High-density miniaturized thermal shift assays as a general strategy for drug discovery.

More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.

Crystallographic and thermodynamic comparison of structurally diverse molecules binding to streptavidin.

Crystallographic structures and thermodynamic binding parameters are compared for three structural classes of streptavidin ligand including d-biotin, 2-[(4'-hydroxyphenyl)-azo] benzoate and the peptide NH(2)-Phe-Ser-His-Pro-Gln-Asn-Thr-COOH. Descriptions of these structural and thermodynamic observations emphasize the diversity of potential strategies for improving ligand affinity.

Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis.

Members of the fibroblast growth factor (FGF) family promote angiogenesis and wound repair, modulate early developmental events and survival of neurons, and have been associated with the pathogenesis of various diseases. FGFs interact with specific FGF receptors (FGFRs) and heparan sulfate proteoglycans on cell surfaces to mediate mitogenesis. Using protein structure-based site-directed mutagenesis of basic FGF (bFGF), we have identified two FGFR binding sites on bFGF which act in concert to initiate signal transduction. Both FGFR binding surfaces are distinct from the heparan sulfate proteoglycan binding domain. The primary, higher affinity, binding interaction comprises a cluster of solvent exposed hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140, and Met-142), and two polar residues (Arg-44 and Asn-101). The hydrophobic contacts dominate the primary binding interaction and provide approximately 75% of the binding affinity. The secondary FGFR binding site on bFGF has an approximately 250-fold lower affinity and is composed of amino acids Lys-110, Tyr-111, and Trp-114 in a surface-exposed type I beta-turn (formerly known as the putative receptor binding loop). Binding of FGFR to both bFGF surfaces in a stoichiometry of 2FGFR:1bFGF is required for growth factor mediated cell proliferation. This represents a mechanism for the fibroblast growth factor/receptor family in which FGF facilitates FGFR dimerization and subsequent signal transduction events as a monomeric ligand.

Multivalent ligand-receptor binding interactions in the fibroblast growth factor system produce a cooperative growth factor and heparin mechanism for receptor dimerization.

The binding interactions for the three primary reactants of the fibroblast growth factor (FGF) system, basic FGF (bFGF), an FGF receptor, FGFR1, and the cofactor heparin/heparan sulfate (HS), were explored by isothermal titrating calorimetry, ultracentrifugation, and molecular modeling. The binding reactions were first dissected into three binary reactions: (1) FGFR1 + bFGF<==>FGFR1/bFGF, K1 = 41 (+/- 12) nM; (2) FGFR1 + HS<==>FGFR1/HS, K2 = 104 (+/- 17) microM; and (3) bFGF + HS<==>bFGF/HS, K3 = 470 (+/- 20) nM, where HS = low MW heparin, approximately 3 kDa. The first, binding of bFGF to FGFR1 in the absence of HS, was found to be a simple binary binding reaction that is enthalpy dominated and characterized by a single equilibrium constant, K1. The conditional reactions of bFGF and FGFR1 in the presence of heparin were then examined under conditions that saturate only the bFGF heparin site (1.5 equiv of HS/bFGF) or saturate the HS binding sites of both bFGF and FGFR1 (1.0 mM HS). Both 3-and 5-kDa low MW heparins increased the affinity for FGFR1 binding to bFGF by approximately 10-fold (Kd = 4.9 +/- 2.0 nM), relative to the reaction with no HS. In addition, HS, at a minimum of 1.5 equiv/bFGF, induced a second FGFR1 molecule to bind to another lower affinity secondary site on bFGF (K4 = 1.9 +/- 0.7 microM) in an entropy-dominated reaction to yield a quaternary complex containing two FGFR1, one bFGF, and at least one HS. Molecular weight estimates by analytical ultracentrifugation of such fully bound complexes were consistent with this proposed composition. To understand these binding reactions in terms of structural components of FGFR1, a three-dimensional model of FGFR1 was constructed using segment match modeling. Electrostatic potential calculations confirmed that an elongated cluster, approximately 15 x 35 A, of nine cationic residues focused positive potential (+2kBT) to the solvent-exposed beta-sheet A, B, E, C' surface of the D(II) domain model, strongly implicating this locus as the HS binding region of FGFR1. Structural models for HS binding to FGFR1, and HS binding to bFGF, were built individually and then assembled to juxtapose adjacent binding sites for receptor and HS on bFGF, against matching proposed growth factor and HS binding sites on FGFR1. The calorimetric binding results and the molecular modeling exercises suggest that bFGF and HS participate in a concerted bridge mechanism for the dimerization of FGFR1 in vitro and presumably for mitogenic signal transduction in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain.

