替莫唑胺多晶型的制备及其热稳定性

替莫唑胺在不同条件下进行了重结晶，经熔点、红外光谱、差热分析和粉末X—射线衍射法鉴定，确认替莫唑胺存在A、B两种晶型和一种粒状固体C，采用差热分析法对A、B和C的热稳定性进行了测定和比较。     

New Fluoropolymers Having Both Low Water Uptake and a Low Dielectric Constant

Polymers with both low water uptake and a low dielectric constant have seen interest from both the electronic and microelectronic industries for several years. They play a crucial role in maintaining the good operating situation of devices, whereas most other materials are unsatisfactory. In this contribution, two new fluoropolymers are reported, which are derived from the thermo‐crosslinking reaction of functional monomers containing perfluorocyclobutane and benzocyclobutene moieties. These polymers show water uptake ranging from 0.11 to 0.31% after immersion in boiling water (near 98 °C) for 96 h, and exhibit dielectric constants (k) of below 2.4 at a range of frequencies from 0.1 MHz to 30 MHz. In contrast, a comparative polymer without perfluorocyclobutane units has a k value of more than 2.70 and water uptake of 0.67%, respectively, suggesting that the low water absorption and dielectric constants of the fluoropolymers can be attributed to the existence of perfluorocyclobutane groups in the molecular backbone. These results also indicate that the properties of the fluoropolymers are superior to these of the most commercial polymeric low‐k materials, implying that these fluoropolymers could be used as the varnish for enameled wire in the electrical industry and as encapsulation resins in the microelectronics industry.

     

用流化床干燥法改善颗粒酶的热稳定性

通过流化床干燥法制备稳定化颗粒酶，提高酶的耐热性。在制备过程中进风温度为40℃，酶活回收率达到90％以上。采用海藻酸钠（SA）作包被剂，随着SA质量浓度的提高，酶的耐干热性先增加后降低，在0.4g/dL最高，而其耐湿热性不断增强。水分活度提高，固态酶的耐热性就随之降低，在Aw≥0．3时流化床干燥酶样品的耐热性都明显高于原酶粉，且SA质量浓度越高，酶对水分的耐受能力越高。在流化床干燥酶制剂中添加硫酸铵、氯化钠、硫酸钠等盐类稳定剂，酶的耐干热性有的增强，有的减弱，而其耐湿热性都有所增强，其中硫酸铵的热稳定化效果最好。酶在高温下的失活速度遵循一级动力学，即它的失活是由酶蛋白变性引起的，流化床干燥样品的失活常数低于原酶粉的失活常数，而不同温度下酶的失活遵循阿累尼乌斯方程。     

Cloning and characterization of a thermostable catfish alphaB-crystallin with chaperone-like activity at high temperatures

We have cloned, expressed and characterized catfish alphaB-crystallin (FalphaB). Genomic sequence comparison has revealed conservation of intron splicing sites and coding regions, however, the two intron sequences, 5'- and 3'-untranslated regions of FalphaB gene are shorter than those reported for other vertebrates. In contrast to mammalian homologues with a subunit association ratio (alphaA-crystallin/alphaB-crystallin) of 3:1, alpha-crystallin from catfish lens showed a ratio of 19:1. The biophysical properties and chaperone-like activity of recombinant FalphaB and porcine alphaB-crystallin (PalphaB) were studied and compared by heat denaturation, circular dichroism, intrinsic and dye-binding fluorescence, gel-filtration, and analytical ultracentrifugation. FalphaB shows 50% precipitation occurring at 72 degrees C that is higher than PalphaB at 66 degrees C. Even though FalphaB also possesses more surface hydrophilic regions than PalphaB, FalphaB still possesses higher chaperone activity to prevent aggregation of alcohol dehydrogenase at 60 degrees C. The molecular mass of FalphaB showed a smaller size (450 kDa) than PalphaB (550 kDa), which is also confirmed by analytical ultracentrifugation. In addition, FalphaB possesses better refolding potential after preheating treatment than PalphaB. FalphaB also exhibits higher chaperone-like activity than PalphaB to prevent insulin aggregation induced by dithiothreitol. In contrast to the prevalent notion that fish crystallins generally denature easily, FalphaB with chaperone-like activity appears to be more stable than mammalian homologues towards thermal and chemical denaturation.     

