Stability Of Unpurified Proteins Research Articles

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.

Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange– and Mass Spectrometry–Based Strategy

Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Described here is use of an H/D exchange- and a matrix assisted laser desorption/ionization (MALDI) mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen 2 chemical libraries, including the 1280-compound LOPAC library and the 9600-compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which is demonstrated here to yield screening rates as fast as 6 s/ligand. The false-positive and false-negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false-positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered, including 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1, 4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[5.2.1.0~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., K(d) values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA-targeted diagnostic imaging or therapeutic agents for lung cancer.

Thermodynamic Analysis of a Molecular Chaperone Binding to Unfolded Protein Substrates

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins to facilitate protein folding and prevent nonspecific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports about the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H-D exchange), an H-D exchange and MALDI-based technique, in studying the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates, including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding-unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX-derived K(d) values for Hsp33 complexes with four different substrates were all found to be within the range of 3-300 nM.

Painting Proteins with Covalent Labels: What's In the Picture?

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

Primary hyperoxaluria type I is a severe kidney stone disease caused by mutations in the protein alanine:glyoxylate aminotransferase (AGT). Many patients have mutations in AGT that are not deleterious alone but act synergistically with a common minor allele polymorphic variant to impair protein folding, dimerization, or localization. Although studies suggest that the minor allele variant itself is destabilized, no direct stability studies have been carried out. In this report, we analyze AGT function and stability using three approaches. First, we describe a yeast complementation growth assay for AGT, in which we show that human AGT can substitute for function of yeast Agx1 and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast. We further examine stability of AGT alleles in vitro using two direct methods, a mass spectrometry-based technique (stability of unpurified proteins from rates of H/D exchange) and differential scanning fluorimetry. We also examine the effect of known ligands pyridoxal 5'-phosphate and aminooxyacetic acid on stability. Our work establishes that the minor allele is destabilized and that pyridoxal 5'-phosphate and aminooxyacetic acid binding significantly stabilizes both alleles. To our knowledge, this is the first work that directly measures relative stabilities of AGT variants and ligand complexes. Because previous studies suggest that stabilizing compounds (i.e. pharmacological chaperones) may be effective for treatment of primary hyperoxaluria, we propose that the methods described here can be used in high throughput screens for compounds that stabilize AGT mutants.

Comparison of two ESI-MS based H/D exchange methods for extracting protein folding energies

In this report, the model proteins staphylococcal nuclease and ubiquitin were used to test the applicability of two new hydrogen/deuterium exchange (HX) electrospray ionization mass spectrometry (ESI-MS) methods for estimating protein folding energies. Both methods use the H/D exchange of globally protected amide protons (amide protons which are buried in the hydrophobic core) to elucidate protein folding energies. One method is a kinetic-based method and the other is equilibrium-based. The first method, the HX ESI-MS kinetic-based approach is conceptually identical to SUPREX (stability of unpurified proteins from rates of H/D exchange) method but is based on ESI-MS rather than MALDI-MS (matrix assisted laser desorption mass spectrometry). This method employs the time-dependence of H/D exchange using various denaturant concentrations to extract folding energies. Like SUPREX, this approach requires the assumption of EX2 exchange kinetics. The second method, which we call a protein equilibrium population snapshot (PEPS) by HX ESI-MS uses data collected only for a single time point (usually the shortest possible) to obtain a snapshot of the open and closed populations of the protein. The PEPS approach requires few assumptions in the derivation of the equations used for calculation of the folding energies. The extraction of folding energies from mass spectral data is simple and straightforward. The PEPS method is applicable for proteins that follow either EX1 or EX2 HX mechanisms. In our experiments the kinetic-based method produced less accurate Δ G H 2 O and m GdHCl values for wild-type staphylococcal nuclease and mutants undergoing H/D exchange by EX1, as would be expected. Better results were obtained for ubiquitin which undergoes HX by an EX2 mechanism. Using the PEPS method we obtained Δ G H 2 O and m GdHCl values that were in good agreement with literature values for both staphylococcal nuclease (EX1) and ubiquitin (EX2). We also show that the observation of straight lines in linear extrapolation method (LEM) plots is not a reliable indicator of the validity of the data obtained using the LEM approach.

