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121.
Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline. 下载免费PDF全文
T. M. Gray E. J. Arnoys S. Blankespoor T. Born R. Jagar R. Everman D. Plowman A. Stair D. Zhang 《Protein science : a publication of the Protein Society》1996,5(4):742-751
A class of temperature-sensitive (ts) mutants of T4 lysozyme with reduced activity at 30 degrees C and no activity at 43 degrees C has been selected. These mutants, designated "tight" ts mutants, differ from most other T4 lysozyme mutants that are active at 43 degrees C, but only manifest their ts lesion by a reduced halo size around phage plaques after exposure of the growth plates to chloroform vapors. For example, in the series of T4 lysozyme mutants at position 157, the original randomly selected mutant, T1571, is the least stable of the series, yet, apart from the halo assay and subsequent in vitro protein stability measurements, this mutant is indistinguishable from wild type (WT) even at 43 degrees C. Two mutants were identified: L91P and L66P. Both insert proline residues into alpha-helical regions of the WT protein structure. The stabilities (delta delta G) as determined by urea denaturation are 8.2 kcal/mol for L91P and 7.1 kcal/mol for L66P. CD spectra indicate that no major conformational changes have occurred in the mutant structures. The structures of the mutants were modeled with a 40-ps molecular dynamics simulation using explicit solvent. For L91P, the reduction of stability appears to be due to an unsatisfied hydrogen bond in the alpha-helix and to a new buried cavity. For L66P, the reduction of stability appears to be due to a disruption of the interdomain alpha-helix, at least two unsatisfied hydrogen bonds, and a newly formed solvent-filled pocket that protrudes into the hydrophobic core, possibly reducing the stabilizing contribution of a partially buried intrachain salt bridge. 相似文献
122.
The proteins encoded by the rbs operon of Escherichia coli: II. Use of chimeric protein constructs to isolate and characterize RbsC. 总被引:2,自引:1,他引:1 下载免费PDF全文
J. Zaitseva H. Zhang R. A. Binnie M. Hermodson 《Protein science : a publication of the Protein Society》1996,5(6):1100-1107
Chimeric genes encoding full-length copies of rbsA and rbsC connected by segments coding for short bridge peptides were constructed and expressed in Escherichia coli. Surprisingly, the chimeric genes complemented the strain in which rbsA and rbsC were deleted. The chimeric proteins were overproduced, and the products were purified by affinity chromatography. In order to obtain highly purified protein, a poly-His leader peptide was incorporated so that Ni-chelate affinity chromatography could be employed. The leader peptide and the bridge peptide were designed with factor Xa-cleavable sites to permit recovery of the individual RbsA and RbsC protein. A rbsC gene encoding a poly-His leader was also constructed and expressed. Both the chimeric RbsA-C species and the poly-HisRbsC were produced at levels that permitted isolation of the equivalent of milligram quantities of RbsC per liter of culture. This is a substantial increase in amounts from any previous RbsC production vectors. All proteins from the rbs operon have now been overproduced and substantially purified. 相似文献
123.
Thermal-induced unfolding domains in aldolase identified by amide hydrogen exchange and mass spectrometry. 总被引:2,自引:2,他引:0 下载免费PDF全文
Amide hydrogen exchange has been measured in short segments of intact rabbit muscle aldolase at temperatures of 14-50 degrees C by the protein fragmentation/mass spectrometry method (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). Deuterium levels in some segments did not change over the temperature range of the measurements, whereas deuterium levels in other segments increased rapidly with temperature. These results demonstrate that the equilibrium constant for local unfolding, Kunf, of some segments increases with temperature in the low temperature range (14-30 degrees C) of this study. Aldolase begins to lose activity at temperatures above 40 degrees C. In the 40-50 degrees C temperature range, Kunf is greater than 10(-4) in some regions and less than 10(-6) in other regions. This wide range of regional stability in the temperature range where aldolase begins to denature is interpreted in terms of cooperative unfolding/folding domains. Regions of highest stability were located along the hydrophobic subunit binding surface. It is proposed that hydrogen exchange might be used to identify unfolding domains in multidomain proteins whose thermodynamic properties have been determined by differential scanning calorimetry. 相似文献
124.
第四纪甲虫的研究在国外已开展多年,形成了一套完整的方法,而在我国几乎为一空白。第四纪甲虫研究方法一般包括如下几个步骤:野外采样,实验室分离,标本保存,标本鉴定,结果分析。第四纪甲虫研究的意义主要表现在如下三个方面:重建古气候、恢复昆虫地理史和第四纪考古。 相似文献
125.
DISCOVERY OF VERTEBRATE FOSSILS AND PALEOLITHIC ARTIFACTS IN DANJIANG SUBMERGING AREA AND ITS IMPLICATIONS 总被引:1,自引:1,他引:0
IntheareatobefloodedinthesecondengineeringstagefortheDanjiangreservoir,wediscovered16vertebratefossillocalitiesand52Paleolithicsitesin1994,andcollected603artifactsandmanyfossils.Ofthel6new1yfoundvertebratefossillocalities,threearereptilesitesrepresentedbydinosaureggsandlimbbones,andl3producemamma1s,including4Pale0gene,2Ne0geneand7Quaternarysites'ThemammalianlocalitiesareofPale0cene,Eocene,EarlyMiocene,andPli0ceneorEarlyPleistocene,MiddleandIntePleistocene,respectively.Theyfillinsomestrat… 相似文献
126.
通过与事国春杂交,利用杂交后代F2和回交后代BC1P1及BC2P2,研究了三个小麦新矮杆品系和矮生性遗传特性。结果表明,0004的矮生性受一对部分显性矮杆基因控制,5746和7539-各受两对部分显性矮杆基因控制。 相似文献
127.
为了阻断家蚕核多角体病毒(BmNPV)的基因表达,以BmNPV的即刻早期蛋白基因(IE)为靶序列,设计了三联ribozyme.体外切割反应表明,该ribozyme能特异地切割靶序列的mRNA;细胞实验表明,细胞中表达的ribozyme也能够特异地切割靶序列,从而使受BmNPV感染的Bm-N细胞中的多角体减少约30%. 相似文献
128.
本文从矩阵的加号逆理论出发,根据求矛盾线性方程组最佳逼近解的方法,建立起一种新的参数估计方法,同时给出了显著性检验方法,这种方法更简单更精确. 相似文献
129.
高维非自治系统的周期解 总被引:1,自引:0,他引:1
本文通过建立并利用齐次线性方程解的估计公式,获得了周期系统(1)的周期解的存在性、唯一性定理,对周期系统(2)给出了一个平稳振荡定理,最后给出了实例。 相似文献
130.