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A novel series of pyrazole sEH inhibitors is reported. Lead optimization efforts to replace the aniline core are also described. In particular, 2-pyridine, 3-pyridine and pyridazine analogs are potent sEH inhibitors with favorable CYP3A4 inhibitory and microsomal stability profiles.

© 2015 Elsevier Inc. The beneficial effects of physical activity (PA) are well documented, yet the mechanisms by which PA prevents disease and improves health outcomes are poorly understood. To identify major gaps in knowledge and potential strategies for catalyzing progress in the field, the NIH convened a workshop in late October 2014 entitled "Understanding the Cellular and Molecular Mechanisms of Physical Activity-Induced Health Benefits." Presentations and discussions emphasized the challenges imposed by the integrative and intermittent nature of PA, the tremendous discovery potential of applying "-omics" technologies to understand interorgan crosstalk and biological networking systems during PA, and the need to establish an infrastructure of clinical trial sites with sufficient expertise to incorporate mechanistic outcome measures into adequately sized human PA trials. Identification of the mechanisms that underlie the link between PA and improved health holds extraordinary promise for discovery of novel therapeutic targets and development of personalized exercise medicine.

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level by coordinate action of a limited number of transcription factors that include regulatory factor X (RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer. A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K(d) = 25 nM) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter, while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of binding for the overall complex. AUC experiments demonstrate that in the absence of additional subunits, monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA, and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays, confirming that preassociation with DNA is not required for forming the macromolecular assemblies that drive MHC class II gene expression.

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.

It has been reported that the diaryl urea class of p38alpha inhibitors binds to p38 map kinase with both high affinity and slow binding kinetics (Pargellis et al. Nat. Struct. Biol. 2002, 9, 268-272). The slow binding kinetics of this class of inhibitors is believed to be the result of binding to an allosteric pocket adjacent to the p38alpha active site. The use of traditional kinetic and equilibrium methods to measure the binding affinity of this class of compounds has created many challenges for determination of structure-activity relationships (SAR). The thermal denaturation method provides a means of measuring high-affinity interactions. In this paper, the method of thermal denaturation will be described as it has been applied to the diaryl urea class of p38 map kinase inhibitors.

Signaling by some TNF receptor family members, including CD40, is mediated by TNF receptor-associated factors (TRAFs) that interact with receptor cytoplasmic domains following ligand-induced receptor oligomerization. Here we have defined the oligomeric structure of recombinant TRAF domains that directly interact with CD40 and quantitated the affinities of TRAF2 and TRAF3 for CD40. Biochemical and biophysical analyses demonstrated that TRAF domains of TRAF1, TRAF2, TRAF3, and TRAF6 formed homo-trimers in solution. N-terminal deletions of TRAF2 and TRAF3 defined minimal amino acid sequences necessary for trimer formation and indicated that the coiled coil TRAF-N region is required for trimerization. Consistent with the idea that TRAF trimerization is required for high-affinity interactions with CD40, monomeric TRAF-C domains bound to CD40 significantly weaker than trimeric TRAFs. In surface plasmon resonance studies, a hierarchy of affinity of trimeric TRAFs for trimeric CD40 was found to be TRAF2 > TRAF3 >> TRAF1 and TRAF6. CD40 trimerization was demonstrated to be sufficient for optimal NF-kappaB and p38 mitogen activated protein kinase activation through wild-type CD40. In contrast, a higher degree of CD40 multimerization was necessary for maximal signaling in a cell line expressing a mutated CD40 (T254A) that signaled only through TRAF6. The affinities of TRAF proteins for oligomerized receptors as well as different requirements for degree of receptor multimerization appear to contribute to the selectivity of TRAF recruitment to receptor cytoplasmic domains.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR ζ ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon ζ ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of ζ chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein–phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein–phosphopeptide complex relative to the protein alone.