Throughput and Efficiency of a Mass Spectrometry-Based Screening Assay for Protein–Ligand Binding Detection

An H/D exchange- and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on stability of unpurified proteins from rates of H/D exchange (SUPREX), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution. The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library (Illkirch, France) could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a two-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX.

Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

Ex Vivo Analysis of Synergistic Anion Binding to FbpA in Gram-Negative Bacteria

Ferric binding protein, FbpA, is a member of the transferrin superfamily whose function is to move an essential nutrient, iron, across the periplasm and into the cytosol through formation of a ternary complex containing Fe (3+) and a synergistic anion, X. Here we utilize SUPREX ( stability of unpurified proteins from rates of H/D exchange) to determine the identification and distribution of the synergistic anion in FeFbpA-X species in periplasmic preparations from Gram-negative bacteria. SUPREX is a mass spectrometry-based technique uniquely suited for thermodynamic analyses of protein-ligand complexes in complex biological mixtures such as periplasmic preparations. Model binary mixtures of FeFbpA-Cit and FeFbpA-PO 4 were initially characterized by SUPREX due to the likely presence of citrate and phosphate ions in the periplasm. Ex vivo SUPREX analyses were performed on FeFbpA-X species overexpressed in an Escherichia coli cell line and on endogenous FeFbpA-X species in Neisseria gonorrheae. Detected in the E. coli periplasmic extract were two distinct populations of FbpA, including one in which the protein was unliganded (i.e., apoFbpA) and one in which the protein was bound to iron and the synergistic anion, phosphate (i.e., FeFbpA-PO 4). FeFbpA-PO 4 was the only population of FbpA molecules detected in the N. gonorrheae periplasmic extract. This work provides the first determination of the identity of the in vivo anion bound to FeFbpA-X in the periplasm and substantiates the hypothesis that the synergistic anion plays a structural and functional role in FbpA-mediated transport of iron across the periplasm and into the cytosol.

H/D Exchange and Mass Spectrometry-Based Method for Biophysical Analysis of Multidomain Proteins at the Domain Level

A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.

H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein−Ligand Binding

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 spectrometry-based technique for measuring the free energy (DeltaGf) and m-value (deltaDeltaGf/delta[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 (> approximately 15 kDa) or multidomain proteins. In the absence of ligand, DeltaGf and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound 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.

Mutational Analysis of Active Site Residues in the Staphylococcus aureus Transpeptidase SrtA

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.

A Mass Spectrometry-Based Probe of Equilibrium Intermediates in Protein-Folding Reactions

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution.

The determination of high‐affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometry

Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKCtheta. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay.

Thermodynamic Analysis of Subunit Interactions in Escherichia coli Molybdopterin Synthase

The molybdopterin (MPT) synthase complex in Escherichia coli consists of two MoaE subunits and two MoaD subunits in a heterotetrameric structure with the two MoaE subunits forming a central dimer. Each MoaD subunit binds to a single MoaE molecule to form two identical MoaE/MoaD interfaces. Here we define the thermodynamic properties of the interaction between MoaE and MoaD in MPT synthase using a H/D exchange and matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy based method termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX-derived protein folding free energies and m values are reported for MoaE in the presence and absence of MoaD and MoaD-SH, the thiocarboxylated form of MoaD that is essential for the catalytic activity of MPT synthase. The protein folding free energy measurements were used to calculate a dissociation constant of 17 +/- 7 microM for the binding of MoaD to MoaE in inactive MPT synthase and a dissociation constant of 2.6 +/- 0.9 microM for the binding of MoaD-SH to MoaE in active MPT synthase. The increased binding affinity of MoaD-SH for MoaE is consistent with a previously proposed mechanism for the MPT synthase reaction. Using the increased m values exhibited by MoaE in the presence of either MoaD subunit, the solvent accessible surface area buried upon formation of the subunit interface in MPT synthase was estimated to be 2378 A(2) for inactive MPT synthase and 4117 A(2) for active MPT synthase.

