Thermal inactivation and conformational lock of bovine carbonic anhydrase

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.     

Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity

The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

Proteolytic dimers of porcine muscle lactate dehydrogenase: characterization, folding, and reconstitution of the truncated and nicked polypeptide chain

Lactate dehydrogenase from porcine skeletal muscle is a "dimer of dimers" that is stabilized in its tetrameric state by an N-terminal "arm" of approximately 20 amino acid residues. Due to the low dissociation constant of the tetramer, the dimer is inaccessible to direct analysis. Limited proteolysis during reconstitution (after dissociation at pH 2.3) yields stable "dimers". As suggested by affinity chromatography, these inactive dimers contain the dinucleotide fold of native LDH. In the presence of structure-making ions, approximately 40% activity is restored in the dimeric state [Girg, R., Jaenicke, R., & Rudolph, R. (1983) Biochem. Int. 7, 443-444]. The cleavage yields about equal amounts of three fragments, F 34, F 21, and F 14 (Mr 33.5K, 21.4K, and 13.5K, respectively). F 34 represents the intact chain lacking the N-terminal 10-11 amino acid residues; its C-terminus is heterogeneous, varying in the range between residues 326 +/- 5. F 21 contains residues 11/12 to 200 +/- 3; F 14 is a mixture of three subfragments: residues 11/12 to approximately 133, 38 to approximately 163, and 208 to approximately 327. After solubilization in 6 M guanidine hydrochloride, F 34 can be reconstituted to partially active dimers. Reactivation is determined by slow subunit refolding with subsequent diffusion-controlled dimerization, in accordance with the monomer-dimer transition in the reconstitution mechanism of the intact tetramer. Reconstitution of F 21 and F 14 is concentration dependent and leads to partially active "nicked dimers", indicating that separate domains are able to reassociate correctly to yield the native subunit arrangement.     

Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer

Lipoprotein lipase (LPL), a key enzyme in the metabolism of triglyceride-rich plasma lipoproteins, is a homodimer. Dissociation to monomers leads to loss of activity. Evidence that LPL dimers rapidly exchange subunits was demonstrated by fluorescence resonance energy transfer between lipase subunits labeled with Oregon Green and tetrametylrhodamine, respectively, and also by formation of heterodimers composed of radiolabeled and biotinylated lipase subunits captured on streptavidine-agarose. Compartmental modeling of the inactivation kinetics confirmed that rapid subunit exchange must occur. Studies of activity loss indicated the existence of a monomer that can form catalytically active dimers, but this intermediate state has not been possible to isolate and remains hypothetical. Differences in solution properties and conformation between the stable but catalytically inactive monomeric form of LPL and the active dimers were studied by static light scattering, intrinsic fluorescence, and probing with 4,4'-dianilino-1,1'-binaphtyl-5,5'-disulfonic acid and acrylamide. The catalytically inactive monomer appeared to have a more flexible and exposed structure than the dimers and to be more prone to aggregation. By limited proteolysis the conformational changes accompanying dissociation of the dimers to inactive monomers were localized mainly to the central part of the subunit, probably corresponding to the region for subunit interaction.     

Light-scattering investigation of the subunit structure and dissociation of Helix pomatia hemocyanin. Effects of salts and ureas

The effects of various salts of the Hofmeister and aliphatic acid salt series and hydrophobic reagents of the urea series on the subunit structure and the dissociation of Helix pomatia alpha-hemocyanin were investigated by employing light-scattering molecular weight methods. In moderate ranges of salt concentrations [0-1.0 M NaClO4, NaSCN, NaI, and guanidinium chloride (GdmCl) and 0-2.0 M NaBr], the dissociation reaction is essentially a two-step process characterized by the dissociation of whole hemocyanin molecules dissociating to half-molecules of decamers followed by the dissociation of the half-molecules to five dimeric fragments. The effectiveness of the salts and relative ineffectiveness of the ureas and GdmCl as dissociating agents in the first step of the dissociation reaction suggest that the stabilization of the contact areas between half-molecules in solution is largely a nonhydrophobic energy process involving polar and ionic interactions. Hydrophobic forces appear to be important, however, for stabilization of the half-molecules through side to side contacts of the five dimeric units that make up each half-molecule. The analysis of our dissociation data by use of equations derived in our previous studies [Herskovits, T. T., & Harrington, J.P. (1975) Biochemistry 14, 4964-4971] gave apparent estimates of amino acid groups of about 60-150 for each of the contact areas between the cylindrically shaped half-molecules and 30-60 for each of the dimers in the half-molecules themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of various acylating agents on the catalytic properties and structure of aspartate aminotransferase]

