations of urea from 0 to 7.6 M. The H/D exchange buffers used for BCAII contained 25 mM PIPES (pD 7.0), 100 mM NaCl, and various concentrations of GdmCl from 0 to 3.0 M. The H/D exchange buffers used for RmlB contained 20 mM sodium acetate (pD 5.8), 20 mM KCl, and various concentrations of GdmCl from 0 to 4.5 M. The H/D exchange buffers for Bcl-xL contained 20 mM deuterated phosphate (pD 7.4) and GdmCl concentrations ranging from 0.7 to 6.3 M. Instrumentation. MALDI mass spectra for CaM, BCAII, and RmlB were acquired on a Voyager DE (PerSeptive Biosystems) or a Voyager DE-PRO (Applied Biosystems, Foster City, CA) Biospectrometry workstation in the linear and positive ion mode using a nitrogen laser (337 nm). Spectra for these three proteins were collected using the following parameters: 25 kV acceleration voltage, 75 V guide wire voltage, 22 kV grid voltage, and 225 or 275 ns delay time. MALDI mass spectra for Bcl-xL 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 parameters: 25 kV ion source 1 voltage, 23.4 kV ion source 2 voltage, 6 kV lens voltage, 100 ns pulsed ion extraction, and matrix gating to 3000 Da. pH measurements were performed using a Jenco 6072 pH meter equipped with a Futura calomel pH electrode from Beckman Instruments (Fullerton, CA). The pD value of each exchange buffer was determined by adding 0.4 to the measured pH value.20 Urea and GdmCl concentrations were determined using a Bausch & Lomb (Rochester, NY) refractometer as previously described.21,22 SUPREX Data Collection. The SUPREX protocol used in this work was similar to that described previously.5 Briefly, the fully protonated protein or protein-ligand complex was diluted at least 10-fold into a series of deuterated H/D exchange buffers (see above for the buffer composition used for each protein system). After a given H/D exchange time, an aliquot of the deuterated sample of the protein or protein-ligand complex was diluted at least 4-fold into an ice-cold and low pH SA MALDI matrix solution. This step quenched the H/D exchange and prepared the sample for MALDI analysis. The saturated SA matrix solution was prepared by dissolving SA in 45% MeCN/54.9% H2O/0.1% TFA (v/v, pH ∼2.0). The matrix solution was kept cold on crushed ice before use. A 1-µL aliquot of the protein or protein-ligand sample in the quenched H/D exchange buffer was spotted onto a stainless steel MALDI target and allowed to dry under a gentle flow of air or N2 before the sample was introduced into the vacuum of the MALDI mass spectrometer. The Bcl-xL experiments required a two-layer sample preparation method in which the quenched protein or protein-ligand complex was spotted onto a predeposited layer of matrix crystals.23 This layer was preformed on the MALDI target by depositing ∼1 µL of a saturated SA solution prepared in a 40% acetone and 60% methanol (v/v) mixture and allowing the solvent to evaporate. Each mass spectrum collected was the sum of the signal obtained from 25 to 100 laser shots. A total of 8-10 replicate mass spectra were collected for the sample in each H/D exchange buffer to obtain an average mass value for the respective protein in the buffer. (20) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190. (21) Nozaki, Y. Methods Enzymol. 1972, 26, 43-50. (22) Pace, C. N. Methods Enzymol. 1986, 131, 266-280. (23) Dai, Y. Q.; Whittal, R. M.; Li, L. Anal. Chem. 1999, 71 (5), 1087-1091.

