Biomedical subjects

Michael C Fitzgerald

Publications and source records attributed to Michael C Fitzgerald.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Direct analysis of backbone-backbone hydrogen bond formation in protein folding transition states.

Here we investigate the role of backbone-backbone hydrogen bonding interactions in stabilizing the protein folding transition states of two model protein systems, the B1 domain of protein L (ProtL) and the P22 Arc repressor. A backbone modified analogue of ProtL containing an amide-to-ester bond substitution between residues 105 and 106 was prepared by total chemical synthesis, and the thermodynamic and kinetic parameters associated with its folding reaction were evaluated. Ultimately, these parameters were used in a Phi-value analysis to determine if the native backbone-backbone hydrogen bonding interaction perturbed in this analogue (i.e. a hydrogen bond in the first beta-turn of ProtL's beta-beta-alpha-beta-beta fold) was formed in the transition state of ProtL's folding reaction. Also determined were the kinetic parameters associated with the folding reactions of two Arc repressor analogues, each containing an amide-to-ester bond substitution in the backbone of their polypeptide chains. These parameters were used together with previously established thermodynamic parameters for the folding of these analogues in Phi-value analyses to determine if the native backbone-backbone hydrogen bonding interactions perturbed in these analogues (i.e. a hydrogen bond at the end of the intersubunit beta-sheet interface and hydrogen bonds at the beginning of the second alpha-helix in Arc repressor's beta-alpha-alpha structure) were formed in the transition state of Arc repressor's folding reaction. Our results reveal that backbone-backbone hydrogen bonding interactions are formed in the beta-turn and alpha-helical transition state structures of ProtL and Arc repressor, respectively; and they were not formed in the intersubunit beta-sheet interface of Arc repressor, a region of Arc repressor's polypeptide chain previously shown to have other non-native-like conformations in Arc's protein folding transition state.

Amino Acid Sequence↗

PubMed2006

Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions.

Described here is the impact of so-called non-EX2 exchange behavior on the accuracy of protein unfolding free energies (i.e., DeltaG u values) and m values (i.e.,-deltaDeltaG u/delta[denaturant] values) determined by an H/D exchange and mass spectrometry-based technique termed stability of unpurified proteins from rates of H/D exchange (SUPREX). Both experimental and theoretical results on a model protein, ubiquitin, reveal that reasonably accurate thermodynamic parameters for its folding reaction can be determined by SUPREX even when H/D exchange data is collected in a non-EX2 regime. Not surprisingly, the theoretical results reported here on a series of hypothetical protein systems with a wide range of biophysical properties show that the accuracy of SUPREX-derived DeltaG u and m values is compromised for many proteins when analyses are performed at high pH (e.g., pH 9) and for selected proteins with specific biophysical parameters (e.g., slow folding rates) when analyses are performed at lower pH. Of more significance is that the experimental and theoretical results reveal a means by which problems with non-EX2 exchange behavior can be detected in the SUPREX experiment without prior knowledge of the protein's biophysical properties. The results of this work also reveal that such problems with non-EX2 exchange behavior can generally be minimized if appropriate H/D exchange times are employed in the SUPREX experiment to yield SUPREX curve transition midpoints at chemical denaturant concentrations less than 2 M.

Deuterium Exchange Measurement↗

PubMed2006

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus.

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.

Amino Acid Sequence↗

PubMed2006

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold.

Backbone-backbone hydrogen-bonding interactions are a ubiquitous and highly conserved structural feature of proteins that adopt the same fold (i.e., have the same overall backbone topology). This work addresses the question of whether or not this structural conservation is also reflected as a thermodynamic conservation. Reported here is a comparative thermodynamic analysis of backbone hydrogen bonds in two proteins that adopt the same fold but are unrelated at the primary amino acid sequence level. With amide-to-ester bond mutations introduced by total chemical synthesis methods, the thermodynamic consequences of backbone-backbone hydrogen-bond deletions at five different structurally equivalent positions throughout the beta-alpha-alpha fold of Arc repressor and CopG were assessed. The ester bond-containing analogues all folded into native-like three-dimensional structures that were destabilized from 2.5 to 6.0 kcal/(mol dimer) compared with wild-type controls. Remarkably, the five paired analogues with amide-to-ester bond mutations at structurally equivalent positions were destabilized to exactly the same degree, regardless of the degree to which the mutation site was buried in the structure. The results are interpreted as evidence that the thermodynamics of backbone-backbone hydrogen-bonding interactions in a protein fold are conserved.

