Molecular Replacement Studies of Cucurmosin from Cucurbita Moschata:Structure Homology with Trichosanthin

1 INTRODUCTION Many plants contain proteins that are capable of inactivating ribosomes and therefore are called ribosome-inactivating proteins or RIPs[1]. RIPs are RNA N-glycosidases that inactivate ribosomes through a site-specific deadenylation of the large ribosomal RNA[2, 3]. RIPs are also capable of inactivating many nonribosomal nucleic acid substrates and can be considered as polynucleotide: adenosine glycosidases[4～6]. There are two types of RIPs: type I, single chain pr…     

Exact rotamer optimization for protein design

Computational methods play a central role in the rational design of novel proteins. The present work describes a new hybrid exact rotamer optimization (HERO) method that builds on previous dead-end elimination algorithms to yield dramatic performance enhancements. Measured on experimentally validated physical models, these improvements make it possible to perform previously intractable designs of entire protein core, surface, or boundary regions. Computational demonstrations include a full core design of the variable domains of the light and heavy chains of catalytic antibody 48G7 FAB with 74 residues and 10(128) conformations, a full core/boundary design of the beta1 domain of protein G with 25 residues and 10(53) conformations, and a full surface design of the beta1 domain of protein G with 27 residues and 10(60) conformations. In addition, a full sequence design of the beta1 domain of protein G is used to demonstrate the strong dependence of algorithm performance on the exact form of the potential function and the fidelity of the rotamer library. These results emphasize that search algorithm performance for protein design can only be meaningfully evaluated on physical models that have been subjected to experimental scrutiny. The new algorithm greatly facilitates ongoing efforts to engineer increasingly complex protein features.     

一种基于形成杂多核络合物的荧光增敏效应的研究 Ⅲ.锰(Ⅱ)、钴(Ⅱ)的混合荧光增敏及其连续测定

本文研究了Mn~(2+)和Co~(2+)对7-(8-羟基-3,6-二磺基萘偶氮)-8-羟基喹啉-5-磺酸-硼砂反应体系的混合荧光增敏作用。实验条件下,荧光增敏强度满足线性加和关系的范围是:Mn~(2+)浓度0～2.9×10~(-7)mol/L;Co~(2+)浓度0～8.8×10~(-7)mol/L;总浓度不超过1.0×10~(-6)mol/L。检出限量为Mn~(2+)4.5×10~(-9)mol/L和Co~(2+)1.4×10~(-8)mol/L。实现了Mn~(2+)和Co~(2+)的连续测定。     

Signaling molecularly imprinted polymers: molecular recognition-based sensing materials

Molecular imprinting is a template polymerization technique that can easily provide synthetic polymers capable of molecular recognition for given target molecules. In addition to their highly specific recognition ability, we are attempting to introduce signaling functions to molecularly imprinted polymers, enabling them to respond according to specific binding events. Some of our work regarding such signaling molecularly imprinted polymers is presented here, including molecularly imprinted polymers that induce spectral shifts of target compounds because of binding. Such compounds include hydrogen-bonding-based fluorescent imprinted polymers and metalloporphyrin-based signaling molecularly imprinted polymers.     

Purification of recombinant green fluorescent protein by three-phase partitioning

The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature.     

Quantitative study on La~(3+) influx mediated by sodium-calcium exchanger in human lymphocytes

Whether La3+ can enter human peripheral blood lymphocytes by the Na+/Ca2+ exchanger or not and the effect of La3+on the Na+/Ca2+ exchanger activity are examined by fura-2 technique. And that whether La3+ is sequestered by intracellular organelles (mainly endoplasmic reticulum and mitochondria) is studied by this method. La3+uptake is obviously stimulated by pre-treating the cells with ouabain and by removing extracellular Na+, and intracellular La3+concentration ([La3+]i) is directly proportional to its extracellular concentration ([La3+]o). But when [La3+]o exceeds 0.4 mmol/L, the 340/380 nm ratio of fluorescence is no longer varied and the maximum [La3+], is 1.5×10-12 mol · L-1. The higher concentration of La3+ (0.1 mmol/L) increases Na+/Ca2+ exchange-mediated calcium influx, but lower concentration (10 μmol/L) appears to block calcium influx. The results also suggest that cytosolic La3+ is transported by the ATP-dependent Ca2+ pump. Intracellular Ca2+ stores are depleted by ionomycin, and then ion     