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5871

The average mass values determined at 8-12 different denaturant concentrations were converted to ∆mass values (see below) and used to generate each SUPREX curve in this work. The SUPREX protocol employed for RmlB included a microchromatography step to concentrate and desalt the RmlB sample.24 This step was performed after the specified exchange time and prior to the MALDI mass spectral analysis. The H/D exchange reaction of each RmlB sample was quenched by mixing each sample with an appropriate amount of a 3% TFA solution (v/v) to lower the solution pH to 3. A C4 ZipTip, pretreated according to the manufacturer’s instructions, was used to extract protein from the quenched sample. The tip containing the preacidified sample was washed with a 0.1% TFA solution to remove the salt. Protein was then eluted from the tip using the matrix solution. The eluted protein solution was directly deposited onto a MALDI target on which SA matrix and mass standards had been predeposited. The ZipTips used here facilitated our SUPREX analyses of RmlB in two ways: (1) they reduced the amount of protein needed to complete our studies, and (2) they increased the overall quality (i.e., signal intensity and resolution) of the protein ion signals in our MALDI-MS analyses. SUPREX Data Analysis. SUPREX data analysis was performed as previously described.5 Briefly, the raw data in each MALDI mass spectrum were analyzed using an in-house Microsoft Excel Macro or MATLAB program to determine the mass of the sample at each denaturant concentration. The mass increase (i.e., the ∆mass value) of the protein due to deuterium uptake was calculated by subtracting the mass of the fully protonated protein from the mass of the protein determined in our MALDI analyses after the protein was subjected to H/D exchange. The average ∆mass values obtained from 8 to 10 replicate spectra were plotted as a function of the denaturant concentration to generate a SUPREX curve at each H/D exchange time used in this work. The standard deviations associated with our ∆mass measurements were typically less than 10%. This was consistent with the ∼0.050.1% MALDI-TOF mass accuracies observed for the proteins in 1/2 this study. A CSUPREX value (i.e., the concentration of the denaturant at the transition midpoint of each SUPREX curve) was obtained by fitting the ∆mass versus [denaturant] data to a fourparameter sigmoidal equation using a nonlinear regression routine in SigmaPlot (SYSTAT Software, Inc., San Jose, CA). Equation 1 was used to determine the m- and ∆Gf values from the SUPREX data. In eq 1, R is the gas constant, T is the

For each protein and protein-ligand complex, a series of SUPREX curves was generated using different exchange times. 1/2 The CSUPREX values obtained at each H/D exchange time were fit to eq 1 using a linear least-squares analysis. The y-intercept and the slope of the best-fit line were taken as the ∆Gf value and the m-value, respectively. The values used in this work were predicted using either the SPHERE program25,26 or the relationship ) 10pH-5 min-1.7 To determine with SPHERE, the protein’s amino acid sequence, the test pH, and the temperature were input into the SPHERE program. In theory, the ∆Gf values, as determined above, could be used to determine ∆∆Gf values (i.e., the change in folding free energy induced by ligand binding). However, in this study, we used ∆Gf,av values to calculate ∆∆Gf,av values (see discussion) for quantifying the binding affinity of ligands for the proteins. Calculations of ∆Gf,av values involved averaging the m-values from multiple ∆Gapp versus 1/2 CSUPREX value plots for a given protein system. The average m-value was used in eq 1 to directly calculate a ∆Gf value from 1/2 value determined for a given protein system. In each CSUPREX this manner, at least four independent ∆Gf values were calculated for each protein complex. These values were ultimately averaged to generate ∆Gf,av values. The Kd values were calculated using eq 2. In eq 2, [L] is the

< kint > t -1 0.693 1/2 -RT ln ) mCSUPREX + ∆Gf nn n-1 [P] 2n-1

concentration of ligand and Ptotal is the concentration of protein. In cases where the ligand was present in less than a 10-fold excess over protein, eqs 2 and 3 were combined to give eq 4, which was used to evaluate Kd.

[

]

(1)

temperature in kelvin, is the average intrinsic exchange rate of an unprotected amide proton, t is the H/D exchange time, m is defined as δ∆Gf/δ[denaturant], ∆Gf is the folding free energy of the protein in the absence of denaturant, n is the number of protein subunits involved in the folding reaction (e.g., n ) 1 for CaM, BCAII, and Bcl-xL; n ) 2 for RmlB), and [P] is the protein concentration expressed in n-mer equivalents. For simplicity, the left side of the equality in eq 1 is hereafter referred to as ∆Gapp. (24) Powell, K. D.; Fitzgerald, M. C. Anal. Chem. 2001, 73 (14), 3300-3304.

5872

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Kd )

[L] e

-∆∆Gf,av/NRT

-1

(2)

concentration of free ligand, and N is the number of independent equivalent binding sites. For all of the model protein systems in this study, N ) 1. In our experiments with BCAII-Zn(II)-SULFA, Bcl-xL-Bak1, and Bcl-xL-Bak2, the SULFA, Bak1, or Bak2 was present in 10-fold excess over the protein concentration; therefore, [L] was taken as the total concentration of SULFA, Bak1, or Bak2 in solution. For the other protein-ligand complexes studied in this work, the ligands were present at less than a 10-fold excess over the protein concentration. Therefore, eq 3 was needed to calculate the free ligand concentration, [L].27 In eq 3, Ltotal is the

[L] ) Ltotal -

(

)

Ptotal + Ltotal + Kd - x(Ptotal + Ltotal + Kd)2 - 4PtotalLtotal 2 (3)

Kd )

