Conformational properties of native sperm whale apomyoglobin in solution.

Apomyoglobin from sperm whale is often used for studies of ligand binding, protein folding, and protein stability. In an effort to describe its conformational properties in solution, homonuclear and heteronuclear (13C and 15N) NMR methods were applied to the protein in its native state. Assignments were confirmed for nuclear Overhauser effects (NOEs) involving side chain and backbone protons in the folded regions of the structure. These NOEs were used to derive distance restraints. The shifts induced by the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS) were inspected in the regions remote from its binding site and served as an indicator of conformational flexibility. 3JalphaH-NH values were obtained to assess dihedral angle averaging and to provide additional restraints. A family of structures was calculated with X-PLOR and an ab initio simulated annealing protocol using holomyoglobin as a template. Where the structure appeared well defined by chemical shift, line width, ANS perturbation, and density of NOEs, the low resolution model of apomyoglobin provides a valid approximation for the structure. The new model offers an improved representation of the folded regions of the protein, which encompass the A, B, E, helices as well as parts of the G and H helices. Regions that are less well defined at this stage of calculations include the CD corner and the end of the H-helix. The EF-F-FG segment remains uncharacterized.     

The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.     

Effect of succinylation of antibodies on their conformation and interaction with the antigen

Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 ± 6% modification was prepared and its physicochemical and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation, which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified. Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to the antigen. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1642–1647.     

On the role of some conserved and nonconserved amino acid residues in the transitional state and intermediate of apomyoglobin folding

The contributions of some amino acid residues in the A, B, G, and H helices to the formation of the folding nucleus and folding intermediate of apomyoglobin were estimated. The effects of point substitutions of Ala for hydrophobic amino acid residues on the structural stability of the native (N) protein and its folding intermediate (I), as well as on the folding/unfolding rates for four mutant apomyoglobin forms, were studied. The equilibrium and kinetic studies of the folding/unfolding rates of these mutant proteins in a wide range of urea concentrations demonstrated that their native state was considerably destabilized as compared with the wild-type protein, whereas the stability of the intermediate state changed moderately. It was shown that the amino acid residues in the A, G, and H helices contributed insignificantly to the stabilization of the apomyoglobin folding nucleus in the rate-limiting I ? N transition, taking place after the formation of the intermediate, whereas the residue of the B helix was of great importance in the formation of the folding nucleus in this transition.     

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: Effect of the hydrophobic chain length of the surfactants on the structural changes

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion ofa-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, thea-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.     

Hydrodynamic modelling of protein conformation in solution: ELLIPS and HYDRO

The last three decades has seen some important advances in our ability to represent the conformation of proteins in solution on the basis of hydrodynamic measurements. Advances in theoretical modeling capabilities have been matched by commensurate advances in the precision of hydrodynamic measurements. We consider the advances in whole-body (simple ellipsoid-based) modeling—still useful for providing an overall idea of molecular shape, particularly for those systems where only a limited amount of data is available—and outline the ELLIPS suite of algorithms which facilitates the use of this approach. We then focus on bead modeling strategies, particularly the surface or shell–bead approaches and the HYDRO suite of algorithms. We demonstrate how these are providing great insights into complex issues such as the conformation of immunoglobulins and other multi-domain complexes.     

New methods for estimating the numbers of synonymous and nonsynonymous substitutions

New methods for estimating the numbers of synonymous and nonsynonymous substitutions per site were developed. The methods are unweighted pathway methods based on Kimura's two-parameter model. Computer simulations were conducted to evaluate the accuracies of the new methods, Nei and Gojobori's (NG) method, Miyata and Yasunaga's (MY) method, Li, Wu, and Luo's (LWL) method, and Pamilo, Bianchi, and Li's (PBL) method. The following results were obtained: (1) The NG, MY, and LWL methods give overestimates of the number of synonymous substitutions and underestimates of the number of nonsynonymous substitutions. The major cause for the biased estimation is that these three methods underestimate the number of synonymous sites and overestimate the number of nonsynonymous sites. (2) The PBL method gives better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. (3) The new methods also give better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. In addition, estimates of the numbers of synonymous and nonsynonymous sites obtained by the new methods are reasonably accurate. (4) In some cases, the new methods and the PBL method give biased estimates of substitution numbers. However, from the number of nucleotide substitutions at the third position of codons, we can examine whether estimates obtained by the new methods are good or not, whereas we cannot make an examination of estimates obtained by the PBL method. (5) When there are strong transition/transversion and nucleotide-frequency biases like mitochondrial genes, all of the above methods give biased estimates of substitution numbers. In such cases, Kondo et al.'s method is recommended to be used for estimating the number of synonymous substitutions, although their method cannot estimate the number of nonsynonymous substitutions and is time-consuming. These results, particularly result (1), call for reexaminations of some genes. This is because evolutionary pictures of genes have often been discussed on the basis of results obtained by the NG, MY, and LWL methods, which are favorable for the neutral theory of molecular evolution.     

