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PubMed2003

Tryptophanyl substitutions in apomyoglobin affect conformation and dynamic properties of AGH subdomain.

The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W --> F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule.

Acrylamide↗
PubMed2003

Protein side-chain rearrangement in regions of point mutations.

A major problem in predicting amino acid side-chain rearrangements following point mutations is the potentially large search space. We analyzed a nonredundant data set of 393 Protein Data Bank protein pairs, each consisting of structures differing in one amino acid, to determine the number of residues changing conformation in the region of mutation. In 91-95% of cases, two or fewer residues underwent side-chain conformational change. If mutation sites with backbone displacements were excluded, the number increased to 97%. The majority of rearrangements (over 60%) were due to the inherent flexibility of side-chains, as derived from analysis of a control set of protein subunits whose crystal structures were determined more than once. Different amino acids demonstrated different degrees of flexibility near mutation sites. Large polar or charged residues, and serine, are more flexible, while the aromatic amino acids, and cysteine, are less so. This pattern is common to the inherent side-chain flexibility, as well as the increased flexibility at ligand binding sites and mutation sites. The probability for conformational change was correlated with B-factor, frequency of the side-chain conformation in proteins and solvent accessibility. The last trend was stronger for aromatic and hydrophilic residues than for hydrophobic ones. We conclude that the search space for predicting side-chain conformations in the region of mutation can be effectively restricted. However, the overall ability to predict a particular side-chain conformation, or to check predictions according to individual existing structures, is limited. These findings may be useful in deriving empirical rules for modeling side-chain conformations.

Binding Sites↗
PubMed1997

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.

1-Deoxynojirimycin↗
PubMed1995

Modeling protein-substrate interactions in the heme domain of cytochrome P450(BM-3).

The crystal structure of heme domain of the fatty acid monooxygenase, cytochrome P450(BM-3), consisting of residues 1-455 has been independently solved to R = 0.18 at 2.0 A. The crystal form used, space group P2(1) with two molecules per asymmetric unit, is isomorphous with that form with residues 1-471 first described by Boddupalli et al. [Boddupalli, Hasemann, Ravinchandran, Lu, Goldsmith, Deisenhofer & Peterson (1992). Proc. Natl Acad. Sci. USA, 89, 5567-5571] and used by Ravichandran, Boddupalli, Hasemann, Peterson & Deisenhofer [(1993). Science, 261, 731-736] to determine the crystal structure. The substrate-access channel consists of a large, hydrophobic cleft that appears to be the most likely route taken by fatty acid substrates. Attempts to soak crystals in mother liquor containing a variety of fatty acid substrates yielded featureless difference Fouriers even though fatty acid substrates are known to bind with dissociation constants in the micro M range. Modeling substrate-enzyme interactions reveals few contacts between the enzyme and substrate. More detailed modeling was carried out by subjecting both molecules in the asymmetric unit to extensive energy minimization. These studies reveal that the heme-domain active-site cleft can undergo a large conformational change that closes the access channel thereby providing enhanced protein-substrate interactions. These conformational changes are prevented from occurring by intermolecular contacts in the crystal lattice which lock the protein in the 'open' conformation.

Journal Article↗
PubMed1992

Modeling the antigen combining site of an anti-dinitrophenyl antibody, ANO2.

A model structure has been constructed for a monoclonal anti-dinitrophenyl antibody. The antibody, ANO2, has been sequenced and cloned (Anglister, J., Frey, T., & McConnell, H.M., 1984, Biochemistry 23, 1138-1142). Its amino acid sequence shows striking homology with the anti-lysozyme Fab fragments HyHel5 (83%) and HyHel10 (73%). Based on this homology, a model for the ANO2 variable heavy and variable light chain framework was constructed using a hybrid of the HyHel5 light chain and the HyHel10 heavy chain backbone, omitting the hypervariable loops. These coordinates were used as scaffolds for the model building of ANO2. The CONGEN conformational sampling algorithm (Bruccoleri, R.E. & Karplus, M., 1987, Biopolymers 26, 127-196) was used to model the six hypervariable loops that contain the antigen-combining site. All the possible conformations of the loop backbones were constructed and the best loop structures were selected using a combination of the CHARMM potential energy function and evaluation of the solvent-accessible surface area of the conformers. The order in which the loops were searched was carried out based on the relative locations of the loops with reference to the framework of the beta-barrel, namely, L2-H1-L3-H2-H3-L1. The model structures thus obtained were compared to the high resolution X-ray structure (Brünger, A.T., Leahy, D.J., Hynes, T.R., & Fox, R.O., 1991, J. Mol. Biol. 221, 239-256).

