Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.     

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.     

双水相电泳分离蛋白质的研究

近几年来,随着生物技术的迅速发展,制备型电泳技术的研究得到了重视。然而由于技术上的原因,大规模的制备型电泳技术的研究还未能取得突破。阻碍电泳放大的一个主要问题是由于电加热作用而导致的热对流对电泳分离的破坏。为解决这一问题,人们提出了许多方法。例如,在太空的微重力环境下进行电泳,应力稳定自由流动电泳,循环等电聚焦和区带电泳,色谱电泳和等电膜等电聚焦等。这些方法在电泳放大上都取得了一定的进展,但各有其局限性。最近,Clark提出利用双水相的液液界面阻止热对流的设想,为开发大规模的制备型电泳技术开辟了一条新途径、Raghava Rao等在两种双水相体系上施加电场后成倍地缩短了分相时间。Levine和Bier采用U型管电泳装置研究了双水相体系中血红蛋白的电泳迁移率,观测到界面有阻滞作用。Clark在柱型电泳装置中进行了一组双水相萃取肌红蛋白的简单实验。在10mA的恒电流下电泳40min之后,肌红蛋白的分配系数为7.5,而当电场反向后,分配系数变为0.04,界面阻力并不显著,两者结论并不一致。     

六种鼠苹果酸脱氢酶,血浆蛋白质和血红蛋白的变异分析

本文分析了家鼠属（Ｒａｔｔｕｓ）４种鼠和姬鼠属（Ａｐｏｄｅｍｕｓ）２种鼠的苹果酸脱氢酶、血浆中β１区和β２区蛋白质、血浆清蛋白、前清蛋白以及红细胞中血红蛋白的变异。结果表明,ＭＤＨ在进化上是比较保守的,其中ＭＤＨｍ和ＭＤＨｓ２带在各鼠间位于同一泳动线上,仅ＭＤＨｓ１带的泳动速度出现属间和种间的微小差异;血浆中β１区和β２区的蛋白质带、清蛋白带以及前清蛋白带各鼠间出现明显的变异,表现为黑线姬鼠和社鼠在β１区出现２种多态型区带,褐家鼠在β２区也有２种多态型带;各鼠间的血红蛋白变异比较明显,主要表现在区带数和主成分的泳动度显著不同,而且社鼠的Ｈｂ出现３种多态型,黄毛鼠出现２种多态型,其余各鼠的Ｈｂ皆为单态性。上述１６项生化特性按相似和相异进行配对比较,初步表明黄毛鼠与褐家鼠亲缘上比较相近,白腹巨鼠要比其他３种鼠亲缘上更接近于黑线姬鼠,而社鼠与白腹巨鼠之间进化分歧相对地较大。     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Expression of a human cytomegalovirus gp58 antigenic domain fused to the hepatitis B virus nucleocapsid protein

Abstract Hepatitis B virus core antigen (HBcAg) has been used as a carrier for expression and presentation of a variety of heterologous viral epitopes in particulate form. The aim of this study was to produce hybrid antigens comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions in order to investigate the influence of position of the foreign epitope on hybrid core particle formation, antigenicity and immunogenicity. HCMV DNA encoding a neutralising epitope of the surface glycoprotein gp58 was either inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg and expressed in Escherichia coli . The carboxyl terminal fusion (HBc_3–144-HCMV) was expressed at high levels and assembled into core like particles resembling native HBcAg. Protein with a similar fusion at the amino terminus (HCMV-HBc_1–183) could not be purified or characterised immunologically, although it formed core like particles. HBc_3–144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected by radioimmunoassay or western blotting using anti-HCMV monoclonal antibody 7–17 or an anti-HCMV human polyclonal antiserum. Following immunisation of rabbits with HBc_3–144-HCMV, a high titre of anti-HBc specific antibody was produced along with lower titres of HCMV/gp58 specific antibody.     

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

Summary Protein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)₁₀ to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. ¹H, ¹⁵N and ¹³C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.Abbreviations Rho116, Rho130 protein containing the first 116 (130) residues of rho - CSD cold shock domain - RRM RNA recognition motif - RBD RNA-binding domain - IPTG isopropyl -D-thiogalactopyranoside - EDTA ethylenediaminetetraacetic acid - NOE nuclear Overhauser enhancement     

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

Abstract The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.