Orientational, conformational stability and surface potential of immunoglobulins in monomolecular layers

Orientation, resistance to surface denaturation and surface potential of bovine and horse immunoglobulin G (IgG) and also of pig IgG-antibodies against DNP and DNS groups on the surface of NaCl solutions of various concentrations have been studied by monomolecular layer method. High conformational stability of IgG molecules of all the species was confirmed. At the surface of NaCl solutions with concentrations 0.15--0.5 M immunoglobulins of all species from monolayers consisting of practically non-unfolded molecules with such an orientation, that all the fragments of IgG molecule are located horizontally. Beyond the limits of this native structure stability zone the rate of surface denaturation is directly proportional to NaCl concentration in the solution. The values of surface potentials of all immunoglobulins under study are close to each other and change little with the surface denaturation.     

土壤食物网:结构、能流及稳定性

在国外4个土壤生态学项目的基础上,结合其他学者的研究成果,从连通网、能流网及功能网3个层次分别阐述了土壤食物网的结构、能流与稳定性。在连通网中,主要描述土壤食物网的功能群、营养位及格局;在能流网中,主要描述了面向过程食物网模型及其应用,主要涉及到土壤中的碳流与氮流;在功能网中,以作用强度为中心,描述了土壤食物网的稳定性问题。     

Crystal structure, conformation, and potential energy calculations of the chemotactic peptide N-formyl-L-Met-L-Leu-L-Phe-OMe

The tripeptide N-formyl-L-Met-L-Leu-L-Phe-OMe (FMLP-OMe) crystallizes in the orthorhombic system, space group P 2(1)2(1)2(1), with the following unit-cell parameters: a = 21.727, b = 21.836, c = 5.133 A, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I greater than 2 omega (I). The peptide backbone is folded at the Leu residue (phi L = -67.7, psi L = -49.1 degrees) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side-chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP-OMe and the anionic form of N-formyl-L-Met-L-Leu-L-Phe-OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have been carried out in order to compare the energies of the various backbone conformers.     

Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ₂, χ₃, χ₂′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ₂～ ? 60°, χ₃～90, χ₂′～ ? 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.     

Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe C

Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.     

The secondary structure of staphylococcal enterotoxins A,B and C

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215–218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little α-helix as analyzed with CD models. A secondary structure of enterotoxin B computed from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in α-helices, 71 in β-pleated sheets, 88 in β-turns and 55 in aperiodic conformation.     

3D antibody immobilization on a planar matrix surface

The amount of active capture antibodies immobilized per unit square is crucial to developing effective antibody chips, biosensors, immunoassays and other molecular recognition technologies. In this study, we present a novel yet simple method for oriented antibody immobilization at high density based on the formation of an orderly, organized aggregation of immunoglobulin G (IgG) and staphylococcal protein A (SPA). Following the chelation of His-tag with Ni(2+), antibodies were immobilized on a solid surface in a three-dimensional (3D) manner and exposed the analyte receptor sites well, which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in detection sensitivity. Pull-down assays confirmed that IgG antibody can only bind to Ni(2+) matrix indirectly via a SPA linkage, where the His-tag is responsible for binding Ni(2+) and homologous domains are responsible for binding IgG Fc. The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.     

Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most of the B chain

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.     

Variations of coxsackievirus B3 capsid primary structure, ligands, and stability are selected for in a coxsackievirus and adenovirus receptor-limited environment

While group B coxsackieviruses (CVB) use the coxsackievirus and adenovirus receptor (CAR) as the receptor through which they infect susceptible cells, some CVB strains are known for their acquired capacity to bind other molecules. The CVB3/RD strain that emerged from a CVB3/Nancy population sequentially passaged in the CAR-poor RD cell line binds decay-accelerating factor (DAF) (CD55) and CAR. A new strain, CVB3/RDVa, has been isolated from RD cells chronically infected with CVB3/RD and binds multiple molecules in addition to DAF and CAR. The capsid proteins of CVB3/RD differ from those of CVB3/28, a cloned strain that binds only CAR, by only four amino acids, including a glutamate/glutamine dimorphism in the DAF-binding region of the capsid. The capsid proteins of CVB3/RD and CVB3/RDVa differ by seven amino acids. The ability of CVB3/RDVa to bind ligands in addition to CAR and DAF may be attributed to lysine residues near the icosahedral 5-fold axes of symmetry. Considered with differences in the stability of the CVB3 strains, these traits suggest that in vitro selection in a CAR-limited environment selects for virus populations that can associate with molecules on the cell surface and survive until CAR becomes available to support infection.     