4Ltotale-∆∆Gf,av/NRT - 4Ptotal(e-∆∆Gf,av/NRT - 1) (2e- ∆∆Gf,av/NRT - 1)2 - 1

(4)

RESULTS AND DISCUSSION SUPREX Analysis of Apoproteins. SUPREX analyses were performed on apo-CaM, apo-BCAII, apo-RmlB, and apo-Bcl-xL (25) Bai, Y. W.; Milne, J. S.; Mayne, L.; Englander, S. W. Proteins Struct. Funct. Genet. 1993, 17 (1), 75-86. (26) Zhang, Y. Z. Structural Biology and Molecular Biophysics, University of Pennsylvania, PA, 1995. (27) Segel, I. H. Enzyme Kinetics; John Wiley & Sons: New York, 1975.

Figure 1. SUPREX analyses of apoproteins. (A) Apo-CaM SUPREX data collected using H/D exchange times of 15 (open circles), 25 (filled circles), and 60 min (filled squares). (B) Apo-RmlB SUPREX data collected using H/D exchange times of 10 (filled circles), 30 (filled triangles), and 60 min (open squares). (C) Apo-BCAII SUPREX data collected using H/D exchange times of 2 (filled circles), 4 (open squares), and 5 h (open diamonds). (D) Apo-Bcl-xL SUPREX data collected using H/D exchange times of 80 (filled squares), 160 (filled circles), and 240 min (open circles). The solid lines represent the best fit of the data in each SUPREX data set to a four-parameter sigmoidal equation using SigmaPlot. 1/2 1/2 The dotted lines in (A) mark the CSUPREX value for each curve (see Supporting Information for a summary of the CSUPREX values extracted for each apoprotein). The standard deviations associated with our ∆mass measurements were typically less than 10%.

using H/D exchange times that ranged from 5 min to 96 h. The 1/2 values for specific H/D exchange times and resulting CSUPREX each protein are included in the Supporting Information. The H/D exchange times used in our experiments were different for each protein system, but in each case, they were chosen such that a SUPREX transition could be detected (see Figure 1). In theory, any H/D exchange time can be used in the SUPREX experiment. However, in practice, the H/D exchange times that can be used for a given protein system are limited to those that generate 1/2 experimentally accessible CSUPREX values. For example, the H/D 1/2 exchange time cannot be so long as to yield a CSUPREX value at negative chemical denaturant concentrations, and it cannot be so 1/2 short as to yield a CSUPREX value at a denaturant concentration at which the denaturant is not soluble. The useful range of H/D exchange times varies for different protein systems (as it did in this work) depending on the folding thermodynamics of the system. For example, relatively long exchange times can be used 1/2 to generate experimentally measurable CSUPREX values if the protein has a large negative ∆Gf or large m-value associated with its folding reaction (see eq 1 above). Typical SUPREX curves obtained for each apoprotein are shown in Figure 1. A single cooperative transition was detected in all of the SUPREX curves except for those obtained on the BCAII system. The SUPREX curves obtained for apo-BCAII were difficult to interpret. It was not clear whether they exhibited two distinct transitions or one broad transition (see Figure 1C). Nonetheless, it was apparent that the SUPREX curve transition midpoints for apo-BCAII were relatively constant with H/D exchange time, as were the transitions obtained for RmlB and 1/2 Bcl-xL. In contrast, the CSUPREX values obtained for apo-CaM were shifted to lower denaturant concentrations when the H/D exchange time was increased.

1/2 values of well-behaved proteins in the SUPREX The CSUPREX experiment are expected to move to lower denaturant concentrations when the H/D exchange time is increased. Well-behaved protein systems for SUPREX include those that exhibit reversible, two-state unfolding properties and EX2 (and not EX1) exchange behavior. In the case of such well-behaved proteins, eq 1 can be 1/2 used to describe the relationship between the CSUPREX value and the H/D exchange time used in SUPREX.5 Equation 1 predicts 1/2 that a plot of ∆Gapp versus CSUPREX will be linear and that the y-intercept and slope of the resulting plot will correspond to the ∆Gf and m-value, respectively, of the protein’s folding/unfolding reaction. Equation 1 clearly does not describe the SUPREX behavior of apo-RmlB, apo-BCAII, or apo-Bcl-xL because the SUPREX curve transition midpoints for these proteins did not 1/2 significantly change with exchange time. While the CSUPREX values determined for apo-CaM were shifted to measurably lower denaturant concentrations with increasing H/D exchange times in SUPREX, it is noteworthy that the SUPREX data obtained for 1/2 apo-CaM (i.e., the CSUPREX values recorded at different H/D exchange times) did not fit well to eq 1 as judged by the correlation coefficient, 0.4926 (data not shown). 1/2 value shifts in the apo-CaM SUPREX The apparent CSUPREX curves appear to be an artifact of the pretransition baseline movement to higher ∆mass values at longer H/D exchange times. Such pretransition baseline shifts have been previously observed with well-behaved proteins. The shifts are observed because H/D exchange reactions resulting from local unfolding reactions in the protein largely define the ∆mass value in the pretransition baseline. As the H/D exchange time is increased, the local H/D exchange reactions proceed closer to completion; therefore, the ∆mass values in the pretransition region increase. The unique aspect of the pretransition baseline shifts observed for apo-CaM

