NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)-III of the squash family and comparison with those of counterparts of CMTI-V of the potato I family.

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value.     

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.     

脑型一氧化氮合成酶的钙调蛋白结合区的表达及活性鉴定

用PCR法克隆出nNOS的CaM结合区基因(nNOS 2455～2988bp),并在大肠杆菌中进行了高效表达。经金属离子螯合亲和层析得到纯度为90％以上的重组蛋白．分子量为22kDa,CaM Oveday assay证实该蛋白具有CaM的结合活性。由于所表达的重组蛋白既具有序列特异性又具有CaM的结合活性．因此。可将它作为筛选nNOS特异性抑制肽的靶蛋白,亦可用于特异性抗体的制备。     

三链DNA的形成抑制DNA结合蛋白与启动子的结合

电泳迁移分析方法及DNaseⅠ足迹实验表明21nt脱氧寡核苷酸G3TG2T GT2G5TG2TGT(CP1)与乙肝病毒(HBV)核心启动子(Cp)片段之间三链DNA的形成有较高的特异性及稳定性.凝胶滞留实验显示, 在大鼠肝细胞核提取物体外转录系统中, CP1可特异地抑制DNA结合蛋白与Cp片段的结合, 而不能与Cp结合形成三链DNA的脱氧寡核苷酸CP3(TGTG2TG5T2GTG2TG3)对蛋白与Cp的结合并无抑制作用.这些结果表明, 三链DNA的形成有可能抑制HBV DNA的转录.     

不同籼稻品种的矮生性与内源ABA水平及其结合蛋白的关系

试验研究了内源脱落酸及其结合蛋白与含有不同矮秆主基因的籼稻矮生性状的关系。结果表明：籼稻的矮生性也受内源ＡＢＡ及其结合蛋白协同调控。矮秆品种幼苗内源ＡＢＡ及其幼芽膜上ＡＢＡ结合蛋白水平显著高于离秆品种,且含有强矮化效应的ｓｄ－ｇ基因的新桂矮、双矮的内源ＡＢＡ及其结合蛋白水平又明显高于含ｓｄ－１半矮秆基因的广场矮。外源ＡＢＡ能显著抑制籼稻幼苗伸长,苗高下降率与ＡＢＡ结合蛋白水平有一定的关系,且矮秆品     

Iron Binding and Autoreduction by Citrate: Are These Involved in Signalling by Iron Regulatory Protein-l?

Ferric ions bind to citrate and undergo an autoreduction to form a ferrous-citrate complex, greatly increasing the redox activity of the iron complex. Ferrous ions and citrate are also essential for the enzymic activity of aconitase. Aconitase, with its iron-sulphur cluster has a versatile structure which allows it to act as an iron regulatory protein (IW-1). The purpose of this study was to see whether iron binding, and its autoreduction by citrate, could play a physiological signalling role in iron regulation. Significant amounts of ferrous ions were associated with citrate, when measured using ferrozine, however, these did not appear to activate iron-requiring aconitase.     

Molecular Changes in Entamoeba histolytica in Response to Bacteria1

Entamoeba histolytica, the protozoan parasite, is the causative agent of amoebiasis. The degree of virulence, as inferred from invasiveness, of potentially pathogenic strains may be regulated by both host and parasite factors that determine the gut environment. One such factor that plays an important role is the bacterial flora in the gut. Previous studies have clearly shown that bacterial flora is an important determinant of virulence in E. histolytica. However, the exact nature of changes induced in E. histolytica in response to bacteria and their role in virulence is not clear. In this study the levels of a number of molecules potentially important in virulence mechanisms were determined in E. histolytica cells grown with and without normal human bacterial flora, using enzyme-linked immunosorbent assay. Significant changes were observed only after the E. histolytica cells had been adapted to grow with bacterial flora for a number of generations, and not in short term culture.     

The haemoglobin system of the mudfish, Labeo capensis: adaptations to temperature and hypoxia

The effect of temperature and hypoxic acclimation on the haemoglobin system and intraerythrocytic organic phosphate concentrations in the South African mudfish, Labeo capensis, have been investigated. Exposure to hypoxia or increased temperature raised haemoglobin concentration and decreased NTP/Hb ratio. Temperature acclimation did not effect the oxygenation characteristics of the haemolysate or haemoglobin multiplicity, as evident from isoelectric focussing experiments that showed one cathodic (Hb I) and three anodic haemoglobins (Hb II, III and IV). Oxygen equilibria of the isolated components showed a smaller Bohr effect and lower temperature and organic phosphate sensitivities in the cathodic than in the anodic haemoglobins. Unlike the trout and eel haemoglobin systems, both the anodic and cathodic haemoglobins from L. capensis exhibited sensitivity to organic phosphates but the effect was smaller in the latter. The results indicate that oxygen transport in mudfish blood is supported by variations in the red cell organic phosphate\haemoglobin ratio and the functional differentiation between anodic and cathodic haemoglobins.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

From Tumor Necrosis Factor Receptor to RANK, from the Selectins and Link Proteins to CD44: New Molecular Models of Cell Surface Receptors and Analysis of Specificity Determinants

Modeling and structure-function studies on two cell surface proteins are presented, which are implicated in the regulation of immune responses and cell adhesion. In the first part, model building of RANK, a new member of the tumor necrosis factor receptor (TNFR) superfamily (TNFRSF), is reported. The model is analyzed in light of structural studies on the TNFR-ligand complex and molecular model-based mutagenesis analyses of CD40-ligand and Fas-ligand interactions. The study makes it possible to predict residues important for ligand binding to RANK and further rationalizes differences in specificity between TNFR-like cell surface receptors. In the second part, recent investigations on the structure and carbohydrate binding site of CD44, a member of the link protein family, are discussed. The binding site in CD44 is compared to calcium-dependent (C-type) lectins, which include the selectins, another family of cell adhesion molecules. The studies on TNFRSF members and link proteins reported herein complement a recent review article in this journal, which focused on modeling and binding site analysis of immune cell surface proteins.Electronic Supplementary Material available.