Molecular recognition of a model globular protein apomyoglobin by synthetic receptor cyclodextrin: effect of fluorescence modification of the protein and cavity size of the receptor in the interaction

Labelling of proteins with some extrinsic probe is unavoidable in molecular biology research. Particularly, spectroscopic studies in the optical region require fluorescence modification of native proteins by attaching polycyclic aromatic fluoroprobe with the proteins under investigation. Our present study aims to address the consequence of the attachment of a fluoroprobe at the protein surface in the molecular recognition of the protein by selectively small model receptor. A spectroscopic study involving apomyoglobin (Apo‐Mb) and cyclodextrin (CyD) of various cavity sizes as model globular protein and synthetic receptors, respectively, using steady‐state and picosecond‐resolved techniques, is detailed here. A study involving Förster resonance energy transfer, between intrinsic amino acid tryptophan (donor) and N, N‐dimethyl naphthalene moiety of the extrinsic dansyl probes at the surface of Apo‐Mb, precisely monitor changes in donor acceptor distance as a consequence of interaction of the protein with CyD having different cavity sizes (β and γ variety). Molecular modelling studies on the interaction of tryptophan and dansyl probe with β‐CyD is reported here and found to be consistent with the experimental observations. In order to investigate structural aspects of the interacting protein, we have used circular dichroism spectroscopy. Temperature‐dependent circular dichroism studies explore the change in the secondary structure of Apo‐Mb in association with CyD, before and after fluorescence modification of the protein. Overall, the study well exemplifies approaches to protein recognition by CyD as a synthetic receptor and offers a cautionary note on the use of hydrophobic fluorescent labels for proteins in biochemical studies involving recognition of molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity

Summary Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.     

Fibrillogenesis of apomyoglobin facilitated by aggregation sequence of yeast Sup35 in various regions

To examine the effect of aggregation sequence QGGYQQQYNP from yeast Sup35 on fibril formation of sperm whale apomyoglobin (apoMb), we constructed several mutants via substitution. Urea-induced unfolding of apoMb confirms that the substitution of the aggregation sequence does not significantly affect the stability of the mutants compared to wild type (WT) at pH 4.2. Under this condition, however, despite the difference in rate most apoMb mutants form fibrils more readily than WT with distinct morphology. These results suggest that the aggregation sequence facilitates fibril assembly of apoMb at acidic pH in vitro and this facilitation depends on the regions replaced.     

Amino acid substitutions in transmembrane domains 9 and 10 of GerVB that affect the germination properties of Bacillus megaterium spores

The molecular basis for differences in germinant recognition of Bacillus megaterium QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants. Kinetic analyses of mutants with different amino acid substitutions at these positions indicate that Ser319 and Leu345 are not involved directly in the binding of germinants, but probably reside in regions of the receptor where structural perturbations can affect the conformation of, or access to, germinant binding sites. Position 345 is also shown to be of importance in GerUB, where the F345A mutation severely impairs receptor function. Functionality is restored in the GerUB Ala345 background by substituting putative outer-loop residues adjacent to TM10 for the corresponding residues in GerVB, indicating that a degree of structural coordination between these regions is important to receptor function.     