Algorithms↗
PubMed1993

Insights into interfacial activation from an open structure of Candida rugosa lipase.

The structure of the Candida rugosa lipase determined at 2.06-A resolution reveals a conformation with a solvent-accessible active site. Comparison with the crystal structure of the homologous lipase from Geotrichum candidum, in which the active site is covered by surface loops and is inaccessible from the solvent, shows that the largest structural differences occur in the vicinity of the active site. Three loops in this region differ significantly in conformation, and the interfacial activation of these lipases is likely to be associated with conformational rearrangements of these loops. The "open" structure provides a new image of the substrate binding region and active site access, which is different from that inferred from the structure of the "closed" form of the G. candidum lipase.

Amino Acid Sequence↗
PubMed1992

Hydrophobic interaction of lysozyme and alpha-lactalbumin from equine milk whey.

From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca(2+)-dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca2+ and in 2 mM Ca2+ are also performed with equine alpha-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp----Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca2+ conformers is clearly influenced by the mutation. The Ca(2+)-dependent exclusion of a hydrophobic domain is used in a new and simplified method for preparing lysozyme and alpha-lactalbumins simultaneously from equine milk whey.

Anilino Naphthalenesulfonates↗
PubMed2000

From agonist to antagonist: Fab fragments of an agonist-like monoclonal anti-beta(2)-adrenoceptor antibody behave as antagonists.

We previously demonstrated that the monoclonal antibody Mab6H8 raised against the second extracellular loop of the beta(2)-adrenoceptor (beta(2)-AR) had an agonist-like activity, mediated by the activation of L-type Ca(2+) channels by protein kinase A through the adenylyl cyclase pathway. We suggested that this Mab acts by stabilizing an active dimeric conformation of the beta(2)-AR. To substantiate this hypothesis, we prepared monomeric Fab fragments of Mab6H8. Comparison of the physicochemical parameters of antigen interaction with both the Mab and its Fab fragments were determined by surface plasmon resonance, showing a 5- to 10-fold lower affinity of the fragments compared with the bivalent antibody. We determined the biological activity of antibody and Fab fragments in two systems: spontaneous beating neonatal rat cardiomyocytes to study the chronotropic effects and isolated guinea pig cardiomyocytes to study L-type Ca(2+) channel activation. Fab fragments as such had no "agonist-like" effects in both systems but inhibited receptor activation with the beta(2)-specific agonist clenbuterol. Addition of a cross-linking rabbit anti-mouse IgG restored the agonist-like effect of the Fab fragments. These results suggest that Fab fragments induce a conformational change in the receptor, inhibiting the accessibility of the pharmacophore pocket to clenbuterol. Dimerization of this receptor conformation induces an agonist-like effect. Antireceptor antibodies can thus act both as agonist in the dimeric state and as antagonist in the monomeric state.

Adrenergic beta-Antagonists↗
PubMed1993

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes stabilized by the formation of G.A base pairs.

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes which are stabilized by the formation of G.A pairs. Three base pairings are known to occur between adenine and guanine: AH+ (anti).G(syn), A(anti).G(anti) and A(syn).G(anti). Protonation of the adenine residues is not involved in the stabilization of this structure, since it is observed at any pH value from 8.3 to 4.5; at pH < or = 4.0 antiparallel stranded d(GA.GA) DNA is destabilized. The results reported in this paper strongly suggest that antiparallel stranded d(GA.GA) homoduplexes are stabilized by the formation of alternating A(anti).G(anti) and G(anti).A(syn) pairs. In this structure, all guanine residues are in the anti conformation with their N7 position freely accessible to DMS methylation. On the other hand, adenines in one strand adopt the anti conformation, with their N7 position also free for reaction, while those of the opposite strand are in the syn conformation, with their N7 position hydrogen bonded to the guanine N1 group of the opposite strand. A regular right-handed helix can be generated using alternating G(anti).A(syn) and A(anti).G(anti) pairs.