Changes of quaternary structure and conformation of molecule concomitant with inactivation were observed in the course of aspartate transaminase acylation by maleic, citraconic, dimethylmaleic and succinic anhydrides. It was established that acylation of 10-12 xi-amino groups of lysine did not induce the dissociation of transaminase into subunits. Further acylation of amino groups (2 groups if dimethylmaleic anhydrade was used as acylating agent) induced dissociation of transaminase dimer into subunits. These data were obtained by sedimentation analysis. The dissociation was accompanied with a sharp decrease of correlation time (from 18 nsec to 9 nsec) of the paramagnetic label covalently bound to the protein. The obtained results allow us to distinguish three types of xi-aminogroups of aspartate transaminase: exposed (about 12 residues), "contact" (2 residues) located in the vicinity to complementary surfaces of subunits and buried (about 6 residues). The stepwise inactivation occurred during the acylation as a result of conformational changes or appearance of sterical hindrances in the cataytic site of the enzyme. The thiol groups were not modified in transaminase molecule under experimental conditions used. Aspartate transaminase treated with citraconic or dimethylmaleic anhydride may be deacylated under mild conditions. After reacylation the quaternary structure was reconstituted and catalytic activity was almost fully restored.     

Subunit dissociation of Busycon canaliculatum hemocyanin

The hemocyanin of the channeled whelk, Busycon canaliculatum, is a multisubunit protein with a molecular weight close to 9 X 10(6). The increase in pH above neutrality and the addition of 0-5 M urea and 0-2 M GdnHCl is found to dissociate the whole molecules to half-molecules and smaller dimeric and monomeric fragments of one-tenth and one-twentieth mass of the parent hemocyanin. The molecular weight transitions investigated at constant protein concentration of 5 X 10(-2) g X l-1 show no clearly discernible plateau regions, where essentially only half-molecules and one-tenth molecules are present. The ultracentrifugation patterns in much of the dissociation region produced by urea at pH 6.9 suggests the presence of three distinct components consisting of whole molecules, half-molecules and largely one-tenth molecular weight fragments. At pH 8.2 and higher, where whole molecules are largely absent, the effects of urea on the dissociation of half-molecules to tenths and tenth-molecules to twentieth molecule was investigated by means of light scattering. Analysis of the urea data based on a decamer to dimer and dimer to monomer scheme of dissociation used in our earlier studies gave apparent estimates of about 90 amino acid groups at the contact areas of the dimers in the half-molecules and 110 groups at the monomer contacts forming the dimers. The latter relatively large estimate of groups suggests that the dissociation of the tenth molecules or dimers must occur by longitudinal splitting of the contact areas along both the folded domains and the connecting chain segments of the twentieth molecules. Circular dichroism, absorbance and viscosity data suggest that the secondary structure and conformation of the folded domains of the hemocyanin subunits are largely retained at both high pH and in 3-8 M urea solutions. The molecular weights at pH 9.0-10.6 and in 3-8 M urea are found to be (4.2-4.7) X 10(5), close to one-twentieth of the mass of the parent hemocyanin. Denaturation and unfolding of the subunit domains is observed between 3 and 6 M GdnHCl solutions, as evidenced by the abolition of the characteristic copper absorbance in the neighborhood of 346 nm and the relatively pronounced changes in circular dichroism at 222 nm and intrinsic viscosity. The further decrease in molecular weights to about (2.6-3.2) X 10(5), below one-twentieth of the mass of hemocyanin suggests the presence of hidden breaks or scissions in the polypeptide chains suffered during isolation, which become exposed as a result of complete unfolding in GdnHCl solutions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antipeptide antibodies to the porcine lactate dehydrogenase isoenzyme M4 active center fragment as a probe for the structural analysis of active centers of human lactate dehydrogenase isoforms

Antipeptide antibodies (AB) to the fragment of the active center of porcine lactate dehydrogenase M4 isoform were used for the analysis of antigenic properties and structural comparison of active centers of human lactate dehydrogenase isoforms. Selective precipitation of the M-subunit-containing isoforms using an immunoadsorbent based on antipeptide AB as well as selective inhibition of the enzymic activity of the M4 isoform by antipeptide AB testify to the specific binding of isoforms to antipeptide AB. The experimental results confirm the literary data on conformational changes in the structure of the active centers of corresponding human lactate dehydrogenase isoforms. The specific interaction of antipeptide AB with human lactate dehydrogenase isoforms suggests that the site of the amino acid sequence (residues 180-214) in both human and porcine M4 isoenzymes is immunochemically identical. The data obtained suggest that antipeptide AB are convenient probes for detecting differences (including minor ones) in the primary and spatial structure of enzymes.     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.     

