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PubMed1995

Interaction of polycyclic aromatic hydrocarbons and flavones with cytochromes P450 in the endoplasmic reticulum: effect on CO binding kinetics.

The flash photolysis technique was used to examine the kinetics of CO binding to cytochromes P450 in rat liver microsomes. The effect of polycyclic aromatic hydrocarbons (PAHs) and flavones was used to distinguish the kinetic behavior of the PAH-metabolizing P450 1A1 from that of the remaining multiple microsomal P450s. Applying this approach to microsomes from 3-methylcholanthrene-treated rats showed that although all tested PAHs accelerated CO binding to P450 1A1, the extent varied markedly for different PAHs. The tricyclic PAHs phenanthrene and anthracene enhanced CO binding by 37- and 49-fold, respectively, while several tetracyclic and pentacyclic PAHs increased the rate by 3-16-fold. The results indicate that PAHs exert a dual effect on the rate of CO binding to P450 1A1: a general enhancement via widening of the CO access channel and a reduction that is dependent on PAH size. Although 5,6-benzoflavone increased the rate of CO binding to P450 1A1 by 3.5-fold, it additionally decelerated binding to a constitutive P450 by 15-fold. This flavone thus exerts markedly different effects on two P450s within the same microsomal sample. In contrast, the sole effect of 7,8-benzoflavone was acceleration of CO binding to P450 1A1 by 18-fold. The divergent effects of these isomeric flavones, which only differ in positioning of an aromatic ring, illustrate the sensitivity of CO binding to substrate structure. The varying effects of these PAHs and flavones on CO binding kinetics show that they differentially modulate P450 conformation and access of ligands to the P450 heme and demonstrate that binding of carcinogens to a specific target P450 can be evaluated in its native microsomal milieu.

Animals↗
PubMed2006

HARMONY: a server for the assessment of protein structures.

Protein structure validation is an important step in computational modeling and structure determination. Stereochemical assessment of protein structures examine internal parameters such as bond lengths and Ramachandran (varphi,psi) angles. Gross structure prediction methods such as inverse folding procedure and structure determination especially at low resolution can sometimes give rise to models that are incorrect due to assignment of misfolds or mistracing of electron density maps. Such errors are not reflected as strain in internal parameters. HARMONY is a procedure that examines the compatibility between the sequence and the structure of a protein by assigning scores to individual residues and their amino acid exchange patterns after considering their local environments. Local environments are described by the backbone conformation, solvent accessibility and hydrogen bonding patterns. We are now providing HARMONY through a web server such that users can submit their protein structure files and, if required, the alignment of homologous sequences. Scores are mapped on the structure for subsequent examination that is useful to also recognize regions of possible local errors in protein structures. HARMONY server is located at http://caps.ncbs.res.in/harmony/

Algorithms↗
PubMed1997

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli.

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.

ATP-Binding Cassette Transporters↗
PubMed2005

Molecular dynamics simulation of paracetamol molecules ordering around glycogen.

By the use of classical atomistic molecular dynamics simulations, we demonstrate that paracetamol molecules exist in a highly ordered phase in the presence of a glycogen substrate at 317 K whereas the paracetamol fluid exists in an isotropic phase in the absence of the glycogen substrate at the same temperature. This result further validates the studies made on polysaccharide regarding its abilities to promote nucleation of paracetamol via liquid preordering. As little is known regarding liquid ordering induced by a polymeric substrate, we seek to explore the ordering mechanism from an energy perspective. This is accomplished using conformation mappings. Our analysis shows that the conformation space accessible to the paracetamol molecule at 317 K in the vicinity of glycogen is smaller than the one in the absence of glycogen. An investigation on the orientation of the dipole moments of the glycogen monomers and paracetamol molecules were carried out as well. From the investigations, we show that dipolar interactions play an important role in the ordering process. These studies bear significance to the understanding of the ordering process as well as the promotion and effective control of the nucleation rate.

Acetaminophen↗
PubMed1990

Cyclic hexapeptides related to somatostatin. Conformational analysis employing 1H-NMR and molecular dynamics.

