Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

This work studies the structure-function relationships of Asn¹³⁵, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn¹³⁵ side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN 135D/D 138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn¹³⁵ is important for the correct folding of the nucleotide binding site and that EF-Tu·kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.     

Trypanosoma brucei: Maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis

A continuous dialysis technique capable of maintaining concentrated suspensions of bloodstream Trypanosoma brucei (up to 4 × 10⁸/ml) at 25 C for 2 hr without loss of viability has been developed in order to measure endocytosis under controlled conditions in vitro. Using this technique, the kinetics and mechanism of uptake of the metabolically inert macromolecule, ¹²⁵I-labelled polyvinylpyrrolidone (¹²⁵I-PVP), have been investigated. Binding to the plasma membrane and the rate of uptake of ¹²⁵I-PVP from the extracellular medium by the trypanosome are both decreased by the addition of unlabelled PVP and human serum albumin. A mechanism for uptake of ¹²⁵I-PVP by a combination of fluid-phase and adsorptive endocytosis from the flagellar pocket of the trypanosome is proposed. In the presence of serum albumin and unlabelled PVP, endocytosis of ¹²⁵I-PVP occurs in the fluid phase only, with endocytic indices of 14.5 ± 0.9 and 54.1 ± 11.3 nl/hr/mg protein in vitro at 25 C and in vivo at 37 C, respectively.     

肝素C5异构酶的表达优化及分子改造

肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase,C5,EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶,催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5号位羧基翻转生成L-艾杜糖醛酸 (L-iduronic acid,L-IdoA)。文中以大肠杆菌Escherichia coli为宿主对斑马鱼来源的肝素葡萄糖醛酸C5异构酶基因Glce进行重组表达优化与分子改造。比较了3种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5表达的差异情况,其中以嗜冷启动型载体pCold Ⅲ表达酶活最高,达到(1 873.61±5.42) U/L。为了进一步提高C5的可溶表达量,在N端融合促溶标签SET2后,可溶蛋白表达量比对照提高了50%,酶活达到 (2 409.25±6.43) U/L。在此基础上,通过理性设计对底物结合口袋进行定点突变,获得最优突变体 (V153R) 的酶活和比酶活分别为 (5 804.32±5.63) U/L和(145.14±2.33) U/mg,是原始酶的2.41倍和2.28倍。肝素C5异构酶改造与表达优化为酶法催化合成肝素奠定了基础。     

Acidic biphenyl derivatives: Synthesis and biological activity of a new series of potent 5-HT4 receptor antagonists

Serotonin (5-hydroxytryptamine, 5-HT) is an important signaling molecule in the central nervous system (CNS) and in non-neuronal tissues and organs. Serotonin mediates a positive chronotropic and inotropic response through 5-HT₄ receptors in the atrium and ventricle of the heart. Recent investigations have revealed increased expression of the 5-HT_4(b) isoform in cardiomyocytes of chronic arrhythmic and failing hearts, and that the use of 5-HT₄ receptor antagonists may be beneficial for treating these conditions. The 5-HT₄ receptor possesses a transmembrane (TM) binding site important for ligand affinity and recognition, as well as a capacity to accommodate bulky ligands. A new series of peripherally-acting 5-HT₄ receptor antagonists were prepared by combining the acidic biphenyl group from the class of angiotensin II receptor blockers (ARBs) with the SB207266 (piboserod) scaffold. The new compounds were pharmacologically evaluated and carboxylic acid 21 was identified as a potent and promising 5-HT₄ receptor antagonist with moderate affinity for the AT₁ receptor. The permeability of carboxylic acid 21 in a Caco-2 assay was low and the corresponding prodrug esters 23a–f were therefore prepared. The pharmacokinetics of methyl ester 20 and n-butyl ester 23c were evaluated in a rat model, revealing incomplete metabolism to carboxylic acid 21. However, methyl ester 20 is a potent 5-HT₄ receptor antagonist with binding affinities in the low picomolar range. Methyl ester 20 has promising oral bioavailability and pharmacokinetics and may target 5-HT₄ receptors in both CNS and peripheral organs.     