Amino Acid Sequence↗

PubMed2006

Theoretical and experimental determination on two substrates turned over by 4-oxalocrotonate tautomerase.

Quantum mechanical/molecular mechanical (QM/MM) calculations and experimental kinetic studies have been performed on 4-oxalocrotonate tautomerase (4OT) for two different substrates, 2-hydroxymuconate (2HM) and 2-oxo-4-hexenedioate (2o4hex). Potential (deltaE) and free energy (deltaG) paths for both steps of the reaction using both substrates were calculated to determine the free energy barriers and compared to the experimental values obtained from the kinetic studies via the transition state theory. In the first step, a proton from the hydroxyl oxygen on the second carbon of 2HM, or from the third carbon of 2o4hex, is abstracted by Pro-1. In the second step, the proton is transferred to the fifth carbon of the substrate to form the product, 2-oxo-3-hexenedioate (2o3hex). For both substrates we obtain a calculated deltaG of approximately 13 kcal/mol, in agreement with experimental determinations. The calculated free energy barrier difference deltaG2o4hex - deltaG2HM (deltadeltaG) is 0.87 kcal/mol. We obtained an experimental deltadeltaG of 0.85 kcal/mol. These results suggest that 2HM is turned over faster than 2o4hex by 4OT. However, these energy differences are so small that both 2HM and 2o4hex need to be taken into account in considering the mechanism of catalysis of 4OT.

Isomerases↗

PubMed2005

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.

Coenzymes↗

PubMed2005

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.

Deuterium Exchange Measurement↗

PubMed2004

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.

Anions↗

PubMed2004

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

Cyclophilin A↗

PubMed2004

The protein backbone makes important contributions to 4-oxalocrotonate tautomerase enzyme catalysis: understanding from theory and experiment.

The role of polypeptide backbone interactions in 4-oxalocrotonate tautomerase (4OT) catalysis has been investigated using a combination of site-directed mutagenesis experiments with unnatural amino acids and quantum mechanical/molecular mechanical (QM/MM) calculations of the 4OT reaction mechanism. Energy barriers for the wild-type enzyme (wt-4OT) and for a 4OT analogue containing a backbone amide to ester bond mutation between Ile-7 and Leu-8 [(OL8)4OT] were determined by both theory and experiment. The amide to ester bond mutation in (OL8)4OT effectively deleted a putative hydrogen bonding interaction between the enzyme's polypeptide backbone and its substrate. Recent theoretical calculations for the 4OT reaction mechanism suggested that this hydrogen bonding interaction helps properly position the substrate in the active site [Cisneros, G. A., et al. (2003) J. Am. Chem. Soc. 125, 10384-10393]. Our experimental results for (OL8)4OT reveal that the energy barrier for the (OL8)4OT-catalyzed reaction was increased 1.8 kcal/mol over that of the wild-type enzyme. This increase was in good agreement with the 1.0 kcal/mol increase obtained from QM/MM calculations for this analogue. Our theoretical calculations further suggest the hydrogen bond deletion in (OL8)4OT results in a rearrangement of the substrate in the active site. In this rearrangement, an ordered water molecule loses its ability to stabilize the transition state (TS), and Arg-61 gains the ability to stabilize the TS. The predicted role of Arg-61 in (OL8)4OT catalysis was confirmed in kinetic experiments with an analogue of (OL8)4OT containing an Arg to Ala mutation at position 61.

Arginine↗

PubMed2004

Facile chemical synthesis and equilibrium unfolding properties of CopG.

The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, DeltaG(f) and m-values of -13.42 +/- 0.04 kcal/mole dimer and 1.92 +/- 0.01 kcal/(mole M) were calculated for CopG.