We report the conformational analysis of a series of cyclic hexapeptides related to the hormone somatostatin utilizing 1H NMR spectroscopy and NOE restrained molecular dynamics. The conformational preferences and results from biological analysis of these analogs (previous paper) allow for refinement of the current understanding of the structure-activity relationship of somatostatin. For most of the molecules examined, a beta II' turn about the D-tryptophan-lysine residues, postulated to be required for biological activity, was present. From the NOE restrained molecular dynamics, it can be seen that the turn structure is important for the maintenance of the proper orientation of the side chains of the adjacent phenylalanine, tryptophan and lysine. The biologically active analogs have the side chains of lysine and D-tryptophan extended away from the 18-membered ring in close proximity to each other for a significant portion of the dynamic simulations. Although other conformations are accessible and monitored during the simulations, we believe this is important for biological recognition. The absence of the beta II' turn at the D-tryptophan-lysine disrupts this side chain array producing inactive molecules. The role of the bridging region, the Phe-Pro dipeptide, is to stabilize the beta II' turn and help maintain the proper orientation of the biologically important side chains.

Amino Acid Sequence↗
PubMed1993

1H-NMR spectroscopy of beta B2-crystallin from bovine eye lens. Conformation of the N- and C-terminal extensions.

1H-NMR spectroscopic studies of a 46-kDa homodimer, beta B2-crystallin, from bovine eye lens are presented. beta B2-crystallin has terminal extensions extending from globular N- and C-terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two-dimensional NMR methods on beta B2-crystallin, its synthesised terminal extensions and a proteolysed sample of beta B2-crystallin with a portion of its C-terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One-dimensional experiments at various pH values provided H-2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of beta B2-crystallin are in excellent agreement with those for the X-ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776-780]. No change in the spectrum of beta B2-crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.

Animals↗
PubMed1977

Haemoglobin--a frustrated oxidase? Implications for red cell metabolism.

The haem proteins can be considered, in one aspect of their function, as machines for activating oxygen. In the case of oxygen-carriers such as haemoglobin, the globin has evolved so that its conformation limits access to the haem group, with resultant reversible release of oxygen. However, distortion of the globin may allow either the discharge of oxygen as the activated product superoxide or, more threateningly, allow direct function of the haemoglobin as an oxidative enzyme. Support for this is shown by the reaction with acetylphenylhydrazine where haemoglobin functions as both an oxidase and oxygenase. An implication of oxidase activity is the potential to initiate free radical formation particularly with unsaturated lipids. Observations of the acetylphenylhydrazine reaction emphasize the role of glutathione as a free radical scavenger.

Ascorbic Acid↗
PubMed1994

An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3.

The monoclonal antibody PR1A3 has been used successfully for in vivo imaging of colorectal cancers, and several properties associated with this antibody, including minimal reactions of the antibody with circulating antigen in patients' sera, differentiate it from anti-carcinoembryonic antigen (CEA) antibodies used in similar studies. However, the antigen bound by PR1A3 was identified as CEA by analysis of somatic cell hybrids and by antigen expression from yeast artificial chromosomes, cosmids, and cDNA clones. The molecular weight, presence of a glycosyl-phosphatidylinositol anchor, elevation of surface expression by gamma-interferon, and N-terminal amino acid sequence all confirmed the antigen identification as CEA. A series of biliary glycoprotein-CEA hybrid proteins was produced which demonstrated that the epitope bound by the antibody was at the site of membrane attachment and involved parts of the glycosyl-phosphatidylinositol anchor and the B3 domain of CEA to form a conformational epitope. Access to this epitope, although possible when the antigen was on the cell surface, appeared to be blocked when CEA was released from the cell. The nature and location of the epitope on CEA are proposed to be responsible for the unique properties of the antibody.

Animals↗
PubMed1984

Probing the conformation of 26S rRNA in yeast 60S ribosomal subunits with kethoxal.