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5873

Figure 2. SUPREX analyses of holoproteins. (A) SUPREX data recorded using an H/D exchange time of 150 min for CaM in the presence of calcium (open circles) and for CaM in the presence of both calcium and melittin (closed circles). (B) SUPREX data recorded using an H/D exchange time of 24 h for BCAII in the presence of zinc (open circles), for BCAII in the presence of both zinc and SULFA (open triangles), and for BCAII in the presence of both zinc and CBS (filled circles). (C) SUPREX data recorded using an H/D exchange time of 110 min for Bcl-xL in the presence of Bak1 (filled circles) and using an H/D exchange time of 130 min for Bcl-xL in the presence of Bak2 (open circles). The solid lines represent the best fit of the data in each SUPREX curve to a four-parameter sigmoidal equation using 1/2 SigmaPlot. The dotted lines mark the CSUPREX value for each curve 1/2 (see Supporting Information for a summary of the CSUPREX values extracted for each protein-ligand complex). The standard deviations associated with our ∆mass measurements were typically less than 10%.

(and to some extent for apo-RmlB and apo-BCAII) is that they are 1/2 not accompanied by the CSUPREX value shifts predicted by eq 1. SUPREX Analysis of Holoproteins. SUPREX analyses were performed on CaM, BCAII, RmlB, and Bcl-xL in the presence of known ligands of each protein. CaM was analyzed in the presence of Ca2+ and in the presence of both Ca2+ and melittin (see Figure 2A). BCAII was analyzed in the presence of Zn2+ and in the presence of both Zn2+ and either one of two ligands, SULFA or CBS (see Figure 2B). Bcl-xL was analyzed in the presence of the Bak1 peptide and in the presence of the Bak2 peptide (see Figure 2C). RmlB was analyzed in the presence of NAD+, in the presence 5874 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

Figure 3. SUPREX analyses of RmlB. SUPREX data collected for (A) apo-RmlB (filled circles) and RmlB in the presence of NAD+ (filled triangles); (B) apo-RmlB (filled circles) and RmlB in the presence of TMP (filled squares); and (C) apo-RmlB (filled circles) and RmlB in the presence of both NAD+ and TMP (filled squares). All SUPREX curves were recorded using an H/D exchange time of 10 min. The solid lines represent the best fit of the data in each SUPREX curve to a four-parameter sigmoidal equation using SigmaPlot. See Sup1/2 porting Information for the CSUPREX values obtained for each RmlB SUPREX curve.

of TMP, and in the presence of both NAD+ and TMP (see Figure 3). It is interesting to note that the pretransition baselines of the holoproteins were generally shifted to lower ∆mass values compared to the pretransition baselines of SUPREX curves obtained in the absence of ligand at comparable H/D exchange times. The magnitude of the shifts varied depending on the protein system. For example, the shift was smallest in the case of TMP binding to RmlB (Figure 3B), and it was the largest in the case of melittin binding to calcium-loaded CaM (Figure 2A). Such pretransition baseline shifts result from the increased global protection of amide protons upon ligand binding. The presence of such pretransition baseline shifts upon ligand binding has been noted in other SUPREX studies,5 and the shifts generally do not impact quantitative analyses of protein-ligand binding. The

1/2 Figure 4. Plots of ∆Gapp versus CSUPREX for (A) CaM-4Ca(II) (open circles) and CaM-4Ca(II)-melittin (filled circles); (B) BCAII-Zn(II) (open circles), BCAII-Zn(II)-SULFA (filled squares), and BCAII-Zn(II)-CBS (filled circles); (C) RmlB in the presence of both NAD+ and TMP; (D) Bcl-xL in the presence of Bak1 (filled circles) and Bcl-xL in the presence of Bak2 (open circles). The solid lines are the results of linear leastsquares fitting of the data to eq 1. The correlation coefficients obtained for these data sets ranged from 0.9120 to 0.9814.