微波溶样肉桂基荧光酮和表面活性剂连续荧光测定葡萄酒中铁和钴

观察到肉桂基荧光酮(略作CF)在pH≈8与Fc(Ⅲ)配位后荧光增强,CPB的加入使荧光更强,络合物与试剂荧光强度之比F_(FeR)/F_R随0～0.01μg/ml Fe(Ⅲ)线性变化,λ_(ex)=410nm,λ_(ex)=480nm;而CF在pH≈10与Co(Ⅲ)配位后荧光却减弱,乳化剂OP存在时使试剂荧光增强,若以荧光熄灭Co(Ⅲ)灵敏度便提高,试剂与络合物荧光强度之比F_R/F_(CoR)对0～0.016 μg/ml Co(Ⅲ)呈直线关系,λ_(ex)=300nm,L_(em)=352nm。控制化学条件和激发、发射波长,可不经分离测定葡萄酒(微波溶样)中铁和钴。     

A Novel Real-time Fluorescence Mutant-allele-specific Amplification Method for Rapid Single Nucleotide Polymorphism Analysis

Current methods for single nucleotide polymorphism (SNP) analysis are time-consuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-rar oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.     

Selective formation of AAB- and ABC-type heterotrimeric alpha-helical coiled coils

The alpha-helical coiled coils have a representative amino acid sequence of (abcdefg)(n) heptad repeats. We previously reported that two peptides named IZ-2A and IZ-2W formed an (IZ-2A)(2)/IZ-2W heterotrimer with an Ala-Ala-Trp interaction in the hydrophobic core. In this paper, we describe the selective formation of AAB- and ABC-type heterotrimers. To increase the selectivity of the AAB-type heterotrimeric formation, Lys residues at the f position were mutated to either an Ala or a Gln residue to form IZ-2A(fA) or IZ-2W(fQ). Separately, both IZ-2A(fA) and IZ-2W(fQ) have a random structure at pH 7 and 20 degrees C. However, together IZ-2A(fA) and IZ-2W(fQ) form a 2:1 complex with a thermal transition midpoint (Tm) of 48 degrees C. This procedure was applied to prepare the ABC-type heterotrimer, in which two sets of Ala-Ala-Trp interactions were designed in the hydrophobic core. Interhelical interaction between the e and g positions and the alpha-helical propensity of the amino acid at the f position were also considered in the design. The resultant three peptides selectively formed the ABC-type heterotrimer with a Tm of 51 degrees C. Other peptide combinations had random coil properties.     

A comparative study of various computational approaches in calculating the structure of pyridoxal 5'-phosphate (PLP)-dependent beta-lyase protein. The importance of protein environment

Various computational approaches, using molecular mechanics (Amber), semiempirical (AM1), density functional (B3LYP), and various ONIOM methods, have been comparatively investigated for the structure of Escherichia coli NifS CsdB protein. The structure of the entire monomer containing 407 amino acid residues and 579 surrounding water molecules has been optimized. The full geometry optimization in the "active site-only" approach (including only active site atoms) has been found to give the largest root-mean-square (RMS) deviation from the X-ray structure; a much better agreement has been achieved by keeping the atoms leading to the backbones of some amino acids frozen in their positions in the X-ray structure. The best agreement has been attained by including the surrounding protein in the calculations using the two-layer ONIOM (B3LYP:Amber) approach. The results presented in this study conclusively demonstrate the importance of the protein/active-site interaction on the active-site structure of the enzyme. The present theoretical study represents the largest system studied at the ONIOM level to date, containing 7992 atoms, including 84 atoms in the QM region and rest in the MM region.