Leishmania infantum 5’-Methylthioadenosine Phosphorylase presents relevant structural divergence to constitute a potential drug target

Background

So far, little is known about the molecular mechanisms of amyotrophic lateral sclerosis onset and progression caused by SOD1 mutations. One of the hypotheses is based on SOD1 misfolding resulting from mutations and subsequent deposition of its cytotoxic aggregates. This hypothesis is complicated by the fact that known SOD1 mutations of similar clinical effect could be distributed over the whole protein structure.

Results

In this work, a measure of hydrogen bond stability in conformational states was studied with elastic network analysis of 35 SOD1 mutants. Twenty-eight hydrogen bonds were detected in nine of 35 mutants with their stability being significantly different from that with the wild-type. These hydrogen bonds were formed by the amino acid residues known from the literature to be located in contact between SOD1 aggregates. Additionally, residues disposed between copper binding sites of both protein subunits were found from the models to form a stiff core, which can be involved in mechanical impulse transduction between these active centres.

Conclusions

The modelling highlights that both stability of the copper binding site and stability of the dimer can play an important role in ALS progression.

     

Classification of amino acids based on comparative analysis of contacts in DNA-protein complexes and specific DNA-protein interactions

We propose a classification of amino acid residues based on the events of contact formation between particular residues and DNA nucleotides, i.e., using the most integral properties that characterize interactions organizing DNA-protein complexes. We apply the Voronoi-Delaunay tessellation to draw statistics of contacts and of contact areas for a set of 1937 DNA-protein complexes. Similarity of amino acid residues is defined upon comparison of corresponding rows and matrices of contacts and areas of contacts. Nine measures of distance have been used to estimate the closeness of rows. Residues have been grouped by three hierarchical and two nonhierarchical clustering methods. In a total tree built using nine metrics with three hierarchical methods, we show that clustering centers (pairs of amino acids) in the main groups are always constant while other relationships between objects vary. Major classes of up to six amino acids correspond to certain local structures of the polypeptide chain. These data can be taken into account when designing DNA-protein ligands.     

Physical investigations of the hemocyanin of the chiton, Cryptochiton stelleri (Middendorff)

The hemocyanin of the giant Pacific chiton, Cryptochiton stelleri has a molecular weight of 4.2 +/- 0.3 X 10(6), determined by light-scattering, and a sedimentation coefficient of 60S. The fully dissociated subunits in nondenaturing solvents, at pH 10.6, 1 X 10(-2)M EDTA and in 8.0 M urea, pH 7.4 have molecular weights of 4.10 X 10(5) and 4.35 X 10(5), close to one-tenth of the molecular mass of the parent hemocyanin decamers. In the pH region from about 3.5 to 11 the molecular weight (Mw), determined at constant protein concentration of 0.10 g1(-1) exhibits a bell-shaped molecular weight profile centering about the physiological pH of the hemolymph of 7.2. The pH-Mw profile is best accounted for in terms of a three state, decamer-dimer-monomer dissociation scheme. Analysis of the Mg2+ and Ca2+ effects on the molecular weight transitions suggest stabilization of the hemocyanin decamers through one bound divalent ion per hemocyanin monomer or dimer. Urea, GdmCl, and the higher members of the chaotropic salt series are effective dissociating agents for Cryptochiton stelleri hemocyanin. The dissociation profile obtained with urea at pH 8.5, 0.01 M Mg2+, 0.01 M Ca2+ has been analyzed in terms of both the two- and three-species schemes of subunit-dissociation. Hydrophobic stabilization of the subunit contacts is suggested by the large number of apparent amino acid groups (Napp), of the order of 30 between dimers stabilizing the decamers, and 120 apparent amino acid groups between each monomer forming the constituent dimers.     

The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds

It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.     

Kinetics of deoxyhemoglobin subunit dissociation determined by haptoglobin binding: estimation of the equilibrium constant from forward and reverse rates.

