小远志多糖一级结构的初步研究

从药用植物小远志中提取、精制得多糖PP-1,PP-2,采用纸层析、薄层层析和气相色谱等方法确定了多糖中的单糖组成.通过高碘酸氧化、Smith降解和甲基化分析等方法确定了相邻单糖基连接方式,初步推测了两个小远志多糖可能的一级结构,为进一步合成和研究其生物活性提供了数据支持.     

沙冬青抗冻蛋白热滞活性的DSC研究

在某些极地鱼类的血清以及某些昆虫和植物中 ,人们发现了一类特殊的物质 .它们的共同特点是能通过直接与冰晶核相互作用,抑制冰晶核的形成和生长,从而降低溶液冰点 .现已发现的这类抗冻物质都属于蛋白质,被统称为抗冻蛋白（ Antifreeze Proteins, AFPs） .抗冻蛋白能以非依数性形式大大降低溶液的冰点,但对熔点的影响很微弱,而且遵从依数性的原则,使冰点低于平衡态的熔点,溶液处于反常的非平衡相变状态 .这种冰点低于熔点的特性称为热滞 (Hysteresis).因此,抗冻蛋白也叫热滞蛋白或温度迟滞蛋白（ Thermal Hysteresis Proteins, …     

猪精浆中附睾特异性蛋白质的纯化及其一级结构的研究

哺乳动物体中附睾内精子的成熟过程要经过配子融合而形成合子的过程,其中,精子聚合到附睾分泌的特异蛋白质上的过程是合子形成的重要环节,目前已证明附睾中分泌出来的一些特异蛋白质和精子成熟之间的相互关系,但精子动能的获得或与合子的结合能力的研究尚不十分清楚。     

抗冻蛋白抗冻机制的分子模拟研究

抗冻蛋白能使生物体在寒冷环境下生存,具有极大的潜在应用价值。近年来,人们对抗冻蛋白开展了广泛的研究,但其抗冻机理还未明确。本文阐述了抗冻蛋白的功能特性和结构特征,并从结构的角度对其抗冻机制方面的分子模拟研究成果进行了综述。另一方面,对目前已知晶体结构的29个野生型抗冻蛋白的结构特性进行了分析,发现在整个抗冻蛋白表面和在冰结合位点处都存在亲水残基与水形成氢键和疏水残基与类冰结构特异性结合的特点。然后,探讨了抗冻蛋白的二级结构、冰结合位点残基的疏水性与抗冻活性之间的关系。最后,从结构的角度讨论了抗冻蛋白的机制和影响抗冻活性的因素并简要总结了仿生抗冻材料设计和应用的研究进展。     

短小芽孢杆菌碱性蛋白酶的结构研究

测定了短小芽孢杆菌碱性蛋白酶BP的分子量为19 500,分析了它的氨基酸组成。用N-溴代琥珀酰亚胺对酶进行了化学修饰,以紫外光谱法测得此酶含有2个色氨酸残基,其中一个残基为酶表现活性的必需基团,且位于分子表面。用荧光表面猝灭剂法推测另一个残基也位于分子表面或临近分子表面。通过其园二色谱证明此酶为无规则卷曲构象。     

操控冰水:仿生抗冻蛋白研究进展*

抗冻蛋白是自然界娴熟操控(冰)水的分子识别的典范之一。抗冻蛋白的抗冻活性与其特殊结构有着十分密切的关系。作为目前最高效的生物抗冻剂,抗冻蛋白因其含量低、易变性失活,导致产量过低,亟待开发新的来源。近年来,模拟抗冻蛋白的研究工作吸引了科学家们的广泛关注,抗冻蛋白关键的结构特质:氢键作用、疏水性、冰晶吸附、“结构水”在各类仿生抗结冰材料中相继得以体现,对深入理解抗冻蛋白作用机制起到了重要的推动作用。综述了仿生抗冻蛋白在仿生抗结冰材料领域的研究进展,对基于仿生抗冻蛋白的仿生抗结冰材料的发展做出了展望。     