Fibroblast growth factors (FGF's) interact on cell surfaces with "low-affinity" heparan sulfate proteoglycans (HSPG) and "high-affinity" FGF receptors (FGFR) to initiate cell proliferation. Previous reports have implicated the binding of heparin, or heparan sulfate, to FGF as essential for FGF-mediated signal transduction and mitogenicity. However, the molecular recognition events which dictate the specificity of this interaction have remained elusive. Amino acid residues on the surface of basic FGF (bFGF) were targeted as potential heparin contacts on the basis of the position of sulfate anions in the X-ray crystal structure of bFGF and of a modeled pentasaccharide heparin-bFGF complex. Each identified amino acid was replaced individually with alanine by site-directed mutagenesis, and the resulting mutant proteins were characterized for differences in binding to a low molecular weight heparin (approximately 3000) by isothermal titrating calorimetry and also for differences in [NaCl] elution from a heparin-Sepharose affinity resin. The combination of site-directed mutagenesis and titrating calorimetry permitted an analysis of the energetic contributions of individual bFGF residues in the binding of heparin to bFGF. The key amino acids which comprise the heparin binding domain on bFGF constitute a discontinuous binding epitope and include K26, N27, R81, K119, R120, T121, Q123, K125, K129, Q134, and K135. Addition of the observed delta delta G degrees of binding for each single site mutant accounts for 8.56 kcal/mol (> 95%) of the free energy of binding. The delta delta G degrees values for N27A, R120A, K125A, and Q134A are all greater than 1 kcal/mol each, and these four amino acids together contribute 4.8 kcal/mol (56%) to the total binding free energy. Amino acid residues K119 through K135 reside in the C-terminal domain of bFGF and collectively contribute 6.6 kcal/mol (76%) of the binding free energy. Although 7 out of the 11 identified amino acids in the heparin binding domain are positively charged, a 7-fold increase in [NaCl] decreases the affinity of wild-type bFGF binding to heparin only 37-fold (Kd at 0.1 M NaCl = 470 nM vs Kd at 0.7 M NaCl = 17.2 microM). This indicates that pure electrostatic interactions contribute only 30% of the binding free energy as analyzed by polyelectrolyte theory and that more specific nonionic interactions, such as hydrogen bonding and van der Waals packing, contribute the majority of the free energy for this binding reaction.

Crystal structure and ligand-binding studies of a screened peptide complexed with streptavidin.

The thermodynamic binding parameters and crystal structure for streptavidin-peptide complexes where the peptide sequences were obtained by random screening methods are reported. The affinities between streptavidin and two heptapeptides were determined by titrating calorimetric methods [Phe-Ser-His-Pro-Gln-Asn-Thr, Ka = 7944 (+/- 224) M-1, delta G degrees = -5.32 (+/- 0.01) kcal/mol, and delta H degrees = -19.34 (+/- 0.48) kcal/mol; His-Asp-His-Pro-Gln-Asn-Leu, Ka = 3542 (+/- 146) M-1, delta G degrees = -4.84 (+/- 0.03) kcal/mol, and delta H degrees = -19.00 (+/- 0.64) kcal/mol]. The crystal structure of streptavidin complexed with one of these peptides has been determined at 2.0-A resolution. The peptide (Phe-Ser-His-Pro-Gln-Asn-Thr) binds in a turn conformation with the histidine, proline, and glutamine side chains oriented inward at the biotin-binding site. A water molecule is immobilized between the histidine and glutamine side chains of the peptide and an aspartic acid side chain of the protein. Although some of the residues that participate in binding biotin also interact with the screened peptide, the peptide adopts an alternate method of utilizing binding determinants in the biotin-binding site of streptavidin.

Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli.

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.

Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

In vivo tumor targeting of a recombinant single-chain antigen-binding protein.

We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.

Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)

The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case.