Adenine↗
PubMed2002

Intrinsic conformational energetics associated with the glycosyl torsion in DNA: a quantum mechanical study.

The glycosyl torsion (chi) in nucleic acids has long been recognized to be a major determinant of their conformational properties. chi torsional energetics were systematically mapped in deoxyribonucleosides using high-level quantum mechanical methods, for north and south sugar puckers and with gamma in the g(+) and trans conformations. In all cases, the syn conformation is found higher in energy than the anti. When gamma is changed from g(+) to trans, the anti orientation of the base is strongly destabilized, and the energy difference and barrier between anti and syn are significantly decreased. The barrier between anti and syn in deoxyribonucleosides is found to be less than 10 kcal/mol and tends to be lower with purines than with pyrimidines. With gamma = g(+)/chi = anti, a south sugar yields a significantly broader energy well than a north sugar with no energy barrier between chi values typical of A or B DNA. Contrary to the prevailing view, the syn orientation is not more stable with south puckers than with north puckers. The syn conformation is significantly more energetically accessible with guanine than with adenine in 5-nucleotides but not in nucleosides. Analysis of nucleic acid crystal structures shows that gamma = trans/chi = anti is a minor but not negligible conformation. Overall, chi appears to be a very malleable structural parameter with the experimental chi distributions reflecting, to a large extent, the associated intrinsic torsional energetics.

Adenine↗
PubMed2000

Features of F(1)-ATPase catalytic and noncatalytic sites revealed by fluorescence lifetimes and acrylamide quenching of specifically inserted tryptophan residues.

Catalytic and noncatalytic nucleotide sites of the F(1) sector of ATP synthase were characterized by tryptophan fluorescence techniques. Seven Trp residues inserted in varied microenvironments in the catalytic sites, and one in the noncatalytic sites, were studied in mutant F(1) enzymes which were otherwise devoid of Trp. Parameters measured were fluorescence lifetimes and dynamic and static quenching by acrylamide in the absence or presence of nucleotide. The results indicated that the solution structures of the mutant enzymes were consistent with reported crystal structures. In enzyme with three empty noncatalytic sites, all sites were relatively inaccessible to acrylamide, indicating a closed conformation. In contrast, when the three catalytic sites were empty, they were relatively and equally accessible to acrylamide, indicating an open conformation. This was the case in the presence or absence of Mg(2+). Residue beta-Trp-331 has been extensively used previously to determine nucleotide binding parameters in F(1). Results here showed that in betaY331W mutant F(1), each of the three beta-Trp-331 residues has an unusually long fluorescence lifetime, confirming that each contributes equally to the overall fluorescence signal.

Acrylamide↗
PubMed1992

Immunoprecipitation of alpha 2a-adrenergic receptor-GTP-binding protein complexes using GTP-binding protein selective antisera. Changes in receptor/GTP-binding protein interaction following agonist binding.

The nature of the interaction of cloned alpha 2a-adrenergic receptors from LLC-PK1-O clone cells with G proteins was investigated using an immunoprecipitation approach. Following solubilization of the alpha 2a receptors, antiserum 8730, which is directed against the C-terminal region of Gi alpha, immunoprecipitated alpha 2a receptor-Gi alpha complexes. The immunoprecipitation was specific since it could be blocked by the peptide to which antiserum 8730 was generated. Antisera 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3) immunoprecipitated solubilized alpha 2a receptor-Gi alpha complexes, indicating that all three Gi alpha subtypes couple with the alpha 2a receptor. Antiserum 9072, which is directed against the C-terminal region of G(o)alpha, immunoprecipitated solubilized alpha 2a receptor-G alpha complexes indicating that these receptors are also coupled to G(o)alpha. Antiserum 8132, which is directed against G beta 36, immunoprecipitated solubilized alpha 2a receptors while the G beta 35 antiserum 8129, did not, indicating that alpha 2a receptors selectively associate with G beta 36. The binding of the partial agonist p-aminoclonidine to the solubilized alpha 2a receptor alters the association of the receptor with G proteins. Following p-aminoclonidine binding to the solubilized alpha 2a receptor, the ability of the C-terminal directed G alpha antisera 8730 and 9072 to coimmunoprecipitate the alpha 2a receptor-G alpha complex was greatly reduced. The effect of p-aminoclonidine was concentration dependent, mimicked by the full agonist UK 14304 and blocked by the alpha 2 receptor antagonist yohimbine. In contrast, antisera directed against internal regions of Gi alpha and G(o)alpha, immunoprecipitated the agonist-bound and agonist-free alpha 2a receptor equally well. These findings indicate that following the binding of agonists to the alpha 2a receptor, Gi alpha and G(o)alpha remain physically associated with the receptor but either the conformation of G alpha linked to the receptor or the conformation of the receptor itself is modified such that the epitope for the C-terminal directed anti-Gi alpha and anti-G(o)alpha antisera are not accessible. These agonist-induced conformational changes in the alpha 2a receptor-G alpha complex may be important for the activation of the G protein and the stimulation of the alpha 2a receptor signal transduction pathway.