SUPREX analysis of protein-ligand binding is solely dependent 1/2 value. Shifts in the on measurements of a protein’s CSUPREX 1/2 CSUPREX value result from the fundamental link between protein stability and ligand binding, and they occur whether or not the ligand binding event results in the increased global protection of amide protons. Unlike some other H/D exchange- and mass spectrometry-based methods for the quantitative analysis of protein-ligand binding, SUPREX can be used to study proteinligand binding events regardless of whether the number of protected amide protons is altered. This is in contrast to PLIMSTEX, which requires the ligand binding event to protect a measurable number of amide protons in the protein.6 The Supporting Information includes a summary of the 1/2 CSUPREX values extracted from all of the SUPREX curves generated at the selected H/D exchanges times for the different protein-ligand complexes in this work. Similar to the apoprotein SUPREX analyses, the range of H/D exchange times selected in this work varied for each protein-ligand complex (see above). 1/2 The CSUPREX values obtained for the holoproteins were all at 1/2 higher denaturant concentrations than the CSUPREX values obtained for the respective apoproteins for a given H/D exchange time (see Figures 1-3 and Tables S-1-S-4 in the Supporting 1/2 Information). Such CSUPREX value shifts are consistent with the ligand-induced stabilization of each protein that would be expected for tight-binding ligands such as the ones employed in this study. 1/2 Shown in Figure 4 are ∆Gapp versus CSUPREX plots for all of the protein-ligand complexes in this study with the exception of the RmlB-NAD+ and RmlB-TMP complexes. Remarkably, the 1/2 CSUPREX values obtained for each of the protein-ligand complexes in Figure 4 fit well to eq 1. This is in sharp contrast to the 1/2 CSUPREX values obtained for the apoproteins, which did not fit well to eq 1 (see above). The reasonably good fits of the SUPREX data shown in Figure 4 made it possible to generate a ∆Gf and m-value for each combination of protein and ligand studied. These ∆Gf and m-values are summarized in Table 1.

The accurate measurement of ∆Gf and m-values using eq 1 requires an assumption of reversible, two-state folding.5 The biophysical properties of CaM and BCAII have been previously studied, and the chemical denaturant-induced equilibrium unfolding reactions of these proteins were not found to be two-state.28-30 While biophysical studies on a related Bcl-xL construct suggest a possible two-state folding mechanism for Bcl-xL,31 it is noteworthy that the SUPREX curve transitions recorded on our Bcl-xL construct were significantly less cooperative than would be expected for a two-state folding protein the size of Bcl-xL. The low cooperativity of the SUPREX transition suggests that our BclxL construct is not a two-state folder. The biophysical properties of the unfolding/refolding reactions of RmlB have not been studied, but given the size of this protein it is unlikely that it is a two-state folding protein. Given the non-two-state folding properties of the proteins in this study, it is unlikely that the ∆Gf and m-values in Table 1 accurately describe the biophysical properties of their folding behaviors. The motivation for deriving such ∆Gf and m-values for the proteins in this study was to determine if they could be used in comparative analyses to quantify the binding affinities of different ligands to these protein systems (see below). The m-values recorded for each protein system were not expected to change with ligand. Indeed, significant changes (i.e., differences greater than 30%) were not detected in our experiments (see Table 1). Protein folding m-values have been shown to correlate with the amount of hydrophobic surface area that is buried in a protein folding reaction.32 Large proteins with more (28) Andersson, D.; Hammarstrom, P.; Carlsson, U. Biochemistry 2001, 40 (9), 2653-2661. (29) Henkens, R. W.; Kitchell, B. B.; Lottich, S. C.; Stein, P. J.; Williams, T. J. Biochemistry 1982, 21 (23), 5918-5923. (30) Masino, L.; Martin, S. R.; Bayley, P. M. Protein Sci. 2000, 9 (8), 15191529. (31) Thuduppathy, G. R.; Hill, R. B. Protein Sci. 2006, 15 (2), 248-257. (32) Myers, J. K.; Pace, C. N.; Scholtz, J. M. Protein Sci. 1995, 4 (10), 21382148.

Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5875

Table 1. SUPREX-Derived Thermodynamic Parameters Obtained on the Model Protein Systems in This Study protein complex

∆Gfa (kcal mol-1)

ma (kcal mol-1 M-1)

∆Gf,avb (kcal mol-1)

CaM-Ca(II) CaM-Ca(II-melittin) BCAII-Zn(II) BCAII-Zn(II)-SULFA BCAII-Zn(II)-CBS RmlB-NAD+-TMP Bcl-xL-Bak1 Bcl-xL-Bak2

-7.5 ( 0.2 -9.5 ( 0.3 -16.1 ( 0.6 -16.6 ( 0.7 -17.5 ( 0.9 -13.6 ( 0.3 -11.7 ( 1.2 -13.0 ( 0.8

1.0 ( 0.1 0.7 ( 0.1 6.1 ( 0.4 6.1 ( 0.5 6.1 ( 0.5 2.6 ( 0.3 1.9 ( 0.4 1.8 ( 0.3

-7.1 ( 0.2 -10.2 ( 0.2 -16.0 ( 0.1 -16.6 ( 0.1 -17.5 ( 0.1

-3.1 ( 0.2c 0d -0.6 ( 0.2d -1.5 ( 0.2d

-11.5 ( 0.1 -13.1 ( 0.2

0e -1.6 ( 0.2e

∆∆Gf,av (kcal mol-1) 0c

SUPREXderived Kdf

literature Kd

53 ( 20 nM

110g/18.4h nM

93 ( 49 µM 2.6 ( 1.0 µM

71 µMi 0.730/0.760 µMj 49 nMk 1.5 nMk

a The ∆G and m-values were taken from the linear least-squares analysis of the data using eq 1 (see Figure 4). Errors reported for the ∆G and f f m-values are the fitting errors of the linear least-squares analyses. b ∆Gf,av values were the average of folding free energy values calculated using eq 1 and an established m-value (i.e., the m-value averaged from the m-values of the complexes of each protein). The errors reported for the ∆Gf,av values are the standard deviations of the folding free energy values calculated using eq 1 and the established m-value. c Value relative to CaMCa(II). Propagated error is reported. d Value relative to BCAII-Zn(II). Propagated error is reported. e Value relative to Bcl-xL-Bak1. Propagated error is reported. f Kd values were determined using either eq 2 or 4. The reported errors were propagated through the equations. g Value derived from binding free energy measurement, -9.5 kcal/mol, reported in ref 33. h Value is the reciprocal of the PLIMSTEX binding constant reported in ref 6. i Value is the reciprocal of the binding constant reported in ref 34. j Values are from ref 35. k Value is the Ki from ref 40.

buried hydrophobic surface area in their native three-dimensional structures have larger m-values than small proteins with less buried hydrophobic surface area. The protein-ligand interactions described in this work do involve some burial of hydrophobic surface area. However, the amount of hydrophobic surface area that is buried in these binding interactions is small compared to the thousands of square angstroms that would be required to produce a change in m-value of more than 30%.32 Indeed, the m-value differences between different complexes for the BCAII and Bcl-xL systems were clearly within the experimental error (see Table 1). The ∼30% difference in the m-values measured in our CaM experiments with and without melittin (see Table 1) is also likely due to experimental error. Quantitative Analysis of Ligand Binding. Our SUPREX analyses on CaM, BCAII, and Bcl-xL enabled a quantitative analysis of ligand binding. SUPREX-derived ∆∆Gf,av values were determined for calcium-loaded CaM binding melittin, for zinc-loaded BCAII binding SULFA or CBS and for Bcl-xL binding the Bak2 peptide (see Table 1). The SUPREX-derived ∆∆Gf,av values for the CaM and BCAII systems could also be used in eq 2 or 4 to calculate Kd values for each protein-ligand complex. These SUPREXderived Kd values were within 4-fold of Kd values previously reported under similar conditions (i.e., similar buffer pH and composition) for the same protein-ligand complexes using other techniques (see Table 1).6,33-35 The most significant differences between the SUPREX buffers and the buffer systems used in the literature were that the SUPREX buffers contained a chemical denaturant (i.e., GdmCl or urea) and that they were deuterated. It was possible to calculate Kd values for ligands binding to the BCAII and CaM systems because the two ligands under study in each system had different binding sites in their respective proteins.36-39 The binding of the first ligand rendered each protein (33) Yao, Y. H.; Squier, T. C. Biochemistry 1996, 35 (21), 6815-6827. (34) Matulis, D.; Kranz, J. K.; Salemme, F. R.; Todd, M. J. Biochemistry 2005, 44 (13), 5258-5266. (35) Day, Y. S. N.; Baird, C. L.; Rich, R. L.; Myszka, D. G. Protein Sci. 2002, 11 (5), 1017-1025. (36) Babu, Y. S.; Bugg, C. E.; Cook, W. J. J. Mol. Biol. 1988, 204 (1), 191-204. (37) Gruneberg, S.; Stubbs, M. T.; Klebe, G. J. Med. Chem. 2002, 45 (17), 35883602. (38) Saito, R.; Sato, T.; Ikai, A.; Tanaka, N. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 792-795.