α-Amylases from Thermoactinomyces vulgaris: characteristics,primary structure and structure prediction

Two amylolytic active protein fractions (named α-amylase 1 and α-amylase 2) were isolated from the bacterium Thermoactinomyces vulgaris strain 94-2A. α-Amylase 1 had a molecular mass of 51.6 kDa, whereas α-amylase 2 consists of two fragments which have molecular masses of 17.0 and 34.6 kDa, respectively. These two fragments are products from a proteolytic cleavage of a-amylase 1 at amino acid position 303 (tryptophan) by a serine protease (thermitase) which is also produced by T. vulgaris. The purified α-amylase 1 and 2 follow the Michaelis-Menten kinetics in the presence of starch as substrate with K_m values of 1.37 ± 0.07 and 1.29 ± 0.18 mg/mL, respectively. In effect they differ in their stability characteristics. The amino acid sequence of α-amylase from T. vulgaris derived from DNA sequence (1) was compared with those of other α-amylases. It reveals high homologies to α-amylases from other microorganisms (e.g. B. polymyxa, A. oryzae, S. occidentalis and S.fibuligera). A three-dimensional structure model for α-amylase 1 on the basis of the 3 Å X-ray structure of Taka-amylase was constructed.     

Enhancing protein stability with extended disulfide bonds

Disulfide bonds play an important role in protein folding and stability. However, the cross-linking of sites within proteins by cysteine disulfides has significant distance and dihedral angle constraints. Here we report the genetic encoding of noncanonical amino acids containing long side-chain thiols that are readily incorporated into both bacterial and mammalian proteins in good yields and with excellent fidelity. These amino acids can pair with cysteines to afford extended disulfide bonds and allow cross-linking of more distant sites and distinct domains of proteins. To demonstrate this notion, we preformed growth-based selection experiments at nonpermissive temperatures using a library of random β-lactamase mutants containing these noncanonical amino acids. A mutant enzyme that is cross-linked by one such extended disulfide bond and is stabilized by ∼9 °C was identified. This result indicates that an expanded set of building blocks beyond the canonical 20 amino acids can lead to proteins with improved properties by unique mechanisms, distinct from those possible through conventional mutagenesis schemes.We are developing strategies that begin to address the question of whether an expanded genetic code provides an evolutionary advantage to an organism. For example, it has been recently shown that a unique noncanonical amino acid mutation in TEM-1 β-lactamase significantly increases the enzyme’s catalytic activity for the substrate cephalexin, a result that cannot be recapitulated by substitution of canonical amino acids at this site (1). This same enzyme has been reengineered to be dependent on a noncanonical active site residue for activity, a dependency that was maintained for hundreds of generations without escape (2). Furthermore, addition of noncanonical amino acid building blocks to an unbiased library of ribosomally synthesized cyclic peptides provided a selective advantage in the evolution of inhibitors of cytotoxic intracellular proteases (3). Here we begin to explore whether noncanonical amino acids can provide Escherichia coli a selective growth advantage by increasing the thermal stability of essential proteins.Cysteine is unique among the 20 canonical amino acids in that it can form reversible covalent cross-links in proteins. Disulfide bonds can stabilize monomeric and multisubunit proteins (4), play a role in catalysis (5, 6), and regulate protein activity (7); because of these unique properties, disulfide bonds are highly conserved in protein evolution (8, 9). There has been considerable success in the use of structure-based design to engineer disulfide bonds into proteins for both biopharmaceutical and industrial applications (10, 11). However, the sites in proteins that can be cross-linked by a cystine disulfide are typically constrained to a distance between the two β-carbons of ∼5.5 Å (10) and a near 90° dihedral angle for the disulfide bond (12). Thus, the relatively long distances that might be required to bridge distinct protein domains or subunits, or steric constraints at specific sites may preclude the introduction of a natural disulfide bond. These challenges led us to explore whether we could overcome the geometrical constraints of the cysteine disulfide by genetically encoding noncanonical amino acids (NCAAs) with longer thiol-containing side chains.To this end, we designed a series of tyrosine derivatives (SetY, SprY, and SbuY) with para-substituted aliphatic thiols of various lengths (Fig. 1A). The calculated length between the two β-carbons of the SbuY-Cys disulfide bond is 14 Å when fully extended, which is significantly longer than a natural cystine cross-link. We genetically encoded these NCAAs in bacterial and mammalian cells by suppressing the nonsense codon TAG with an orthogonal, amber suppressor aminoacyl-tRNA synthetase (RS)/tRNA pair (13). Because statistical analyses of proteins have suggested that stabilizing disulfide mutations are most often found in regions of higher mobility near the protein surface and associated with longer loop lengths (>25 residues) (10, 14), we reasoned that these more flexible, extended disulfides might facilitate the introduction of stabilizing disulfide bonds into proteins. Using a N-terminal truncated β-lactamase as a model system (15), we carried out a growth-based selection under nonpermissive temperatures with a library of mutants in which the thiol-containing NCAAs were randomly incorporated, and identified a mutant enzyme cross-linked by an extended disulfide bond that is stabilized by ∼9 °C.Open in a separate windowFig. 1.Genetic incorporation of NCAAs containing long-side-chain thiols. (A) Structure of O-(2-mercaptoethyl)-l-tyrosine (SetY), O-(3-mercaptopropyl)-l-tyrosine (SprY), and O-(4-mercaptobutyl)-l-tyrosine (SbuY). (B) SDS/PAGE analysis of purified GFP (134TAG) expressed in E. coli DH10B using the MbXYRS/tRNA^pyl pair in the presence or absence of 1 mM NCAAs. GFP mutants were expressed in LB medium and purified by standard Ni-NTA affinity chromatography. (C) Mass spectral analysis of GFP mutants containing the corresponding NCAAs. The calculated masses for the mutant GFPs containing SetY, SprY, and SbuY are 28,005 Da, 28,019 Da, and 28,033 Da, respectively. (D) Fluorescence microscopy (10×) of 293T cells expressing an EGFP mutant (Tyr39TAG) in the presence or absence of NCAAs. 293T cells were transiently cotransfected with pCMV-XYRS and pEGFP-Tyr39TAG and were grown in DMEM supplemented with 10 FBS in the presence or absence of 250 μM NCAAs.     