Adrenergic alpha-Agonists↗
PubMed2003

Determination of conformer-specific partition coefficients in octanol/water systems.

The first conformer-specific experimental partition coefficients are presented for octanol/water, the most widespread solvent system to predict lipophilicity of drugs. Rotamer populations in octanol and water were elucidated from 1H NMR vicinal coupling constants and were combined with classical partition coefficients to obtain the conformer-specific ones. Feasibility of the determination of conformer-specific partition coefficients is exemplified on amphetamine and clenbuterol, two flexible drug molecules. Partition capacities of the amphetamine rotamers have been proven to be essentially equal. The conformers of clenbuterol, however, have been found to be greatly different in partition properties, which could be interpreted in terms of intramolecular interactions between the vicinal polar sites and the solvent-accessibility of the groups. The conformers could be put into order of their membrane-influx and -outflow propensities. Deviations between experimental and calculated log P values could also be interpreted in view of the species-specific partition coefficients.

Algorithms↗
PubMed1997

Transient channel-opening in bacteriorhodopsin: an EPR study.

Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.

Bacteriorhodopsins↗
PubMed1995

X-ray crystal structure, partitioning behavior, and molecular modeling study of piracetam-type nootropics: insights into the pharmacophore.

To detect possible molecular determinants of amnesia-reverting activity, the conformational properties of a number of rigid and flexible piracetam-type cognition enhancers have been assessed by X-ray diffraction, NMR spectroscopy, and ab initio and high-temperature-quenched molecular dynamics (QMD) calculations. The structures of the preferred conformers in solution derived from 1H-NMR spectral analysis were in good agreement with those found by QMD calculations. Interestingly, the calculation of the average molecular lipophilicity potential on the water-accessible surface of the selected conformers was helpful in interpreting the partitioning behavior observed by measuring octanol-water partition coefficients and capacity factors in reversed-phase high-performance liquid chromatography. While lipophilicity does not play a relevant role, the distance between polar groups, accounted for by the distance between carbonyl oxygens, emerges as a factor, among others, which should influence the amnesia-reversal activity of piracetam-type nootropics.

Chemical Phenomena↗
PubMedDate not supplied

Evidence for conformationally different states of interleukin-10: binding of a neutralizing antibody enhances accessibility of a hidden epitope.

We present the mapping of two anti-human interleukin-10 (hIL-10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL-10-derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L-amino acids. The epitope-derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody-hIL-10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X-ray structure of IL-10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL-10 can adopt a conformational state other than that observed in the crystal structure.

Amino Acid Sequence↗
PubMed2000

Structural study of the sodium channel inactivation gate peptide including an isoleucine-phenylalanine-methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles.

In order to gain insight into the gating mechanisms of Na+ channels, in particular their inactivation mechanisms, we studied the structures of the Na+ channel inactivation gate related peptide which includes the IFM (Ile-Phe-Met) motif (Ac-KKKFGGQDIFMTEEQKK-NH2; K1480-K1496 in rat brain type-IIA Na+ channels, MP-3A) and its F/Q(Gln) substituted one (MP-4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and 1H-NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three-dimensional structures of MP-3A and MP-4A were determined using simulated annealing molecular dynamics/ energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP-3A, residues 11488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of 11488 and the hydroxyl oxygen atom of T1491, whereas in MP-4A, F/Q substitution separated them owing to conformational changes. The solvent-accessible surfaces calculated for the structures of MP-3A and MP-4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.

Amino Acid Motifs↗