5876 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

amenable to SUPREX analysis (i.e., a ∆Gf value could be determined using eq 1). A subsequent SUPREX analysis of the protein in the presence of both the first ligand and the second ligand made it possible to determine a binding free energy for the second ligand. Unlike the two ligands in the BCAII and CaM systems, the Bak1 and Bak2 ligands in the Bcl-xL system bind to the same site in the protein.40 Thus, the two ligands cannot simultaneously bind to Bcl-xL during the SUPREX analysis. The two-ligand approach described here for Kd value measurements requires that the stabilizing ligand has a different binding site from the test ligand. Therefore, it was not possible to use the two-ligand approach to calculate Kd values for the Bak1 and Bak2 peptides binding to Bcl-xL. However, we note that the differential binding affinity of the Bak1 and Bak2 peptides for Bcl-xL could be quantified. A comparison of the SUPREX-derived ∆Gf,av values for the Bcl-xLBak1 and Bcl-xL-Bak2 complexes revealed that the binding affinity of Bak2 is 1.6 ( 0.2 kcal/mol greater than the binding affinity of Bak1. This is in reasonable agreement with the differential binding affinity expected for the two peptides (2.0 kcal/ mol) based on their relative Ki values.40 Our calculations of the above ligand binding affinities (i.e., the ∆∆Gf,av and Kd values) employed the ∆Gf,av values in Table 1 rather than the ∆Gf values. In theory, the ∆Gf values could be used to quantify protein-ligand binding affinities (i.e., to calculate ∆∆Gf values). Indeed, ∆∆Gf values calculated in this manner are very similar to the ∆∆Gf,av values reported in Table 1. However, the error associated with such ∆∆Gf calculations is large (∼1 kcal/ mol) and close in magnitude to the ∆∆Gf value itself. The magnitude of this error is not consistent with the raw data collected in our SUPREX curves. For example, the two ∆Gf values obtained for the Bcl-xL complexes are the same within experimental error (see Table 1), but the 0.6 M change in the transition midpoint between the two Bcl-xL SUPREX curves shown in Figure 1/2 value 2C is clearly measurable given the error of our CSUPREX measurements (∼ (0.1 M). (39) Schulz, D. M.; Ihling, C.; Clore, G. M.; Sinz, A. Biochemistry 2004, 43 (16), 4703-4715. (40) Sadowsky, J. D.; Peterson, K. J.; Fairlie, W. D.; Huang, D. C. S.; Tomita, Y.; Gellman, S. H. Universitiy of Wisconsin, Madison, WI. To be submitted for publication.