The chains of the T cell receptor complex play a critical role in the initiation of proximal signaling events upon T cell activation. Three pairs of potential tyrosine phosphorylation sites are located within the cytoplasmic domains of the chains. Subsequent to engagement of the T cell receptor, one or more of these tyrosine residues is phosphorylated. The phosphotyrosine residues, along with flanking amino acids, form an activation motif (and are shared by signaling subunits in the TCR, B cell receptor, and FcγRI) termed tyrosine-based activation motifs (ITAMs). ITAMs serve as binding sites for SH2 domain-containing proteins. Recent evidence suggests that the chains provide docking space for several key signal transduction molecules such as ZAP-70, p56 lck, and Shc. To determine if ZAP-70, p56 lck, and Shc bind to particular chain ITAM sequences, quantitative free-solution measurements of binding affinities (Kd) were obtained by use of surface plasmon resonance technology. The results indicate that binding affinities of distinct SH2 domains to individual and paired phosphorylation sites greatly differ, and may dictate the sequence of signal transduction events.

The development of a sensitive fluorescence binding assay for evaluating the binding of phosphotyrosyl (pY) peptides to the recombinant SH2 domain of lck in solution is described. Several fluorescent peptides containing the consensus sequence of the viral hamster polyoma middle T antigen (pYEEI) were characterized. The peptides contained either the acetamido-anilino-naphthyl sulfonic acid (AANS), acrylodan, or dansyl groups as fluorophores. The spectral features of these probes were characterized in the presence and absence of the lck SH2 domain. The binding affinities (Kd) for the fluorescent peptides studied ranged from 40 to 500 nM. The fluorescent peptide containing the sequence FTATEC(AANS)QpYEEIP exhibited the highest binding affinity (Kd = 3.98 x 10(-8) M) and largest change in emission intensity (approximately 8.7-fold) upon binding the SH2 domain. This probe was subsequently used in competitive binding assays to study the interaction of the lck SH2 domain with a series of phosphopeptides related to the pYEEIP and pYQPQP (the pY505 C-terminal) consensus sequences. The effects of peptide length and substitutions of residues within the pYEEIP sequence are discussed in terms of binding affinities. Comparison between the two peptide series revealed that the contributions of individual substitutions to binding affinity are context-dependent. The data also led to the conclusion that the presence of P at +2 results in a functional "truncation" of the binding sequence; i.e., residues at positions higher than +2 do not participate significantly in binding. This implicit truncation may actually be a desired property for the autoregulatory nature of the pYQPQP sequence, since it retains specificity for the SH2 domain while adjusting the Kd to a value appropriate for maintaining the delicate balance of receptor-ligand interactions that are involved in signal transduction events.

Nevirapine (BI-RG-587) is a potent inhibitor of the polymerase activity of reverse transcriptase of human immunodeficiency virus type-1. Nevirapine, as well as several other non-nucleoside compounds of various structural classes, bind strongly at a site which includes tyrosines 181 and 188 of the p66 subunit of reverse transcriptase. The chromatography which was utilized to explore this binding site is described. BI-RH-448 and BI-RJ-70, two tritiated photoaffinity azido analogues of nevirapine, are each crosslinked to reverse transcriptase. The use of several HPLC-based techniques employing different modes of detection makes it possible to demonstrate a dramatic difference between the two azido analogues in crosslinking behavior. In particular, by comparing HPLC tryptic peptide maps of the photoadducts formed between reverse transcriptase and each azido analogue, it can be shown that crosslinking with BI-RJ-70 but not with BI-RH-448 is more localized, stable, and hence exploitable for the identification of the specifically bonded amino acid residue(s). In addition, comparison of the tryptic maps also makes it feasible to assess which rings of the nevirapine structure are proximal or distal to amino acid side chains of reverse transcriptase. Finally, another feature of the HPLC peptide maps is the application of on-line detection by second order derivative UV absorbance spectroscopy to identify the crosslinked amino acid residue.

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled phytohemagglutinin-stimulated lymphoblasts and peripheral blood neutrophils. In addition, the anti-cytoplasmic domain serum, but not the antiectodomain serum, immunoprecipitate a 6-kD species from PMA activated lymphoblasts and formylmethionylleucylphenylalanine-activated neutrophils. Conversely, the antiectodomain serum but not the anti-cytoplasmic domain serum immunoprecipitate a 68-kD soluble form of L-selectin from the supernatant of PMA-activated lymphoblasts. The appearance of the 6-kD species on activated cells correlated with the disappearance of the intact form of L-selectin and the appearance of the soluble form of L-selectin. A third polyclonal serum generated against the membrane proximal region of the ectodomain also reacted with the 6-kD species, indicating that this is a transmembrane peptide of L-selectin. That the 6-kD species is derived from L-selectin was confirmed by immunoprecipitation of the 6-kD species from L-selectin transfectants but not from mock transfectants. Radiochemical sequence analysis defined a cleavage site between Lys321 and Ser322, which would predict a transmembrane fragment consistent in size with the observed 6-kD fragment. A Ser-Phe-Ser motif adjacent to the cleavage site is conserved between human, mouse, and rat L-selectin, and a related motif is found proximal to transmembrane domains of other downregulated proteins, such as ACE, CD16-II, and TNF-RII, suggesting the possibility of a common recognition motif.

Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.

The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.

Nevirapine (BI-RG-587) is a potent and specific non-nucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is non-competitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues.

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance Ca2+-activated K+(KCa) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular K+to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward K+current and increased KCachannel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[14C]EET methyl ester (Me) was converted to 14,15-[14C]DHET-Me, 14,15-[14C]DHET, and 14,15-[14C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of KCachannels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B==AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.

The unfolding of rabbit fast skeletal muscle troponin C has been examined as a function of pH using differential scanning calorimetry as a probe. Studies were conducted between pH 7.2 and 5.25 in the absence of both Ca2+ and Mg2+. At pH 7.2, a single unfolding transition, with a tm of 58 degrees C, is observed. Decreasing the pH to 5.5 increases the tm for this transition to 67 degrees C. At pH values less than or equal to 5.5, a second unfolding transition is observed, which has a tm of 51 degrees C. These studies suggest that troponin C consists of two types of structural domains: one has a compact structure throughout the pH range examined; the other has a poorly ordered structure at pH 7.2 which becomes more compact as the pH is decreased. We conclude that stabilization of both types of domains occurs through protonation of carboxylate groups clustered in each of the four Ca2+-binding loops. Our data are consistent with assignment of the higher-temperature transition to unfolding of the N-terminal portion of the molecule, which contains the Ca2+-specific domains and of the lower-temperature transition to unfolding of the C-terminal portion, which contains the Ca2+-Mg2+ domains.

Rabbit and bovine cardiac troponin (Tn) subunits and complexes were labeled with iodo[14C]acetamide in the presence and absence of Ca2+ to determine the effect of tertiary and quaternary structure on exposure of Cys SH groups. This procedure serves both to map regions of subunit interaction and the effects of Ca2+-induced conformational change and to indicate which Cys residues should be useful attachment sites for spectroscopic or cross-linking probes. After being labeled, Tn subunits were purified by using reversed-phase HPLC and subjected to tryptic cleavage with or without prior citraconylation. Cys-containing fragments were isolated by RP-HPLC, and the percent labeling was determined. Cys-75 and -92 of TnI were completely accessible to iodoacetamide both when TnI was labeled alone or when in the TnC-TnI complex. Both residues were largely inaccessible when Tn or the TnI-TnT complex was labeled, suggesting burial in the TnI-TnT interface. In contrast, the Cys from the N-terminal region of bovine TnT was stoichiometrically labeled when TnT was labeled alone, in native Tn or in a troponin-tropomyosin complex. Cys-35 and -84 of TnC are located in the nonfunctional Ca2+ binding loop I of cardiac TnC and helix D, respectively. For TnC alone, the percent labelings of Cys-35 and -84 were 11% and 26%, respectively (minus Ca2+), and 16% and 63%, respectively (plus Ca2+). For TnC labeled within Tn, the percent labelings of Cys-35 and -84 were 20% and 52%, respectively (minus Ca2+), and 20% and 78%, respectively (plus Ca2+).(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Escherichia coli, to approximately 90% purity. The purified enzyme at a concentration of approximately 20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 microM.