Amino Acid Substitution↗

PubMed2004

Analysis of protein folding and function using backbone modified proteins.

With the recent development of chemical and biological methods to introduce backbone modifications into the polypeptide chains of proteins, there have been a growing number of site-directed mutagenesis experiments focused on understanding the role of the polypeptide backbone in protein folding and function. The substitution of a main chain amide bond with an ester bond is now a popular mutation to investigate the role of the polypeptide backbone in ligand, binding, enzyme catalysis, and protein folding. Here we review the results of studies on some 25 ester-bond containing analogues from nine different protein systems. The structural, thermodynamic, and functional consequences of introducing backbone amide- to ester-bond mutations into these protein systems are discussed.

Esters↗

PubMed2004

Translating biomarkers into clinical practice: prognostic implications of cyclophilin A and macrophage migratory inhibitory factor identified from protein expression profiles in non-small cell lung cancer.

Biomarkers have the potential to significantly change diagnostic strategies and influence therapeutic management. We developed a MALDI-TOF protein expression profiling platform for biomarker discovery and a proof-of-principle study identified two proteins, cyclophilin A (CyPA) and macrophage migration inhibitory factor (MIF), that were overexpressed in non-small cell lung cancer (NSCLC). The current study focused on evaluating the potential of CyPA and MIF as prognostic markers in patients with a new diagnosis of lung cancer for rapid translation into clinical practice. Two hundred and thirty-four primary NSCLC specimens reflecting a broad range of histologies and stages were examined for CyPA and MIF reactivity by tissue microarray immunohistochemistry (TMA-IHC). The percent tumor cell reactivity, staining intensity and a composite staining score were compared with overall patient survival by Kaplan-Meier curves, log rank test and Cox model statistics. Although both proteins were overexpressed in most NSCLC tumors, neither CypA nor MIF showed a correlation with outcome. This pilot project approach can expedite integration of newly discovered biomarkers into clinical practice, with the goal of improving stratification of patients into appropriate treatment regimens. While both proteins considered in this study were overexpressed in the vast majority of NSCLCs, they were not found to be of prognostic significance.

Adult↗

PubMed2003

Comparative analysis of two different amide-to-ester bond mutations in the beta-sheet of 4-oxalocrotonate tautomerase.

Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.

Amino Acids↗

PubMed2003

Accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic properties of protein-peptide complexes.

A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.

Amino Acid Sequence↗

PubMed2003

Protein expression profiling identifies macrophage migration inhibitory factor and cyclophilin a as potential molecular targets in non-small cell lung cancer.

Current diagnostic and therapeutic strategies for lung cancer have had no significant impact on lung cancer mortality over the last several decades. This study used a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) discovery platform to generate protein expression profiles in search of overexpressed proteins in lung tumors as potentially novel molecular targets. Two differentially expressed protein peaks at m/z 12338 and 17882 in the MALDI-TOF spectra were identified in lung tumor specimens as macrophage migration inhibitory factor and cyclophilin A, respectively. Overexpression of both proteins was confirmed by Western blotting, and cyclophilin A was localized to the tumor cells by immunohistochemistry. These data demonstrate the feasibility of using a MALDI-TOF platform to generate protein expression profiles and identify potential molecular targets for cancer diagnostics and therapeutics.

Amino Acid Sequence↗

PubMed2003

Analysis of human serum proteins by liquid phase isoelectric focusing and matrix-assisted laser desorption/ionization-mass spectrometry.

Direct matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of human serum yielded ion signals from only a fraction of the total number of peptides and proteins expected to be in the sample. We increased the number of peptide and protein ion signals observed in the MALDI-TOF mass spectra analysis of human serum by using a prefractionation protocol based on liquid phase isoelectric focusing electrophoresis. This pre-fractionation technique facilitated the MALDI-TOF MS detection of as many as 262 different peptide and protein ion signals from human serum. The results obtained from three replicate fractionation experiments on the same serum sample indicated that 148 different peptide and protein ion signals were reproducibly detected using our isoelectric focusing and MALDI-TOF MS protocol.

Blood Proteins↗