两种昆虫抗冻蛋白抗冻活性差异的分子基础

云杉蚜虫(Choristoneura fumiferana)体内可产生一种抗冻蛋白(AFP), 能够保护其在寒冷的冬季安全越冬, 这类昆虫抗冻蛋白(简称 CfAFP)存在多种异构体, 其中CfAFP-501和CfAFP-337均呈现相似的左手β-螺旋结构. 实验测定, 相比CfAFP-337仅多出两个插入螺旋环的CfAFP-501的抗冻活性竟然是CfAFP-337的3倍左右. 蛋白异构体CfAFP-501显著增强的抗冻活性和它的插入环数不成比例, 当然也不能简单归因于两个插入环所导致的与冰晶作用部位及接触面积的增加. 为了探讨两种昆虫抗冻蛋白异构体抗冻活性差异的分子基础, 深入了解抗冻蛋白作用特点的普遍规律, 分别使用分子力学、分子动力学模拟和量子力学方法来系统研究蛋白及其切割体与冰晶结合的结构特征及相互作用. 结果表明, CfAFP-501中多数螺旋环比CfAFP-337具有更强的冰晶相互作用和破坏冰晶中水分子的成键的能力, 由于螺旋长度增加导致CfAFP-501中各b-螺旋环之间协同效应的增强, 是其具有显著增强的抗冻活性的主要来源. 这种协同作用对具有b-螺旋结构的抗冻蛋白起十分关键的作用和重要贡献.     

IgG1型单克隆抗体对照品的一级结构解析

对单克隆抗体药物对照品(IgG1型)进行了全面的结构表征。采用Exactive plus EMR质谱测定了去N糖前后抗体的精确分子量,并通过计算N糖含量得出其完整分子量为148 285.0～149 020.8,完整去糖后分子量为145 810.4,N糖含量为1.67%～2.15%。优化了全柱成像实时等电聚焦毛细管电泳(WCID-cIEF)检测方法,结果显示该抗体等电点分布在8.21～8.74之间,其中主成分等电点为8.61,相对含量为61.43%。继而使用离子交换色谱检测了电荷异质性,发现碱性峰含量为4.1%,酸性峰含量为23.9%,主峰含量为72.0%,与WCID-cIEF结果吻合,并证明了方法间良好的重现性。通过切糖前后的肽图分析,获得糖肽分布信息,识别出糖修饰位点及重轻链的互补决定区(CDR),甲硫氨酸(M)的氧化位点及氧化率,并通过抗体还原前后的肽图解析出二硫键连接。研究结果同时提示该抗体对照品无C末端糖化修饰现象。     

滴定量热法研究连串一级反应热动力学

本文用滴定量热法分别建立了滴定期和停滴反应期1-1级连串反应热动力学的数学模型。根据这两种模型,由非线性最小二乘法原理直接拟合单次实验结果可同时求得1-1级连串反应的速率常数（ k ₁和 k ₂）和摩尔反应焓。并用滴定量热法研究了丁二酸二乙酯皂化反应的热动力学,实验结果验证了两种数学模型的正确性。     

苏氨酸在昆虫抗冻蛋白抗冻活性中的作用

设计系列昆虫抗冻蛋白CfAFP突变体, 通过分子动力学模拟确定各突变体与冰晶的最佳作用模式, 并用半经验分子轨道方法AM1和PM3研究了其与冰晶的相互作用. 结果表明, TXT面上的苏氨酸在蛋白与冰晶相互识别和结合过程中十分关键, 对CfAFP与冰晶间相互作用的贡献大, 用其它疏水或亲水氨基酸残基替换都将削弱抗冻蛋白与冰晶的相互作用强度, 从而降低蛋白的抗冻活性. 但是, 在维系蛋白和冰晶结构匹配的基础上, 疏水基团的增加加强了抗冻蛋白与冰晶的结合, 从而增加蛋白的抗冻活性.     

The Effects of Different Doses of ROCK Inhibitor,Antifreeze Protein III,and Boron Added to Semen Extender on Semen Freezeability of Ankara Bucks