Cytochrome c, myoglobin and lysozyme, as well as two synthetic peptides, TM-22 and TM-36, were used to examine denaturation of protein structure on a hydrophobic-interaction column, the Bio-Gel TSK-Phenyl -5-PW high-performance liquid chromatography column. The first three proteins were chosen because they have a monomeric structure while both synthetic peptides, which have the sequence Ac-(Lys-Leu-Glu-Ala-Leu-Glu-Gly)inn-Lys-amide where n  3 for TM-22 and n  5 for TM-36, are dimeric under solvent conditions used for hydrophobic-interaction chromatography. Only TM-36 is dimeric under reversed-phase conditions. Thus, denaturation of both tertiary and quaternary structure can be examined. The column was operated in both reversed-phase and hydrophobic-interaction modes. This, in combination with temperature variation between approximately 0-50°C, provided conditions where denaturing effects of the support could be examined. In reversed-phase mode, cytochrome c, myoglobin, and TM-22 were eluted in a denatured form throughout the temperature range. In contrast, lysozyme and TM-36 eluted primarily in their native conformation at low temperatures, but experienced partial or total denaturation at higher temperatures. Significantly, all of the polypeptides studied were denatured at room temperature by a conventional reversed-phase column, the Altex Ultrapore RPSC C-3 indicating that the Bio-Gel TSK-Phenyl-5-PW column is less denaturing. In the hydrophobic-interaction mode, the dimeric structure of TM-22 was totally disrupted at all temperatures, while TM-36, myoglobin, and cytochrome c underwent various degrees of partial denaturation as the temperature increased. Comparison of the temperatures at which the various polypeptides underwent denaturation on the column with their normal melting temperatures (where half the molecules are unfolded) demonstrated that the hydrophobic column itself, rather than the temperature, was primarily responsible for denaturation. Hence, even relatively “gentle” hydrophobic columns can promote denaturation of protein structure. Since the tertiary and quaternary structures of most proteins are stabilized by hydrophobic interactions, the possibility of denaturation must always be taken into consideration when a hydrophobic column is used.

A kinetic study of the reactions of 4-nitrophenyl 5-nitrosalicylate in aqueous Tris buffer at 25°C has revealed three kinetically significant reactions: Tris aminolysis of the un-ionized substrate, Tris aminolysis of the ionized substrate, and spontaneous hydrolysis of the ionized substrate. Solvent isotope effects have been measured for all three reactions. Mechanisms are discussed, and the conclusion is reached that intramolecular catalysis is operating in only the spontaneous hydrolysis.

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg2+ on the acto-S1 ATPase have been examined. The effect of free Mg2+ on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg2+ concentrations: 2.5 mM Mg2+-2.5 mM ATP, conditions considered to have low free Mg2+; and 5.0 mM Mg2+-2.5 mM ATP, conditions providing a high free Mg2+ concentration of ∼2.5 mM. In the presence of high free Mg2+ (2.5 mM ATP-5.0 mM MgCl2) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40–50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg2+ on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg2+ there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg2+. Similarly, in the presence of TM and Tn, Mg2+ exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg2+. Furthermore, ultracentrifugation binding studies using14C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg2+ concentrations, indicating that faciliation of binding to F-actin by Mg2+ is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg2+ binding and by analogy Ca2+ binding to the Ca2+-Mg2+ sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca2+ binding to the Ca2+-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca2+. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca2+, indicate that the role of TnT involves more than anchoring Tn to the thin filament.

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.

The thermodynamic parameters characterizing the interaction between rabbit fast skeletal muscle troponin and tropomyosin have been determined at 25 degrees C for three solution conditions: buffer containing (A) 1 mM CaCl2, simulating a "turned-on" state, (B) 3 mM MgCl2, simulating a "turned-off" state, and (C) 2 mM ethylenediaminetetraacetic acid, a reference state. The enthalpies were measured in two buffers with different heats of ionization to allow correction for dissociation or uptake of protons. The enthalpies corrected for proton effects are -22.1, -25.4, and -23.5 kcal/mol, respectively, in buffers A, B, and C. The interaction between troponin and tropomyosin in the presence of calcium is accompanied by release of 0.9 mol of proton per mole of complex. Proton effects in the presence of magnesium and in the absence of divalent metal ions were too small to quantitate. The association constants were measured by using tropomyosin labeled with the extrinsic fluorescent probe dansylaziridine, and binding was detected by enhancement of the probe fluorescence. The magnitudes of the association constants for unlabeled troponin are 7.5 X 10(5), 4.2 X 10(5), and 9.5 X 10(5) M-1, respectively, for the three solution conditions corresponding to unitary free energies of -10.4, -10.1, and -10.6 kcal/mol. The unitary entropies for the interaction are -39, -51, and -43 cal/(deg X mol), respectively, for the three solution conditions. Under these conditions, the troponin-tropomyosin interaction is enthalpy driven, and a large unfavorable entropy must be overcome in the formation of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)