The conformation and accessibility of 26S rRNA in yeast 60S ribosomal subunits were probed with kethoxal. Oligonucleotides originating from reactive sites were isolated by diagonal electrophoresis and sequenced. From over 70 oligonucleotide sequences, 26 kethoxal-reactive sites could be placed in the 26S rRNA sequence. These are in close agreement with a proposed secondary structure model for the RNA that is based on comparative sequence analysis. At least seven kethoxal-reactive sites in yeast 26S rRNA are in positions that are exactly homologous to reactive positions in E. coli 23S rRNA; each of these sites has previously been implicated in some aspect of ribosomal function.

Aldehydes↗
PubMed1976

Thiols related to mitochondrial ATPase and transports: unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate.

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.

Adenosine Diphosphate↗
PubMed2025

AlphaFold2, SPINE-X, and Seder on Four Hard CASP Targets.

We analyzed four cases from the CASP15 experiment with low prediction accuracy and compared AlphaFold2, SPINE-X, and Seder on these cases. We find that overall, AlphaFold2 performs better than SPINE-X in predicting secondary structure (SS) and solvent accessible surface area (ASA). For some cases, SPINE-X better predicts sheet and coil regions. We also find that AlphaFold2 is better than Seder in selecting the best matching tertiary structure model for one case and is worse in another case. For two cases Alphafold2 and Seder selected the same models. From the cases presented here, it appears that AlphaFold2 predicts more compact structures than the native one. We find that while, as widely reported, AlphaFold2 significantly improved protein tertiary structure prediction, there are cases, such as the four presented here, for which the tertiary structure prediction could still be significantly enhanced. The source code, license, and documentation for SPINE-X and Seder are available from Research and Information Systems, LLC at http://mamiris.com .

Software↗
PubMed2000

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation.

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.

Animals↗
PubMed2005

Age-associated changes in glycosylation of CD43 and CD45 on mouse CD4 T cells.

We have recently shown that treatment of T cells from aged mice with an endopeptidase specific for O-linked glycoproteins can restore synapse formation and early activation markers to CD4 cells from aged mice. New data show that the sialidase from Clostridium perfringens, but not from Vibrio cholerae, can increase activation of CD4 cells from both old and young mice as measured by calcium signals, expression of CD25 and CD69, and secretion of IL-2. Lectin binding assays showed alterations with age in the levels, accessibility or conformation of multiple glycoproteins on the surface of CD4 cells. While some alterations were due to the accumulation of memory cells with age, others were age sensitive and found exclusively in the naive subset or both naive and memory subsets. Furthermore, analysis of the sialic acid links alpha(2,3)Gal/GalNAc and alpha(2,6)Gal/GalNAc in immunoprecipitated CD43 and CD45 molecules confirm that age alters the glycosylation of specific proteins that regulate TCR interaction with antigen presenting cells. These data support the idea that changes in T cell surface glycosylation could play an important role in immune senescence.

Age Factors↗
PubMed2000

Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays.

A cDNA for a new ubiquitin-specific protease (UBP), AtUBP5, was identified from Arabidopsis thaliana flower mRNA using an oligonucleotide made against the conserved UBP cysteine (Cys) box. The 924-amino-acid AtUBP5 contains the regions characteristic of all UBPs and has 35% identity and 53% similarity overall to a mammalian UBP (Unp), resulting from additional significant similarity outside these regions. AtUBP5 has 48% identity and 58% similarity overall to two uncharacterized Arabidopsis genomic sequences but is distinct outside the UBP conserved regions from two other previously published Arabidopsis UBPs, AtUBP3 and -4. Using in vivo Escherichia coli assays, which allow co-expression of GSTAtUBPs and substrates, we show that all three UBPs were active. AtUBP5 was active without 311 amino acids N-terminal to the active site cysteine, or without 233 nonconserved amino acids between the Cys and His boxes, or without both, indicating the core region was sufficient. In in vivo and in vitro assays, GSTAtUBP3, -4, and -5 exhibited preference for specific Ub-Ub linkages, suggesting accessibility and/or conformation is important and demonstrating that these enzymes cleave post-translationally. A chimeric UBP consisting of the AtUBP5 Cys box with AtUBP3 amino acids was active and exhibited AtUBP3 specificity, indicating that the modular nature of UBPs and specificity for cleavage sites is not determined by the Cys box.

