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PubMed1985

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices.

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.

Circular Dichroism↗
PubMed2006

Structure-function analysis of cytochromes P450 2B.

In the last 4 years, breakthroughs were made in the field of P450 2B (CYP2B) structure-function through determination of one ligand-free and two inhibitor-bound X-ray crystal structures of CYP2B4, which revealed many of the structural features required for binding ligands of different size and shape. Large conformational changes of several plastic regions of CYP2B4 can dramatically reshape the active site of the enzyme to fit the size and shape of the bound ligand without perturbing the overall P450 fold. Solution biophysical studies using isothermal titration calorimetry (ITC) have revealed the large difference in the thermodynamic parameters of CYP2B4 in binding inhibitors of different ring chemistry and side chains. Other studies have revealed that the effects of site-specific mutations on steady-state kinetic parameters and mechanism-based inactivation are often substrate dependent. These findings agree with the structural data that the enzymes adopt different conformations to bind various ligands. Thus, the substrate specificity of an individual enzyme is determined not only by active site residues but also non-active site residues that modulate conformational changes that are important for substrate access and rearrangement of the active site to accommodate the bound substrate.

Animals↗
PubMed2005

Pressure denaturation of apomyoglobin: a molecular dynamics simulation study.

The effect of pressure on the structure and mobility of Sperm Wale Apomyoglobin was studied by Molecular Dynamics computer simulation at 1 bar and 3 kbar (1 atm=1.01325 bar=101.325 kPa). The results are in good agreement with the available experimental data, allowing further analysis of other features of the effect of pressure on the protein solution. From the analysis of Secondary Structures (SS) along the trajectories it is observed that alpha-helixes are favoured under pressure at the expense of bends, turns and 3-helixes. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. The studies of tertiary structure show important conformational changes. The evolution of the Solvent Accessed Surface (SAS) with pressure shows a notorious increase due almost completely to a biased raise in the hydrophobic area exposed, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

Animals↗
PubMed2005

Phosphorylation of the CARMA1 linker controls NF-kappaB activation.

PKC isoforms and CARMA1 play crucial roles in immunoreceptor-dependent NF-kappaB activation. We tested whether PKC-dependent phosphorylation of CARMA1 directly regulates this signaling cascade. B cell antigen receptor (BCR) engagement led to the progressive recruitment of CARMA1 into lipid rafts and to the association of CARMA1 with, and phosphorylation by, PKCbeta. Furthermore, PKCbeta interacted with the serine-rich CARMA1 linker, and both PKCbeta and PKCtheta phosphorylated identical serine residues (S564, S649, and S657) within this linker. Mutation of two of these sites ablated the functional activity of CARMA1. In contrast, deletion of the linker resulted in constitutive, receptor- and PKC-independent NF-kappaB activation. Together, our data support a model whereby CARMA1 phosphorylation controls NF-kappaB activation by triggering a shift from an inactive to an active CARMA1 conformer. This PKC-dependent switch regulates accessibility of the CARD and CC domains and controls assembly and full activation of the membrane-associated IkappaB kinase (IKK) signalosome.

Animals↗
PubMed2004

A new lectin family with structure similarity to actinoporins revealed by the crystal structure of Xerocomus chrysenteron lectin XCL.

A newly defined family of fungal lectins displays no significant sequence similarity to any protein in the databases. These proteins, made of about 140 amino acid residues, have sequence identities ranging from 38% to 65% and share binding specificity to N-acetyl galactosamine. One member of this family, the lectin XCL from Xerocomus chrysenteron, induces drastic changes in the actin cytoskeleton after sugar binding at the cell surface and internalization, and has potent insecticidal activity. The crystal structure of XCL to 1.4 A resolution reveals the architecture of this new lectin family. The fold of the protein is not related to any of the several lectin folds documented so far. Unexpectedly, the structure similarity is significant with actinoporins, a family of pore-forming toxins. The specific structural features and sequence signatures in each protein family suggest a potential sugar binding site in XCL and a possible evolutionary relationship between these proteins. Finally, the tetrameric assembly of XCL reveals a complex network of protomer-protomer interfaces and generates a large, hydrated cavity of 1000 A3, which may become accessible to larger solutes after a small conformational change of the protein.

Amino Acid Sequence↗
PubMed2004

Energy transduction in transmembrane ion pumps.

Recent crystallographic structures of three different ion pumps provide a first view of the mechanisms by which these molecular machines transfer ions across cell membranes against an electrochemical gradient. Each of the structures reinforces the concept that several buried counter ions have central roles in substrate recruitment, substrate binding and energy transduction during ion pumping. The spatial organization of the counter ions suggests that, initially, one or more counter ions lowers the Born energy cost of binding a substrate ion in the low-dielectric interior of the membrane. Subsequently, a ligand-induced conformational change seems to close a charged access gate to prevent backflow from a subsequent, low-affinity state of the pump. A final role of the buried counter ions might be to couple the input of external energy to a small charge separation between the substrate ion and the buried counter ions, thereby decreasing the binding affinity for the substrate ion in preparation for its release on the high-energy side of the membrane.