MM3 potential energy surfaces of trisaccharide models of lambda-, mu-, and nu-carrageenans

The adiabatic potential energy surfaces (PES) of six trisaccharides, sulfated derivatives of alpha-D-Gal p-(1-->3)-beta-D-Gal p-(1-->4)-alpha-D-Gal p and beta-D-Gal p-(1-->4)-alpha-D-Gal p-(1-->3)-beta-D-Gal p representing models of lambda-, mu-, and nu-carrageenans were obtained using the MM3 force-field at epsilon = 3. Each PES was described by a single contour map for which the energy is plotted against the two psi glycosidic angles, given the small variations of the phi glycosidic torsional angle in the low-energy regions of disaccharide maps. Most surfaces appear as expected from the maps of the disaccharidic repeating units of carrageenans, with less important factors altering the additive effect of both linkages. Only small interactions between the first and third monosaccharidic moieties of the trisaccharides are observed. The flexibility of the alpha-linkages appears nearly identical to that in their disaccharide counterparts, with only one exception, where it appears reduced by the presence of the third monosaccharide. On the other hand, the flexibility of the beta-linkage appears to be equal or sometimes even higher than that observed for the corresponding disaccharide.     

Band structure of surface electric potential in growing roots

For growing roots of azuki bean (Phaseolus chrysanthos), an electric potential is measured minutely along the surface of the root, together with the surface pH. It was found that the root begins to display a band-type pattern of potential with a spatial period of about 2 cm in a mature region as soon as it grows to about 10 cm in root length, while the surface potential shows only one convex peak around a position 5-20 mm behind a root tip and a succeeding concave peak around 20-35 mm, providing the length of root is shorter than about 10 cm. Since the surface potential takes a relatively positive value on average at the side of the root base compared with that in an elongation zone near the tip, electric current is supposed to flow into the elongation zone, accompanied by some local current loops in the mature region. The present band-type pattern observed first in multi-cell systems seems to be a kind of dissipative structure appearing far from equilibrium, and hence its relationship to growth is discussed.     

Thermal stability of hepatitis B surface antigen S proteins.

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.     

Molecular cloning, gene localization, and structure of human cyclin B3

Here we describe the molecular cloning of human cyclin B3, its localization, and its structure. It is localized in the subcentromeric region of the X chromosome, still not completely sequenced by the Human Genome Project. This cyclin B3 is unusually large for a mitotic cyclin. Its mRNAs were found in all tissues and were particularly abundant in testis. At least three splice variants were found in the ORF and three variants in the 5'UTR.     

Solution structure of human SUMO-3 C47S and its binding surface for Ubc9

Small ubiquitin-related modifier SUMO-3 is a member of a growing family of ubiquitin-like proteins (Ubls). So far, four isoforms of SUMO have been identified in humans. It is generally known that SUMO modification regulates protein localization and activity. Previous structure and function studies have been mainly focused on SUMO-1. The sequence of SUMO-3 is 46% identical with that of SUMO-1; nevertheless, functional heterogeneity has been found between the two homologues. Here we report the solution structure of SUMO-3 C47S (residues 14-92) featuring the beta-beta-alpha-beta-beta-alpha-beta ubiquitin fold. Structural comparison shows that SUMO-3 C47S resembles ubiquitin more than SUMO-1. On the helix-sheet interface, a strong hydrophobic interaction contributes to formation of the globular and compact fold. A Gly-Gly motif at the C-terminal tail, extending away from the core structure, is accessible to enzymes and substrates. In vivo, SUMO modification proceeds via a multistep pathway, and Ubc9 plays an indispensable role as the SUMO conjugating enzyme (E2) in this process. To develop a better understanding of SUMO-3 conjugation, the Ubc9 binding surface on SUMO-3 C47S has been detected by chemical shift perturbation using NMR spectroscopy. The binding site mainly resides on the hydrophilic side of the beta-sheet. Negatively charged and hydrophobic residues of this region are highly or moderately conserved among SUMO family members. Notably, the negatively charged surface of SUMO-3 C47S is highly complementary in its electrostatic potentials and hydrophobicity to the positively charged surface of Ubc9. This work indicates dissimilarities between SUMO-3 and SUMO-1 in tertiary structure and provides insight into the specific interactions of SUMO-3 with its modifying enzyme.     

Structural characterization of the (MeSH)4 potential energy surface

A random walk on the PES for (MeSH)₄ clusters produced 50 structural isomers held together by hydrogen-bonding networks according to calculations performed at the B3LYP/6–311++G** and MP2/6–311++G** levels. The geometric motifs observed are somewhat similar to those encountered for the methanol tetramer, but the interactions responsible for cluster stabilization are quite different in origin. Cluster stabilization is not related to the number of hydrogen bonds. Two distinct, well-defined types of hydrogen bonds scattered over a wide range of distances are predicted.

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.     

MOLS--a program to explore the potential energy surface of a peptide and locate its low energy conformations

We describe a computer program that uses mutually orthogonal Latin squares (MOLS) to perform an efficient and exhaustive conformational search of the multi-dimensional potential energy hypersurface of an oligopeptide, and locate all its low energy conformations. The software package has been developed with a user-friendly graphical interface using the Fast Light Tool Kit (FLTK)--a cross platform C++ toolkit.