Genetic trends of conformation traits and genetic correlations to osteochondrosis in boars

The objective of our study was to investigate the heritabilities and genetic correlations between traits from a linear exterior assessment system and osteochondrosis (OC) measured by computed tomography (CT), and in addition, to study the genetic trend in a population where the conformation traits have been included in the breeding goal. The data material consisted of phenotypes from a total of 4571 Norsvin Landrace test boars. At the end of the test period, all boars were subjected to a detailed exterior assessment system. Within 10 days of the assessment, the boars were CT scanned for measuring OC. The total score of osteochondrosis (OCT), used in this study, is the sum of phenotypes from the assessment on the medial and lateral condyles at the distal end of both the humerus and the femur of the right and the left leg of the boar based on images from CT. The exterior assessment traits included in the study were; ‘front leg knee’ (FKNE), ‘front leg pasterns’ (FPAS), ‘front leg stance’ (FSTA), ‘front leg twisted pasterns’ (FFLK), ‘hind leg stance’, ‘hind leg pasterns’ (HPAS), ‘hind leg standing under’ (HSTU), ‘hind leg small inner toe’, ‘dipped back’, ‘arched back’ (ARCH) and ‘waddling hindquarters’ (WADL). The estimation of (co)variance components and breeding values were performed using bivariate animal genetic models. Breeding values for HSTU, HPAS, FPAS, WADL and OCT traits were additional outputs from the same bivariate analyses. The lowest heritability was found for FFLK (h²_FFLK=0.05), whereas FPAS was estimated to have the highest heritability (h²_FPAS=0.36), and OCT demonstrating a heritability of 0.29. Significant genetic correlations were found between several traits; the strongest correlation was between FSTA and FFLK (0.94), which was followed by the correlation between FPAS and FKNE (0.69). The traits ARCH and FSTA had significant genetic correlations to OCT, whereas all other genetic correlations between OCT and the conformation traits were low and not significantly different from 0. Our study shows positive genetic trends for the conformation traits included in the breeding goal. In general, low genetic correlations between conformation traits and OC were observed in our study.     

Chain conformation and rheological behavior of an extracellular heteropolysaccharide Erwinia gum in aqueous solution

The chain conformation of a heteropolysaccharide Erwinia gum (EG) consisting of Glc, Gal, Fuc, and GlcA in aqueous solution was investigated by using viscometry and static and dynamic light scattering. The Huggins constants k′ ranging from 0.31 to 0.35, and the larger second virial coefficient A₂ of the order of 10⁻⁴ and even 10⁻³ mol g⁻² cm³ for EG fractions having different molecular weights in 0.03 M NaCl aqueous solution at 25 °C, suggested that 0.03 M NaCl aqueous solution is a good solvent for EG polysaccharide. Smidsrød’s ‘B-value’ characterizing chain stiffness was estimated to be 0.028-0.045 for EG fractions indicating that the backbone of EG polysaccharide is semi-stiff having similar stiffness to the semi-stiff Alginate and CMC. The hydrodynamic factor ρ (1.69-1.89), Flory-Fox factor Φ, and the product of ρΦ/N_A (0.16-0.22) also confirmed the semi-stiffness of EG polysaccharide chains. Compared with general flexible polymers, the first remarkable shear-thinning and then Newtonian flowing behaviors in steady shear tests for EG polysaccharides were ascribed to the alignment of extended semi-stiff chains on shearing. The dynamic oscillatory shear experiments indicated that addition of certain amount of NaCl effectively prohibited its gelation in pure water even at high concentration and low temperature for long time, suggesting that 0.03 M NaCl aqueous solution of EG has good stability and ability of antigelation, and thus is a promising additive in food field.     

Polar group conformation of 1,2-di-O-alkylglycerophosphocholines in the absence and presence of ions