We have previously shown that the error associated with the 1/2 values using eq 1 can be calculation of ∆Gf values from CSUPREX significantly reduced if the m-value is accurately established.11 One way to establish an accurate m-value is to average the m-values 1/2 obtained from multiple ∆Gapp versus CSUPREX value plots. For example, since the m-values were not expected to change with ligand (see above), the m-values reported for each protein system in Table 1 can be used to obtain an average m-value for each protein system. With such established m-values (0.9, 6.1, and 1.9 kcal mol-1 M-1 for CaM, BCAII, and Bcl-xL, respectively), it was possible to use eq 1 for the direct calculation of a ∆Gf value from 1/2 each CSUPREX value (see Tables S-1, S-3, and S-4 in the Supporting Information). In this manner, it was possible to calculate between four and nine independent ∆Gf values for each protein complex and then average the values to generate ∆Gf,av values (see Table 1). We believe that the standard deviations associated with these ∆Gf,av values better reflect the precision of our measurements. We also note that the precision of our ∆Gf,av values is comparable to that of similar measurements using conventional spectroscopy-based methods. The quantitative analyses of ligand binding to the CaM, BCAII, and Bcl-xL systems presented above involve the use of eq 1 to analyze the SUPREX data on these protein-ligand complexes. The derivation of eq 1 (see Appendix 1 in ref 5), assumes EX2 exchange conditions for the protein under study. We have recently reported that the accuracy of SUPREX-derived ∆Gf and m-values can be compromised on protein systems when eq 1 is used to analyze SUPREX data obtained under non-EX2 exchange conditions.41 We also showed that in such cases where the accuracy of SUPREX is compromised due to non-EX2 exchange behavior, there is a characteristic nonlinearity observed in the ∆Gapp versus 1/2 CSUPREX plots at high denaturant concentrations.41 We note that no such nonlinearity was observed in any of the ∆Gapp versus 1/2 CSUPREX plots in Figure 4. It is also important to note that the MALDI mass spectra generated in our SUPREX experiments were consistent with EX2 exchange behavior (i.e., only one population of protein ions was detected). Typical mass spectra obtained in our SUPREX analyses of CaM, BCAII, and Bcl-xL complexed with their ligands are included in the Supporting Information. The two populations of protein ions typically observed in the mass spectral analysis of a protein under EX1 exchange conditions were not observed, although we note the resolution of our mass spectrometers was typically limited to ∼500 (as defined using the full width at half-maximum). Qualitative Analysis of Ligand Binding for RmlB. Our SUPREX experiments on RmlB did not permit a quantitative analysis of NAD+ or TMP binding. Only the SUPREX data obtained on the RmlB-NAD+-TMP complex fit well to eq 1 (see Figure 4C). Thus, it was only possible to extract a ∆Gf value for RmlB in the presence of both the NAD+ and TMP ligands. A ∆Gf value for apo-RmlB or for RmlB in the presence of only one ligand (either the NAD+ or the TMP) could not be ascertained. The inability to extract such ∆Gf values precluded the calculation of ∆∆Gf values for the binding of these ligands to RmlB. However, (41) Dai, S. Y.; Fitzgerald, M. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1535-1542. (42) Allard, S. T. M.; Cleland, W. W.; Holden, H. M. J. Biol. Chem. 2004, 279 (3), 2211-2220. (43) Allard, S. T. M.; Giraud, M. F.; Whitfield, C.; Graninger, M.; Messner, P.; Naismith, J. H. J. Mol. Biol. 2001, 307 (1), 283-295.

it is noteworthy that our SUPREX results on the RmlB system did permit a qualitative analysis of NAD+ and TMP binding to RmlB. When SUPREX curves with the same H/D exchange times 1/2 are compared, the CSUPREX values obtained for RmlB in the presence of either NAD+ or TMP appeared at higher denaturant 1/2 concentrations than those obtained for apo-RmlB. Such CSUPREX value shifts are consistent with ligand-induced stabilization of 1/2 value shifts observed for RmlB RmlB. Significantly, the CSUPREX in the presence of TMP (∼0.5 M) were also slightly greater than the shifts observed for RmlB in the presence of NAD+ (∼0.3 M). These results suggest that the binding affinity of TMP for RmlB is likely greater than that of NAD+. Our observations suggest that RmlB binding to both NAD+ and TMP is required for good SUPREX behavior. RmlB consists of two domains, and the NAD+ and TMP ligands bind to the Nand C-terminal domains, respectively.42,43 Thus, when only one ligand is present, only one domain is stabilized. Local fluctuations from the other domain can still dominate the H/D exchange behavior of the protein. Our results on RmlB suggest that when such local fluctuations in structure preclude SUPREX analysis of a multidomain protein, all the domains need to be stabilized to facilitate quantitative SUPREX analyses. CONCLUSIONS This study demonstrates that the ligand-bound forms of large (>∼15 kDa) or multidomain proteins can be amenable to SUPREX analyses using conventional data acquisition and analysis methods, even when apo forms of the protein are not. Of particular significance is the ability of SUPREX to evaluate binding free energies, and in some cases Kd values, for such protein-ligand complexes. The multiple-ligand approach reported here is expected to be a general technique for the detection of ligand binding and the quantitation of ligand binding affinities in large or multidomain proteins. For quantitative studies, at least one stabilizing ligand is needed to render the protein amenable to SUPREX analysis before the binding affinities of additional ligands can be quantified. We anticipate that the mulitiple-ligand approach described here will be particularly useful in ligand binding studies involving metalloproteins and other proteins that require specific cofactors for their function. ACKNOWLEDGMENT Financial support for this work was from NIH GM56414 granted to S.H.G.; E.D.H. was supported by an NIH training grant from the Duke University Center for Biomolecular and Tissue Engineering. J.D.S. was supported in part by an NSF Graduate Fellowship, and K.J.P. was supported in part by the NIH ChemistryBiology Interface Training grant at UWsMadison. We are grateful to Dr. John Hatten and Prof. Eric Toone at Duke University for providing the RmlB sample and to Prof. Y. Tomita at Georgetown for providing the Bcl-xL plasmid and advice on expression. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 12, 2007. Accepted April 20, 2007. AC0700777 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

5877