Animals↗
PubMed1990

Hydrogen bond stereochemistry in protein structure and function.

Fifty high resolution protein structures from the Brookhaven Protein Data Bank have been analyzed for recurring motifs in hydrogen bond stereochemistry. Although an exhaustive analysis of hydrogen bond statistics has been presented by Baker & Hubbard, a detailed stereochemical analysis of classical donor (N-H, O-H, or S-H) and acceptor (N:, O:, or S:) structure within proteins is lacking. Here, we describe the preferential hydrogen bond stereochemistry for the side-chains of glutamate and aspartate (carboxylate), glutamine and asparagine (carboxamide), arginine (guanidinium), histidine (imidazole/imidazolium), tryptophan (indole), tyrosine (phenolic hydroxyl), lysine (ammonium), serine and threonine (alkyl hydroxyl), cysteine (thiol), methionine (thioether) and cystine (disulfide). Preferential hydrogen bond stereochemistry is governed by (1) the electronic configuration of acceptor atoms, (2) the steric accessibility of donor atoms and (3) the conformation of amino acid side-chains. Applications of hydrogen bond stereochemistry are useful in the interpretation of protein structure, function and stability. Additionally, this stereochemistry is a prerequisite to the interpretation of protein-other molecule recognition and biological catalysis.

Amino Acids↗
PubMed1993

Glycosylation of the major outer capsid protein of bluetongue viruses.

The major outer capsid protein, VP5, of five US bluetongue viruses, was found to be glycosylated. Enzymatic removal of the carbohydrate moiety did not affect the electrophoretic mobility of VP5 in SDS-PAGE, indicating the presence of only short and possibly unbranched oligosaccharide chains. The surface accessibilities and immunogenic specificities of two conformational-dependent antigenic epitopes on VP5 of BTV were not affected by deglycosylation. Selective binding of those lectins which have well-defined sugar specificities suggests that the potential short N-linked carbohydrate chain present on BTV VP5 might be composed of sialic acid alpha(2-6)-N-acetylgalactosamine-beta(1-3)-galactose-beta(1-4)-N- acetylglucosamine-beta(1-2)-mannose-N-acetylglucosamine-Asn. In accordance with the standard nomenclature, BTV-VP5 should now be termed GP5 due to the presence of the carbohydrate moiety.

Antibodies, Monoclonal↗
PubMed1990

Conformations of folded proteins in restricted spaces.

A new method is presented to examine the complete range of folded topologies accessible in the compact state of globular proteins. The procedure is to generate all conformations, with volume exclusion, upon a lattice in a space restricted to the individual protein's known compact conformational space. Using one lattice point per residue, we find 10(2)-10(4) possible compact conformations for the five small globular proteins studied. Subsequently, these conformations are evaluated in terms of residue-specific, pairwise contact energies that favor nonbonded, hydrophobic interactions. Native structures for the five proteins are always found within the best 2% of all conformers generated. This novel method is simple and general and can be used to determine a small group of most favorable overall arrangements for the folding of specific amino acid sequences within a restricted space.

Animals↗
PubMed2002

The three-dimensional structure of the gallium complex of azoverdin, a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants.

In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins.

Gallium↗
PubMed1987

Consequences of detailed balance in a model for sensory adaptation based on ligand-induced receptor modification.

A model for exact sensory adaptation has been published by Segel and co-workers in several papers [e.g., Knox, B. E., Devreotes, P. N., Goldbeter, A. & Segel, L. A. (1986) Proc. Natl. Acad. Sci. USA 83, 2345-2349]. The model comprises a pair of states whose relative probabilities are determined by the binding of a ligand. A second pair of states related by the same ligand binding is accessible as a consequence of either a conformational change or a "covalent modification." By taking proper account of detailed balance, we show that the notion of covalent modification in this context includes three cases, two of which involve dissipation of metabolic energy. The condition for exact adaptation is dependent on metabolite concentrations in all cases of covalent modification. The performance of the model is critically examined on thermodynamic and kinetic grounds.

Adaptation, Physiological↗
PubMed1996

Temperature and pH dependence of fluorescein binding within the monoclonal antibody 9-40 active site as monitored by hydrostatic pressure.

In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten in this active site was solvent accessible. Imperfect packing, which leads to increased conformational dynamics, was determined as a possible cause of the low affinity for mAb 9-40.

Antibodies, Monoclonal↗
PubMed1997

Dissecting the cooperative reassociation of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Role of Trp-196 in the catalytic subunit.