The conformation of the glycerophosphocholine (GPC) group of various 1,2-di-O-alkyl and 1,2-diacylglycerophosphocholines forming small micelles or single-bilayer vesicles in H₂O has been studed by NMR in the absence and presence of lanthanide ions. In the absence of lanthanides the motionally averaged polar group conformation of 1,2-di-O-alkylglycerophosphocholine (dialkyl-GPC) is similar to that of the diacyl compound. The replacement of the ester linkages in diacyl phosphatidylcholine by ether bonds has therefore no significant effect on the conformation and segmental motion of the glycerophosphocholine group. This conformation is found to be independent of the state of aggregation, i.e., the main features are the same below and above the critical micellar concentration (CMC). The determining factor must therefore be the intramolecular energetics. Within the experimental accuracy the conformation of dialkyl-GPC in the presence of lanthanide ions is also the same as that of the corresponding diacyl compound. Furthermore, in the presence of lanthanides the polar group conformation of dialkyl-GPC is the same within experimental accuracy in small micelles and single bilayer vesicles. The conformational change induced by lanthanides leads to a reorientation of the OPN dipole. In the presence of lanthanides the OPN dipole increases its angle of tilt with respect to the bilayer plane from about 0° (coplanar orientation) to an average inclinication of about 45°. This gives rise to a more extended disposition of the polar group with respect to the bilayer normal.     

Solution state conformation and degradation of cyclopeptides containing an NGR motif.

In contrast to the RGD-peptides, head to tail cyclization of LNGRV and LNGRv caused only a marginal change in their integrin receptor affinity as shown by the limited effect and selectivity on the adhesion of endothelial cells to ECM components. Structure determination of the two cyclopeptides by NMR and MD, semiempirical and ab initio methods revealed that both are very flexible and take on multiple stable conformers in solution. This structural diversity, along with the presence of the Asn-Gly peptide bond, enhances succinimide ring formation leading to the hydrolysis of Asn. It has been demonstrated that c(LNGRV) suffers deamidation with time both in solution and during storage. As the isoaspartyl-peptide may co-elute with the asparginyl-peptide in the course of HPLC analysis, MS measurement is necessary to check the purity of peptides containing the NGR sequence. Our stability investigations raise the question whether the NGR motif or its hydrolysis product is effective in in vivo experiments.     

Chain stiffness of heteropolysaccharide from Aeromonas gum in dilute solution by dynamic light scattering

Dynamic light scattering measurements have been made on 15 fractions of aeromonas (A) gum, an extracellular heteropolysaccharide produced by the strain Aeromonas nichidenii, with dimethylsulfoxide containing 0.2M lithium chloride as the solvent at 25 degrees C. Data for the translational diffusion coefficient D covering a molecular weight range from 4.5 x 10(5) to 2.1 x 10(6) and ratios of the z-average radius of gyration (z) (1/2) to the hydrodynamic radius R(H) (calculated with previous (z) data) suggest that the polymer behaves like a semiflexible chain in this solvent similar to the stiffness of cellulose derivatives. Thus the D data are analyzed on the basis of the Yamakawa-Fujii theory for the translational friction coefficient of a wormlike cylinder by coarse-graining the heteropolysaccharide molecule. Excluded-volume effects are taken into account in the quasi-two-parameter scheme, as was done previously for (z) and [eta] (the intrinsic viscosity) of A gum in the same solvent. The molecular weight dependence of R(H) is found to be explained by the perturbed wormlike chain with a persistence length of 10 nm, a linear mass density of 1350 nm(-1), an excluded-volume strength parameter of 1.3 nm, and a chain diameter of 2.8 nm. These parameters are in substantial agreement with those estimated previously from (z) and [eta] data, demonstrating that the solution properties (D, (z), and [eta]) of the heteropolysaccharide are almost quantitatively described by the current theories for wormlike chains in the molecular weight range studied.     

Role of protein conformation and aggregation in pumping water in and out of a cell

Dialysis cassettes containing BSA solutions were used to simulate passive in vivo conditions to assess the effect of protein conformation and aggregation on cell water content. The cassettes were suspended in dextran solutions to provide a range of fixed osmotic stress values simulating blood plasma. The system was placed on a shaker for 24 h to attain equilibrium. Four manipulation methods; pH, cosolute salt concentration, e.g. NaCl, temperature annealing and urea concentration denaturant were varied to produce well-known manipulations of BSA conformation. It was observed that the cell water content varied from +14% to about -13% with changes in protein conformation and aggregation. The findings demonstrate that a change in protein conformation and aggregation, pumps water in and out of a cell to maintain equilibrium % water content matching the protein conformational hydration parameter. This concept supplements existing theories on cell volume regulation.