A weak Ca2+ binding site in the bacterial serine protease subtilisin BPN' (EC 3.4.21.14) was chosen as a model to explore the feasibility of stabilizing a protein by increasing the binding affinity at a metal ion binding site. The existence of this weak Ca2+ binding site was first discovered through a study of the rate of thermal inactivation of wild-type subtilisin BPN' at 65 degrees C as a function of the free [Ca2+]. Increasing the [Ca2+] in the range 0.10-100 mM caused a 100-fold decrease in the rate of thermal inactivation. The data were found to closely fit a theoretical titration curve for a single Ca2+ specific binding site with an apparent log Ka = 1.49. A series of refined X-ray crystal structures (R less than or equal to 0.15, 1.7 A) of subtilisin in the presence of 0.0, 25.0, and 40.0 mM CaCl2 has allowed a detailed structural characterization of this Ca2+ binding site. Negatively charged side chains were introduced in the vicinity of the bound Ca2+ by changing Pro 172 and Gly 131 to Asp residues through site-directed and random mutagenesis techniques, respectively. These changes were found to increase the affinity of the Ca2+ binding site by 3.4- and 2-fold, respectively, when compared with the wild-type protein (ionic strength = 0.10). X-ray studies of these new variants of subtilisin revealed the carboxylate side chains to be 6.8 and 13.2 A, respectively, from the bound Ca2+. These distances and the degree of enhanced binding are consistent with simple electrostatic theory.(ABSTRACT TRUNCATED AT 250 WORDS)

Engineering thermostability in subtilisin BPN' by in vitro mutagenesis.

A procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. The identification and characterization of eight different stabilizing mutations are described. Several mutants containing various combinations of these stabilizing mutations were constructed by oligonucleotide-directed mutagenesis. Combining independent, stabilizing mutations in the same subtilisin molecule has resulted in an approximate multiplicative decrease in the rate of thermal inactivation. In this way, a variant of subtilisin has been constructed which is about 12-fold more stable than wild-type subtilisin, with no radical changes in the tertiary protein structure but rather minor, independent alterations in amino acid sequence. The ultimate goal in these studies is to be able to accurately predict where stabilizing changes can be made in a protein.

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

Proteases of enhanced stability: characterization of a thermostable variant of subtilisin.

A procedure has been developed for the isolation and identification of mutants in the bacterial serine protease subtilisin that exhibit enhanced thermal stability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with bisulfite, a chemical mutagen that deaminates cytosine to uracil in single-stranded DNA. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermal stability were selected by a simple plate assay procedure which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. One thermostable subtilisin variant, designated 7150, has been fully characterized and found to differ from wild-type subtilisin by a single substitution of Ser for Asn at position 218. The 7150 enzyme was found to undergo thermal inactivation at one-fourth the rate of the wild-type enzyme when incubated at elevated temperatures. Moreover, the mid-point in the thermally induced transition from the folded to unfolded state was found to be 2.4-3.9 degrees C higher for 7150 as determined by differential scanning calorimetry under a variety of conditions. The refined, 1.8-A crystal structures of the wild-type and 7150 subtilisin have been compared in detail, leading to the conclusion that slight improvements in hydrogen bond parameters in the vicinity of position 218 result in the enhanced thermal stability of 7150.

Site-directed mutagenesis and the role of the oxyanion hole in subtilisin.

Oligonucleotide-directed mutagenesis was used to investigate the nature of transition state stabilization in the catalytic mechanism of the serine protease, subtilisin BPN'. The gene for this extracellular enzyme from Bacillus amyloliquefaciens has been cloned and expressed in Bacillus subtilis. In the transition state complex, the carbonyl group of the peptide bond to be hydrolyzed is believed to adopt a tetrahedral configuration rather than the ground-state planar configuration. Crystallographic studies suggest that stabilization of this activated complex is accomplished in part through the donation of a hydrogen bond from the amide side group of Asn-155 to the carbonyl oxygen of the peptide substrate. To specifically test this hypothesis, leucine was introduced at position 155. Leucine is isosteric with asparagine but is incapable of donating a hydrogen bond to the tetrahedral intermediate. The Leu-155 variant was found to have an unaltered Km but a greatly reduced catalytic rate constant, kcat, (factor of 200-300 smaller) when assayed with a peptide substrate. These kinetic results are consistent with the Asn-155 mediating stabilization of the activated complex and lend further experimental support for the transition-state stabilization hypothesis of enzyme catalysis.

Affinity chromatographic purification of angiotensin converting enzyme.

The compounds N-[1 (S)-carboxy-5-amino-pentyl]-L-phenylalanylglycine and N-[1 (S)-carboxy-5-aminopentyl]-DL-alanyl-L-proline were synthesized and explored as potential ligands for the affinity chromatography of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1) (ACE), a membrane-bound zinc metalloprotease. The N-alkylated Ala-Pro derivative has an apparent Ki less than 1 nM (at pH 7.5, 0.50 M NaCl) while the Phe-Gly derivative is a much less potent competitive inhibitor with an apparent Ki = 0.20 microM under the same conditions and thus more suitable for use as an affinity ligand. Immobilization of these compounds via a 28-A spacer to agarose yields resins with binding capacities of greater than 7 mg of enzyme/mL of resin, while spacers of 22 A or less result in binding capacities at least 350 times smaller. Immobilized N-[1 (S)-carboxy-5-amino-pentyl]-L-Phe-Gly is superior to the Ala-Pro derivative because elution can be affected by raising the pH to 8.9 with 98% yields compared with only 20% from the latter. Thus, a three-step process involving detergent extraction, concentration by ammonium sulfate precipitation, and affinity chromatography on the resin-immobilized Phe-Gly derivative provides 30 mg of homogeneous ACE from 640 g of rabbit lung tissue. An ACE-like metalloprotease has also been isolated from testicular tissue by this same technique.