The catalytic (C) subunit of cAMP-dependent protein kinase requires two distinct surfaces to form a stable complex with its physiological inhibitors, the regulatory (R) subunits and the heat-stable protein kinase inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. This surface is comprised of the essential phosphorylation site, Thr-197, His-87, Trp-196, and several surrounding basic residues. To probe the role of Trp-196 in the recognition of R, Trp-196 was replaced with Arg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readily with cAMP-free R, they failed to form an inhibited holoenzyme complex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importance of the conformation of R, and, in particular, the accessibility of site A. One of these mutants, rR(R333K), having a defect in cAMP binding site B formed a stable complex with rC(W196R) in the absence of cAMP. However, unlike wild-type holoenzyme, this complex was active.

Catalysis↗
PubMed2000

The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate.

The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium- and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M., Bendahan, A. and Kanner, B. I. (1998) Neuron 21, 623-632). Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2-trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C. Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent on the conformation of the transporter.

ATP-Binding Cassette Transporters↗
PubMed2000

Nitric oxide modulates the catalytic activity of myeloperoxidase.

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin six-coordinate complexes, MPO-Fe(III).NO and MPO-Fe(II).NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

Catalysis↗
PubMed2000

BiP and PDI cooperate in the oxidative folding of antibodies in vitro.

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.

Carrier Proteins↗
PubMed2001

Phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to increase the rate of phosphorylation of Ser(40).

The effect of phosphorylation on the shape of tyrosine hydroxylase (TH) was studied directly using gel filtration and indirectly using electrospray ionization mass spectrometry. Phosphorylation of Ser(19) and Ser(40) produced a TH molecule with a more open conformation than the non-phosphorylated form. The conformational effect of Ser(19) phosphorylation is less pronounced than that of the Ser(40) phosphorylation. The effect of Ser(19) and Ser(40) phosphorylation appears to be additive. Binding of dopamine produced a more compact form when compared with the non-dopamine-bound TH. The interdependence of Ser(19) and Ser(40) phosphorylation was probed using electrospray ionization mass spectrometry. The rate constants for the phosphorylation of Ser(19) and Ser(40) were determined by electrospray ionization mass spectrometry using a consecutive reaction model. The rate constant for the phosphorylation of Ser(40) is approximately 2- to 3-fold higher if Ser(19) is already phosphorylated. These results suggest that phosphorylation of Ser(19) alters the conformation of tyrosine hydroxylase to allow increased accessibility of Ser(40) to kinases.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗
PubMed2002

Determination of the site-specific oligosaccharide distribution of the O-glycans attached to the porcine submaxillary mucin tandem repeat. Further evidence for the modulation of O-glycans side chain structures by peptide sequence.

Little is known of the degree that polypeptide sequence and the local environment modulate the structures of O-linked glycans. Toward this understanding, the site-specific mono- (GalNAc-O-), di- (beta-Gal-1,3-alpha-GalNAc-O-), and trisaccharide (alpha-Fuc-1,2-beta-Gal-1,3-alpha-GalNAc-O-) distributions have been determined for 29 of the 31 O-glycosylated Ser/Thr residues in the tandem repeat domains of blood group A-negative porcine submaxillary gland mucin. The glycosylation patterns obtained from three individual animals are in agreement with earlier incomplete determinations on a pooled mucin (Gerken, T. A., Owens, C. L., and Pasumarthy, M. (1997) J. Biol. Chem. 272, 9709-9719; Gerken, T. A., Owens, C. L., and Pasumarthy, M. (1998) J. Biol. Chem. 273, 26580-26588), confirming that the addition of the peptide-linked GalNAc and its substitution by beta-1,3-Gal are sensitive to local peptide sequence in a highly reproducible manner in vivo. The present data further support earlier suggestions of an inverse correlation of the density of hydroxyamino acid residues (and by inference the density of peptide GalNAc) with the extent of substitution of the peptide-linked GalNAc by beta-1,3-Gal. This effect is highly correlated for Ser-linked glycans but not for Thr-linked glycans. A similar correlation is observed with respect to the in vivo peptide GalNAc glycosylation pattern. In contrast, the addition of alpha-1,2-Fuc to beta-Gal shows no apparent correlation with hydroxyamino acid density, although a marked elevation in the fucosylation of Ser-linked glycans compared with Thr-linked glycans is observed. The above effects may represent both steric and conformational factors acting to alter the relative accessibility and activity of the glycosyltransferases toward substrate. These results demonstrate that the porcine submaxillary gland core 1 beta 3-galactosyltransferase and alpha2-fucosyltransferase exhibit unique peptide/glycopeptide sensitivities that may provide mechanisms for the modulation of O-linked side chain structures.

Amino Acid Sequence↗