A mast‐seeding desert shrub regulates population dynamics and behavior of its heteromyid dispersers

Granivorous rodent populations in deserts are primarily regulated through precipitation‐driven resource pulses rather than pulses associated with mast‐seeding, a pattern more common in mesic habitats. We studied heteromyid responses to mast‐seeding in the desert shrub blackbrush (Coleogyne ramosissima), a regionally dominant species in the Mojave–Great Basin Desert transition zone. In a 5‐year study at Arches National Park, Utah, USA, we quantified spatiotemporal variation in seed resources in mast and intermast years in blackbrush‐dominated and mixed desert vegetation and measured responses of Dipodomys ordii (Ord's kangaroo rat) and Perognathus flavescens (plains pocket mouse). In blackbrush‐dominated vegetation, blackbrush seeds comprised >79% of seed production in a mast year, but 0% in the first postmast year. Kangaroo rat abundance in blackbrush‐dominated vegetation was highest in the mast year, declined sharply at the end of the first postmast summer, and then remained at low levels for 3 years. Pocket mouse abundance was not as strongly associated with blackbrush seed production. In mixed desert vegetation, kangaroo rat abundance was higher and more uniform through time. Kangaroo rats excluded the smaller pocket mice from resource‐rich patches including a pipeline disturbance and also moved their home range centers closer to this disturbance in a year of low blackbrush seed production. Home range size for kangaroo rats was unrelated to seed resource density in the mast year, but resource‐poor home ranges were larger (P < 0.001) in the first postmast year, when resources were limiting. Blackbrush seeds are higher in protein and fat but lower in carbohydrates than the more highly preferred seeds of Indian ricegrass (Achnatherum hymenoides) and have similar energy value per unit of handling time. Kangaroo rats cached seeds of these two species in similar spatial configurations, implying that they were equally valued as stored food resources. Blackbrush mast is a key resource regulating populations of kangaroo rats in this ecosystem.     

A molecular docking study of estrogenically active compounds with 1,2-diarylethane and 1,2-diarylethene pharmacophores

Numerous selective estrogen receptor modulators (SERMs) have been synthesized and assayed in recent years. The focus of this study is to apply coarse-grain molecular docking procedures coupled with fine-grain all-atom force field optimization strategies to shed light on the binding mechanisms of currently available estrogen receptor-active compounds. Although the mechanics of ligand binding in estrogen receptors is generally well understood, there is room for surprises. In this paper computational evidence corroborating the experimentally observed type I agonistic binding mode for estradiol (E2) and diethylstilbesterol (DES) and the type II antagonistic binding mode for 4-hydroxytamoxifen and raloxifen is presented. Included in this type I agonistic mode are the DES derivatives, transstilbene and 1,2-diaryldiaminoethane. In addition, a novel ‘type II agonistic’ binding mode for 2,3-diarylimidazolines, 4,5-diarylimidazoles, 2,3-diarylpiperazines is introduced. This mode is stabilized by suggesting alternative hydrogen bond anchor points in the ligand binding domain as potential leads for future drug design.     

Inferring functional relationships of proteins from local sequence and spatial surface patterns

We describe a novel approach for inferring functional relationship of proteins by detecting sequence and spatial patterns of protein surfaces. Well-formed concave surface regions in the form of pockets and voids are examined to identify similarity relationship that might be directly related to protein function. We first exhaustively identify and measure analytically all 910,379 surface pockets and interior voids on 12,177 protein structures from the Protein Data Bank. The similarity of patterns of residues forming pockets and voids are then assessed in sequence, in spatial arrangement, and in orientational arrangement. Statistical significance in the form of E and p-values is then estimated for each of the three types of similarity measurements. Our method is fully automated without human intervention and can be used without input of query patterns. It does not assume any prior knowledge of functional residues of a protein, and can detect similarity based on surface patterns small and large. It also tolerates, to some extent, conformational flexibility of functional sites. We show with examples that this method can detect functional relationship with specificity for members of the same protein family and superfamily, as well as remotely related functional surfaces from proteins of different fold structures. We envision that this method can be used for discovering novel functional relationship of protein surfaces, for functional annotation of protein structures with unknown biological roles, and for further inquiries on evolutionary origins of structural elements important for protein function.     

Reciprocal "flipping" underlies substrate recognition and catalytic activation by the human 8-oxo-guanine DNA glycosylase

Both 8oxo-guanine and formamidopyrimidines are major products of oxidative DNA damage that can result in the fixation of transversion mutations following replication if left unrepaired. These lesions are targeted by the N-DNA glycosylase hOgg1, which catalyses excision of the aberrant base followed by cleavage of the phosphate backbone directly 5' to the resultant abasic site in a context, dependent manner. We present the crystal structure of native hOgg1 refined to 2.15 A resolution that reveals a number of highly significant conformational changes on association with DNA that are clearly required for substrate recognition and specificity. Changes of this magnitude appear to be unique to hOgg1 and have not been observed in any of the DNA-glycosylase structures analysed to date where both native and DNA-bound forms are available. It has been possible to identify a mechanism whereby the catalytic residue Lys 249 is "primed" for nucleophilic attack of the N-glycosidic bond.     

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH₄)₂SO₄ fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (k_cat/K_m) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25°C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of K_m values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.