In the presented study, the effects of ROCK inhibitor Y-27632, antifreeze protein III, and boron at two different doses were investigated on the spermatological parameters of Ankara buck semen after freeze–thawing. Ejaculates were collected from bucks using an electroejaculator during the breeding season. The ejaculates that showed appropriate characteristics were pooled and used in the dilution and freezing of semen. The extender groups were formed by adding two different doses of three different additives (ROCK inhibitor Y-27632, 5 and 20 µM; antifreeze protein III, 1 and 4 µg/mL; boron, 0.25 and 1 mM) to the control extender. The semen was diluted with the different extenders at 35–37 °C and loaded into straws. Sperm samples frozen in liquid nitrogen vapors, following equilibration, were stored in liquid nitrogen. It was observed that extender supplementation improved post-thaw motility of Ankara buck semen after freeze–thawing. Differences were significant (p < 0.01) for 5 and 10 µM doses of ROCK inhibitor (71.82% and 74.04 % motility), as well as for 0.25 and 1 mM doses of boron (76.36% and 72.08% motility), compared to the control group (66.15% motility). With respect to the evaluation of acrosomal integrity and mitochondrial activity after freeze–thawing, although supplementation provided protection at all doses, the efficacy was not statistically significant (p > 0.05). It was observed that DNA damage was improved by antifreeze protein III at 1 µg/mL (1.23% ± 0.23%) and by boron at all doses (0.25 mM: 1.83% and 1 mM: 1.18%) compared to the control group (3.37%) (p < 0.01), following the thawing process. In the present study, it was determined that some additives added to the extender provided significant improvements in buck spermatozoa motility and DNA damage after thawing.     

Differential Scanning Calorimetric and Circular Dichroistic Studies on Plant Antifreeze Proteins

Antifreeze protein (AFP) can lower the freezing point by inhibiting the growth of ice crystals. In this article, the thermal hysteresis activity (THA) of a plant AFP was measured with differential scanning calorimetry (DSC). As is shown, when the amount of ice in the sample was less than 5% THA of this AFP reached as high as 0.35°C. The secondary structure of this AFP was studied with circular dichroism (CD). The CD spectrum from 195to 240 nm indicated a well-defined secondary structure consisting 11% α-helix, 34%antiparallel β-sheet and 55% random coil. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and Primary Structure Determination of a Novel Polypeptide Isolated from Mistletoe Viscum coloratum

A novel polypeptide was isolated from mistletoe Viscum coloratum. The primarystructure of the polypeptide ‘named viscotoxin B2‘ was determined to be KSCCKNTTGRNIYNT CRFAGGSRERCAKLSGCKIISASTCPSDYPK by Edman degradation. Viscotoxin B2 shared highsequence homology with viscotoxins isolated from Viscum album. Pharmacological experimentsshowed that viscotoxin B2 had distinct cytotoxic activity on tumor cells. Viscotoxin B2 could beused as a leading compound in cancer therapy.     

SPOT-Fold: Fragment-Free Protein Structure Prediction Guided by Predicted Backbone Structure and Contact Map

Protein structure determination has long been one of the most challenging problems in molecular biology for the past 60 years. Here we present an ab initio protein tertiary-structure prediction method assisted by predicted contact maps from SPOT-Contact and predicted dihedral angles from SPIDER 3. These predicted properties were then fed to the crystallography and NMR system (CNS) for restrained structure modeling. The resulted structures are first evaluated by the potential energy calculated by CNS, followed by dDFIRE energy function for model selections. The method called SPOT-Fold has been tested on 241 CASP targets between 67 and 670 amino acid residues, 60 randomly selected globular proteins under 100 amino acids. The method has a comparable accuracy to other contact-map-based modeling techniques. © 2019 Wiley Periodicals, Inc.     

Assessment of AI-Based Protein Structure Prediction for the NLRP3 Target

The recent successes of AlphaFold and RoseTTAFold have demonstrated the value of AI methods in highly accurate protein structure prediction. Despite these advances, the role of these methods in the context of small-molecule drug discovery still needs to be thoroughly explored. In this study, we evaluated whether the AI-based models can reliably reproduce the three-dimensional structures of protein–ligand complexes. The structure we chose was NLRP3, a challenging protein target in terms of obtaining a three-dimensional model both experimentally and computationally. The conformation of the binding pockets generated by the AI models was carefully characterized and compared with experimental structures. Further molecular docking results indicated that AI-predicted protein structures combined with molecular dynamics simulations offers a promising approach in small-molecule drug discovery.     

Comparative Analysis of Hydration Layer Reorientation Dynamics of Antifreeze Protein and Protein Cytochrome P450

Antifreeze proteins (AFPs) inhibit ice recrystallization by a mechanism remaining largely elusive. Dynamics of AFPs' hydration water and its involvement in the antifreeze activity have not been identified conclusively. We herein, by simulation and theory, examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm, Choristoneura fumiferana, compared with a protein cytochrome P450 (CYP). The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules. Therefore, the jump reorientation of water molecules around the AFP active region is accelerated. Due to the mutual coupling and excitation of hydrogen bond exchange, with the acceleration of hydrogen bond exchange, the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated. Therefore, the water reorientation dynamics of AFP is faster than that of CYP. The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.