Protocol for the Thermodynamic Analysis of Some Proteins Using an H/D Exchange- and Mass Spectrometry-Based Technique

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a new H/D exchange- and mass spectrometry-based technique for the measurement of protein folding free energies (i.e., DeltaG values) and protein folding m values (i.e., deltaDeltaG/delta[denaturant]). Robust protocols for the acquisition and analysis of SUPREX data have been established and shown to be useful for the analysis of a number of different protein systems. Here we report on the SUPREX behavior of a special class of proteins that are not amenable to conventional SUPREX analyses using previously established protocols. This class of proteins includes protein systems that require an extended time to reach a folding/unfolding equilibrium in chemical denaturant-induced equilibrium unfolding experiments. As part of this work we use ubiquitin as a model system to highlight the complications that can arise in the conventional SUPREX analysis of such protein systems, and we describe a modified SUPREX protocol that can be used to eliminate these complications.

SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) Analysis of the Thermodynamics of Synergistic Anion Binding by Ferric-Binding Protein (FbpA), a Bacterial Transferrin

SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe(3+)FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe(3+) across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe(3+)FbpA-X is not a so-called "ideal" protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe(3+)FbpA-X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe(3+)FbpA. As part of this work, the SUPREX technique was used to evaluate the DeltaDeltaG(f) values of four synergistic anion-containing complexes of Fe(3+)FbpA (i.e., Fe(3+)FbpA-PO(4), Fe(3+)FbpA-citrate, Fe(3+)FbpA-AsO(4), and Fe(3+)FbpA-SO(4)). The DeltaDeltaG(f) value obtained for Fe(3+)FbpA-citrate relative to Fe(3+)FbpA-PO(4) (1.45 +/- 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe(3+)FbpA-AsO(4) (0.58 +/- 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a DeltaDeltaG(f) value for Fe(3+)FbpA-SO(4), -1.43 +/- 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.

Thermodynamic analysis of cyclosporin a binding to cyclophilin a in a lung tumor tissue lysate.

We report on the application of SUPREX (stability of unpurified proteins from rates of H/D exchange) to the analysis of a protein-ligand binding interaction under the ex vivo solution conditions of a human lung tumor tissue lysate. A SUPREX-derived binding free energy (i.e. DeltaDeltaG(f) value) and dissociation constant (i.e., K(d) value) were determined for the binding of cyclosporin A (CsA) to a cyclophilin A (CypA) sample in which the protein was a component of a tissue lysate derived from fresh frozen lung tumor. The DeltaDeltaG(f) and K(d) values determined by SUPREX for CsA binding to CypA in this unpurified protein sample, 4.7 +/- 0.8 kcal/mol and 77 +/- 17 nM, respectively, were comparable to the those obtained when SUPREX was used to analyze the binding of CsA to a highly purified CypA sample, 4.2 +/- 1.0 kcal/mol and 32 +/- 20 nM, respectively. Moreover, the SUPREX-derived K(d) values determined in this work were both in the range of those previously reported for the CypA-CsA complex. The results of this proof-of-principle work validate the extension of SUPREX to the thermodynamic analysis of proteins and protein-ligand binding interactions in the unpurified, ex vivo conditions of human tissue lysates,and they represent the first K(d) measurement on a protein-ligand complex under such conditions

High-throughput screening assay for the tunable selection of protein ligands.

Here, we describe a new protein-ligand binding assay that is amenable to high-throughput screening applications. The assay involves the use of SUPREX (stability of unpurified proteins from rates of H/D exchange), a new H/D exchange and mass spectrometry-based technique we recently developed for the quantitative analysis of protein-ligand binding interactions. As part of this work, we describe a new high-throughput SUPREX protocol, and we demonstrate that this protocol can be used to efficiently screen peptide ligands in a model combinatorial library for binding to a model protein system, the S-protein. The high-throughput SUPREX protocol developed here is generally applicable to a wide variety of protein ligands, including DNA, small molecules, metals, and other proteins. On the basis of the results of the model study in this work, one person with access to one MALDI mass spectrometer should be able to screen approximately 10 000 compounds per 24-h period using the protocol described here. With full automation and the use of a commercially available MALDI mass spectrometer optimized for high-throughput analyses, we estimate that the SUPREX-based assay described here could be used to screen on the order of 100 000 ligands per day.