Amino Acid Sequence↗
PubMed1998

Characterisation of low free-energy excited states of folded proteins.

It is demonstrated that the identity of residues accessing excited conformational states that are of low free energy relative to the ground state in proteins can be obtained from amide proton NMR chemical shift temperature dependences displaying significant curvature. For the N-terminal domain of phosphoglycerate kinase, hen egg-white lysozyme and BPTI, conformational heterogeneity arises from a number of independent sources, including: structural instability resulting from deletion of part of the protein; a minor conformer generated through disulphide bond isomerisation; an alternative hydrogen bond network associated with buried water molecules; alternative hydrogen bonds involving backbone amides and surface-exposed side-chain hydrogen bond acceptors; and the disruption of loops, ends of secondary structural elements and chain termini. In many of these cases, the conformational heterogeneity at these sites has previously been identified by X-ray and/or NMR studies, but conformational heterogeneity of buried water molecules has hitherto received little attention. These multiple independent low free-energy excited states each involve a small number of residues and are shown to be within 2.5 kcal mol-1 of the ground state. Their relationship with the partially unfolded forms previously characterised using amide proton exchange studies is discussed.

Amides↗
PubMed1985

Lectin binding in human skeletal muscle: a comparison of 15 different lectins.

Fifteen lectin-horseradish peroxidase conjugates have been used in a comprehensive histochemical study of human skeletal muscle. The staining patterns of many lectins were found to be coincident with the known distributions of types I, III, IV and V collagen, fibronectin and laminin. One lectin, Bandeiraea simplicifolia (BSA I), selectively stained capillaries in a blood group-specific manner, the significance of which is unknown. The results show that although lectins are useful cytochemical probes for identifying tissue glycoconjugates, lectin binding is not solely determined by monosaccharide specificity as lectins which interact with the same sugars may have completely different staining patterns. Factors such as accessibility, glycan conformation and oligosaccharide sequence also affect lectin binding in tissues. For these reasons, we conclude that a comprehensive histochemical investigation of tissue glycoconjugates should employ a large number of lectins, preferably with overlapping sugar specificities.

Histocytochemistry↗
PubMed1980

ESR spin label studies of the nucleosome core particle and histone core.

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.

Animals↗
PubMed1988

Probing the assembly of the 3' major domain of 16 S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19.

We have studied the effect of assembly of ribosomal proteins S7, S9 and S19 on the accessibility and conformation of nucleotides in 16 S ribosomal RNA. Complexes formed between 16 S rRNA and S7, S7 + S9, S7 + S19 or S7 + S9 + S19 were subjected to a combination of chemical and enzymatic probes, whose sites of attack in 16 S rRNA were identified by primer extension. The results of this study show that: (1) Protein S7 affects the reactivity of an extensive region in the lower half of the 3' major domain. Inclusion of proteins S9 or S19 with S7 has generally little additional effect on S7-specific protection of the RNA. Clusters of nucleotides that are protected by protein S7 are localized in the 935-945 region, the 950/1230 stem, the 1250/1285 internal loop, and the 1350/1370 stem. (2) Addition of protein S9 in the presence of S7 causes several additional effects principally in two structurally distal regions. We observe strong S9-dependent protection of positions 1278 to 1283, and of several positions in the 1125/1145 internal loop. These findings suggest that interaction of protein S9 with 16 S rRNA results in a structure in which the 1125/1145 and 1280 regions are proximal to each other. (3) Most of the strong S19-dependent effects are clustered in the 950-1050 and 1210-1230 regions, which are joined by base-pairing in the 16 S rRNA secondary structure. The highly conserved 960-975 stemp-loop, which has been implicated in tRNA binding, appears to be destabilized in the presence of S19. (4) Protein S7 causes enhanced reactivity at several sites that become protected upon addition of S9 or S19. This suggests that S7-induced conformational changes in 16 S rRNA play a role in the co-operativity of assembly of the 3' major domain.

Aldehydes↗