Deoxyhemoglobin tetramers dissociate into dimers very slowly, with half-times on the order of several hours. It is demonstrated that absorbance changes in the Soret region which accompany this dissociation and persist upon binding of haptoglobin 1-1 to the dissociated dimers can be used for accurate kinetic determinations over the necessarily long periods required for study. This method of study for the slow reactions depends upon long-term spectral integrity of the reaction mixtures and upon accurate measurement. The variation in rate constants determined by this procedure has been correlated with variations in structural constraints at the dimer-dimer contact region. In the presence of 2,3-diphosphoglycerate the rate constant is decreased, consistent with the role of this effector in binding to both beta chains and stabilizing the constrained deoxy tetramer against dissociation into alphabeta dimers. With hemoglobin specifically modified (des-Arg-141alpha) to eliminate half the constraining salt links within the dimer-dimer contact region, the dissociation rate is increased by approximately three orders of magnitude. In hemoglobin S where the amino acid substitution is not directly in the intersubunit contact region of interest, the dissociation rate is found to be approximately the same as that for hemoglobin A. Combination of the dissociation rate constants determined by haptoglobin binding with stopped-flow determinations of the rate constant for reassociation of dissociated dimers provides an estimate of the equilibrium constant, 0K2, for the deoxyhemoglobin dimer-tetramer equilibrium. This estimate is independent of any assumptions regarding other energetic quantities, and yields a value of 2.54 +/- 0.7 X 10(10)M-1 (heme) in 0.1 M Tris-HCl, 0.1 M NaCl, and 1 mM EDTA, pH 7.4, 21.5 degrees C. Thus the intersubunit contact energy is -14.0 +/- 0.2 kcal/mol of heme. The stabilization energy between deoxy and oxy tetramers is found to be approximately 6.4 kcal/mol, under these conditions.     

Isolation and characteristics of insulin-binding proteins from soybeans

Two soybean insulin-binding proteins were isolated using affinity chromatography on insulin-Sepharose. Both proteins have molecular mass about 39 kDa and consist of two subunits linked by disulphide bonds. According to the amino acid composition and N-terminal sequences of the subunits, these proteins, characterized by the absence of free thiol groups and sugar residues, are variants of the previously described soybean basic 7S globulin. The blotted proteins as well as their subunits were shown to bind 125I-labelled bovine insulin. For one of the proteins and insulin, dissociation constant of 4.10(-9) M was measured. The existence of plant insulin-binding proteins suggests the insulin-like regulation in the plant metabolism.     

Rates of dissociation and recombination of the subunits of bovine thyrotropin

The rates of dissociation and recombination of the subunits of bovine thyrotropin have been measured under a variety of conditions using the fluorescence probe 1,8-anilinonaphthalenesulfonate. The method is based on the fact that the native hormone strongly enhances the fluorescence of 1,8-anilinonaphthalenesulfonate whereas the subunits have very little effect. The hormone can be easily dissociated into subunits, either in dilute acid (pH < 4) or in concentrated (8–10 m) urea solutions at pH 8.O. The rate of dissociation is first order with time and increases strongly with increasing temperature. The hormone is very stable in alkali, showing little tendency to dissociate below pH 12. After dissociation in acid, the subunits can be recombined between pH 7 and 9 at a rate which increases with increasing temperature and subunit concentration. The recombination is intermediate between first and second order suggesting a two-step mechanism: association of the subunits followed by a first-order refolding process in which the subunits acquire the tertiary structure characterisitc of the native hormone. Difference absorption measurements indicate that the dissociation is accompanied by the exposure of a substantial fraction of the 16 tyrosine residues to the more polar aqueous environment, suggesting major conformational changes in one or both subunits.     

Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation.

Calpain is a heterodimeric, intracellular Ca(2+)-dependent, "bio-modulator" that alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-terminus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for the expression of protease activity and/or for in vivo function. Recently, the crystal structure of m-calpain in the absence of Ca(2+) has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large subunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To examine the relationship between autolysis, dissociation, and activation, we made and analysed a series of N-terminal mutants of mCL that mimic the autolysed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta 9), dissociated into subunits even in the absence of Ca(2+). Moreover, both Delta 9 and Delta 19 mutants showed an equivalent reduced Ca(2+) requirement for protease activity. These results indicate that autolysis is necessary for the dissociation of the m-calpain subunits, and that the dissociation occurs after, but is not necessary for, activation.