A pH-dependent superoxide dismutase activity for zinc-free bovine erythrocuprein. Reexamination of the role of zinc in the holoprotein.

The zinc-free derivative of bovine erythrocuprein, Cu2E2BE, was prepared and its superoxide dismutase activity was measured and compared with that of the holoprotein, Cu2Zn2BE. The dismutase activity of these proteins was measured by quantitating their inhibition of the superoxide-mediated autooxidation of 6-hydroxydopamine, dihydroxyfumaric acid, pyrogallol, and epinephrine. It was found that the superoxide dismutase activity of the zinc-free protein is pH dependent, ranging between 82 +/- 5% (relative to Cu2Zn2BE) at pH 5.8, and 25 +/- 10% at pH 10.2. The overlapping range of assays and buffers verified that these measurements are independent of the method of assay, buffer, and ionic strength (in the range of mu = 0.10 to 0.20). The variation in activity with pH is probably due, at least in part, to the migration of Cu(II) at high pH as described previously [J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard, Proc. Natl. Acad. Sci. USA 76, 4245 (1979)], since Cu(II) bound at the zinc binding site has been shown to have little or no dismutase activity. The observation of high activity (82%) for the zinc-free protein at pH 5.8, where Cu(II) is predominantly in the native Cu binding site, and less susceptible to removal by ethylenediaminetetraacetic acid, demonstrates that the presence of Zn(II) in Cu2Zn2BE does not greatly enhance the inherent dismutase activity of Cu(II) in the holoprotein.

Nuclear magnetic resonance study of the exchangeable histidine protons in bovine and wheat germ superoxide dismutases.

Nuclear magnetic resonance studies at 220 MHz of the exchangeable histidine NH protons in bovine erythrocyte superoxide dismutase (BESOD) [EC 1.15.1.1] and the two isoenzymes of wheat germ superoxide dismutase (WGSODI and WGSODII) have been carried out. NMR spectral similarities reveal substantial structural homology of WGSOD with BESOD. Comparison of the spectra of the apoproteins and copper-free, native, and reduced proteins suggests that zinc has a structural role in WGSOD similar to that previously reported for BESOD [Lippard, S. J., Burger, A. R., Ugurbil, K., Pantoliano, M. W., & Valentine, J. S. (1977) Biochemistry 16, 1136]. Four resonances are assigned to conserved histidine residues, three of which are coordinated to the zinc atom while the fourth is nonligating.

pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase.

Bovine erythrocyte superoxide dismutase (Cu(2)Zn(2)SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu(2+) and Zn(2+) in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu(2)E(2)SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zinc-free protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu(2)Cu(2)SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30 degrees C are also consistent with formation of Cu(II)-Im-Cu(II). A plot of changes in the signal amplitude of g perpendicular for Cu(2)E(2)SODase as a function of pH gives an apparent pK(a) of 8.2 for the transition. The long-wavelength absorption with lambda(max) = 700 nm characteristic of Cu(2)E(2)SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu(2)Cu(2)SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO(3), although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu(2+) from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu(2)Zn(2)SODase, and the four-copper protein, Cu(2)Cu(2)SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu(2)E(2)SODase and its thermal stability are reexamined in light of these new findings.

Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure.

Nuclear magnetic resonance (NMR) spectroscopy of the exchangeable protons, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O has been found to be a powerful method to study the active site of the enzyme. This technique has been employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that zinc alone organizes the active site structure. All eight histidines per subunit of apoenzyme react with DEP. The accessibility of these residues to solvent is borne out by the broad, featureless NMR spectrum of the apoprotein. In the holoenzyme only His-19, which is exposed to solvent, can be modified with DEP. The reduced holoenzyme shows a well-resolved NMR spectrum compared with the oxidized form in which the lines are broadened by the paramagnetic copper ion. A spectrum very similar to that of the reduced enzyme is generated by addition of one zinc ion per subunit of apoprotein showing that zinc alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of zinc per subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios greater than or equal to 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.