F/10和G/11木聚糖酶家族的不同热稳定性机制

应用生物信息学方法分析了木聚糖酶初级序列中20种氨基酸同其最适温度之间的关系,发现在F/10家族中有正相关作用的氨基酸是W,负相关作用的是 A,D,H,S;理论上最高最适温度是119 8 ℃在G/11家族中有正相关作用的氨基酸是L,D,P,Y,负相关作用的是 H;理论上最高最适温度为105 6 ℃.惊奇地发现在不同家族中 D起不同的作用,这只能从它们各自不同的蛋白质空间结构上进行解释.从初级序列基础上证明了木聚糖酶的不同蛋白质家族有不同的热稳定性机制.     

FLUORESCENCE PROPERTIES OF NEUTRAL PROTEASE FROM BACILLUS SUBTILIS

The fluorescence properties of neutral protease from B. subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977). In the pH range 5–9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm. At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shifted emission, appear rather buried in the hydrophobic protein interior. Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues. On the other hand, removal of both zinc and calcium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium. Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants. Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60–70°. Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents. The results reveal differences in the fluorescence properties of the tryptophanyl residues of B. subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins.     

Structural basis for Ca2+-independence and activation by homodimerization of tomato subtilase 3

Subtilases are serine proteases found in Archae, Bacteria, yeasts, and higher eukaryotes. Plants possess many more of these subtilisin-like endopeptidases than animals, e.g., 56 identified genes in Arabidopsis compared with only 9 in humans, indicating important roles for subtilases in plant biology. We report the first structure of a plant subtilase, SBT3 from tomato, in the active apo form and complexed with a chloromethylketone (cmk) inhibitor. The domain architecture comprises an N-terminal protease domain displaying a 132 aa protease-associated (PA) domain insertion and a C-terminal seven-stranded jelly-roll fibronectin (Fn) III-like domain. We present the first structural evidence for an explicit function of PA domains in proteases revealing a vital role in the homo-dimerization of SBT3 and in enzyme activation. Although Ca²⁺-binding sites are conserved and critical for stability in other subtilases, SBT3 was found to be Ca²⁺-free and its thermo stability is Ca²⁺-independent.     

Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.The Gram-negative encapsulated bacterium Neisseria meningitidis causes severe sepsis and meningococcal meningitis. Invasive meningococcal disease (IMD) is associated with 5–15% mortality; furthermore, devastating long-term sequelae such as amputations, hearing loss, and neurodevelopmental disabilities are observed in 11–19% of IMD survivors (1). Meningococcal serogroups are distinguished by the composition of their capsular polysaccharides. The five serogroups most commonly associated with invasive disease are A, B, C, W, and Y. (2). Effective mono- or polyvalent-conjugated polysaccharide vaccines against N. meningitidis serogroups A, C, W, and Y have been available since the early 1990s (3). However, serogroup B meningococcus (MenB) is responsible for the majority of endemic and epidemic meningococcal disease in developed countries (4–6). The development of an efficient capsular polysaccharide-based vaccine against MenB has been hampered by potential autoimmunity issues, namely, the structural similarity between the MenB capsular polysaccharide and the neuraminic acid present on the surface of human fetal neural tissues (7).In early 2013 the European Medicines Agency approved 4CMenB, to our knowledge the first broadly protective vaccine against MenB, for the prevention of IMD in all age groups. 4CMenB is a multicomponent vaccine formulation composed of three surface-exposed meningococcal proteins originally identified by the reverse vaccinology approach (8) plus outer membrane vesicles from the New Zealand epidemic clone. The three antigenic proteins are factor H-binding protein (fHbp), neisserial heparin-binding antigen (NHBA), and neisserial adhesin A (NadA) (9, 10).The gene encoding NadA is present in ∼30% of pathogenic meningococcal isolates and is associated mostly with strains that belong to three of the four hypervirulent serogroup B lineages (11–14). NadA expression levels can vary among isolates by more than 100-fold, and its expression is up-regulated in vivo by niche-specific signals (15). NadA induces high levels of bactericidal antibodies in humans (16–18) and is recognized by serum antibodies of children convalescent after IMD (19), suggesting that it is expressed and is immunogenic during IMD. Two main genetically distinct groups of NadA have been identified that share overall amino acid sequence identities of 45–50%. Group I includes the three most common variants (NadA1, NadA2, and NadA3, the latter being the vaccine variant), which share ∼95% sequence identity and are immunologically cross-reactive (11). Group II includes three rarer variants: NadA4, primarily associated with carriage strains (11); NadA5, found mainly in strains of clonal complex 213 (20, 21); and NadA6 (Fig. S1A); these three share ∼90% sequence identity (Fig. S1B) (22).Functionally, NadA3 expressed on the surface of Escherichia coli promotes adhesion to and invasion of Chang epithelial cells (23). This adhesive activity has been mapped, at least partially, to an N-terminal region extending to residue T132 (23, 24). Recently, interactions of NadA3 with β-1 integrin (25) and with the heat shock protein Hsp90 (26) have been reported.Structurally, NadA belongs to the class of trimeric autotransporter adhesins (TAAs) (27, 28), which are known to mediate adhesion through interaction with extracellular matrix proteins and are involved in invasion of target cells (29). TAAs are obligate homotrimers, and accordingly the recombinant NadA3 vaccine antigen, lacking the C-terminal membrane anchor region, forms soluble, stable trimers (23, 30). TAAs generally are made of a conserved C-terminal integral membrane β-barrel, which anchors the proteins to the outer membrane, and an N-terminal “passenger” domain responsible for adhesion (31). The TAA passenger domain typically is made of a central α-helical domain (stalk) that forms coiled-coil structures and a distinct N-terminal domain (head) that is mainly responsible for binding to host cellular receptors.Here we present the X-ray structure of a large ectodomain fragment of NadA5 and a structural analysis by transmission electron microscopy (TEM) of the vaccine variant NadA3. In addition, epitope mapping shows that the head of NadA3 contains immunogenic regions responsible for the generation of a protective bactericidal response.