Detection and characterization of a folding intermediate in barnase by NMR

Protein engineering is being developed for mapping the energetics and pathway of protein folding. From kinetic studies on wild-type and mutant proteins, the sequence and energetics of formation of tertiary interactions of side chains can be mapped and the formation of secondary structure inferred. Here we cross-check and complement results from this approach by using a recently developed procedure that traps and characterizes secondary structure in intermediate states using 1H NMR. The refolding of barnase is shown to be a multiphasic process in which the secondary structure in alpha-helices and beta-sheets and some turns is formed more rapidly than is the overall folding.     

Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.     

NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A

The presence of an early intermediate on the folding pathway of ribonuclease A has been demonstrated by a study of the exchange reaction between the backbone amide protons in the folding protein and solvent protons using rapid mixing techniques. A structural analysis of the intermediate by two-dimensional 1H-NMR is consistent with the framework model of protein folding in which stable secondary structure first forms the framework necessary for the subsequent formation of the complete tertiary structure.     

Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.     

Transient folding intermediates characterized by protein engineering

Kinetic experiments on engineered mutants of barnase detect an intermediate on the folding pathway and allow the mapping of the tertiary interactions of the side chains and their energetics. Many of the interactions present in the final folded state tend to be either fully formed or not formed at all in the intermediate or subsequent transition state for folding, but the hydrophobic core becomes progressively consolidated. These methods in combination with NMR provide extensive structural characterization of the folding intermediate and the sequence of events in the folding pathway.     

谈体育教学直观示范的问题

体育直观教学中,位置的选择的必要性.选择恰当的示范位置,学生看清楚所要学的动作.这是示范前首先要考虑的问题;效果的考虑,恰当的示范位置为示范产生效果创造的重要前提,使示范达到预定目的.     

马来酸氯苯那敏片剂的核磁共振定量测定方法研究

提出测定马来酸氯苯那敏片剂中马来酸氯苯那敏的1H-NMR方法。考察了实验条件的影响,选择重水为溶剂,反丁稀二酸为内标,延迟时间为20 S,脉冲宽度为3.0 μs,采样次数为16次,内标浓度为4?5 mg/mL。在内标与样品质量比为0. 48?2.12范围内,峰面积比与质量比之间具有良好的线性关系,相关系数r>0. 9990,用绝对定量法和标准曲线法测定了 3种马来酸氯苯那敏片剂的含量,并与药典的紫外分光光度法作了比较。     

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.     

拟连续domain的网式刻画

本文在定向完备偏序集上引入网的广义S收敛的概念,并给出了拟连续domain的如下网式刻画:定向完备偏序集是拟连续的当且仅当广义S收敛关于Scott拓扑是拓扑的.该结果推广了Domain理论中关于连续domain的类似刻画.     

Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3

Replacing the isoleucine at amino-acid position three of bacteriophage T4 lysozyme causes changes in the thermodynamic stability of the protein that are directly related to the hydrophobicity of the substituted residue. Structural analysis confirms that the hydrophobic stabilization is proportional to the reduction of the surface area accessible to solvent on folding.     

壳聚糖的溶菌酶降解

用粘度法和还原糖法研究了溶菌酶降解壳聚糖过程中温度、pH值、时间、酶浓度、底物浓度以及金属离子对降解速度的影响.比较合适的降解条件是:温度45℃,pH值5.0,适当增大酶浓度和底物浓度能够加速壳聚糖的降解,在一定的底物浓度下溶菌酶降解壳聚糖的反应不遵循简单的一级反应动力学.一定浓度的Cu2+、Zn2+、Ba2+、Cr3+能够促进壳聚糖在溶菌酶作用下的降解,而K+、Ca2+和高浓度的Zn2+会抑制酶活力的发挥.