小角X-射线散射法解析多结构域蛋白质或复合物的溶液结构

随着同步辐射装置的建设与发展及各种建模方法的产生与完善,小角X-射线散射（small angle X-ray scattering,SAXS）法已经逐渐成为结构生物学中的一种重要的工具。SAXS可以用于研究溶液中生物大分子的结构及构象变化,蛋白质的组装、折叠等动态过程。本文对SAXS的基本原理、常用的研究技术和建模方法及其应用进行了综述。     

应用动态光散射对一些蛋白质结晶性能的研究

动态光散射是研究生物大分子溶液物化行为和结晶性能的重要工具。以溶菌酶为模型蛋白 ,对其溶液的DLS研究表明 ,一定范围的蛋白质浓度不会影响蛋白质聚合性质 ,而起沉淀剂作用的盐对多色散性的影响较大。考虑这些研究结果 ,应用DLS研究对空间微重力下晶体生长样品的质量做了考察 ,这对于提高空间样品准备水平 ,进而提高空间实验成功率是一重要途径。     

同步辐射小角散射实验站探测系统的改进

同步辐射小角X射线散射（SAXS）实验站所用的探测器由原来的NaI闪烁计数器改为位置灵敏探测器,其位置分辨好于100 μm,有效探测面积为50 mm×10 mm,得到了较以往更高质量的SAXS谱,并成功地对液体样品的SAXS谱进行了测试.     

同步辐射圆二色谱及其在糖生物学及生物分子中的应用

同步辐射圆二色谱与普通圆二色谱相比,特点在于向真空紫外波段（〈200nm）拓展,以及同步辐射所提供的高强度紫外和真空紫外光源。糖的圆二色谱结构主要在200nm以下。蛋白质和核酸在200nm以下的真空紫外范围,也具有丰富的光谱结构。因此向真空紫外拓展,伴随新的电子跃迁,对应新的光谱结构,包含更丰富的结构信息,确定的结构种类就越多和越精确。同步辐射高强度的真空紫外光源,是获得高质量真空紫外圆二色谱数据的保证,为糖及糖蛋白、蛋白质和核酸研究提供了溶液中结构探测新的实验方法。综述同步辐射圆二色谱特点及其在结构生物学中的应用,以及新发展的蛋白质圆二色谱数据库（PCDDB）。介绍已对外开放的北京同步辐射实验室同步辐射圆二色谱探测,及其在蛋白质、糖和核酸研究中的应用,以及基于微流控混合芯片的亚毫秒动态探测发展。     

食药用真菌: 天然抗氧化剂的重要来源

生物体在新陈代谢过程中不断产生自由基。自由基在机体内的生成和去除通常处于平衡状态,不会对机体造成严重损伤,但当机体内的自由基过剩时,常引起生物大分子如脂类、蛋白质和核酸的氧化损伤,进而引发机体衰老以及癌症、动脉粥样硬化、风湿性关节炎、肺气肿等疾病。过去20年中,自由基在细胞损     

劳埃晶体学时代的到来

近年来,人们对古老的劳埃衍射技术重产生了浓厚的兴趣,这主要是因为世界各地新的同步辐射光源的建成;各种先进的插入件,诸如波形器（wiggler）、波荡器（undulator)的飞速发展;以及利用这类光源通过时间分辨晶体学来研究分子研究动态变化的前景,在过去的十年中,理论研究已经阐明了多波长衍射几何学的特征,在很大幅度上加深了我们对劳埃法的认识。劳埃数据处理方法及其软件开发也因此有所创新。曾在相当长的时间内限制这项技术应用的劳埃数据处理中的大部分问题存在已经得到解决;同步辐射光源,束线光学器件及X射线探测器等方面也都取得了显著的进步。表态劳埃实验得到的结构因子振幅在质量上已同单波长数据相当。晶体中反应易于启动的时间分辨劳埃实验已经开始在一系列生物分子体系中得到成功的实践,由此得到的关于结构动力学的信息是任何其它传统的衍射方法所无能为力的。这些静态的及时间分辨的实验说明了劳埃已经开始走向成熟,并指明了今后发展的方向, 即对晶体的镶嵌度和劳埃衍射点的相应能量宽度的正确处理、扩散散射的考虑,时间分辨实验中低浓度瞬态中间物结构的测定等等。     

试论生物信息流与激光生物效应的相关性--激光生物效应分子机理研究

为了探索激光生物学效应的分子机理,以便更好的掌控和利用激光生物效应,用XeCl（308 nm）准分子紫外激光以相同变化的激光参量直接辐照有生物活性的生物大分子BTG DNA、BSA（v）蛋白质和糖Mannan.实验结果分析告知：电镜法观察到受辐照的三类生物大分子的表观结构、构象（含结构信息）和光谱法（IR、Vis-UV、FR、CD）分析指出生物大分子的内在结构部件的相关的特征峰的峰位、峰值都受影响,其变迁都与激光参量的变化相呼应;与三类生物大分子中分子内、分子间沟通与信息传递相关的氢键、糖苷键等的形成与否的类似或相同的结构部件（如-C-H、-N-H、-CH-OH、-C-O、-C =O等）其特征峰的变迁都更敏感于激光参量的改变.激光辐照生物体时,激光似生物信号分子通过它的能量以粒子性、电磁波相干性影响生物大分子的分子结构、构象（含结构信息）的稳定性、增加分子内、分子间相互沟通、信息传递,亦增加了结构部件的被修饰的可能性.进而影响着生物信息流的流量与流向和细胞信号转导的协同与整体表达,产生相应的生物效应.掌握获得功能生物大分子结构构象信息与使用适宜的激光参量的相关的关系值（阈值）是重要的关键.     

细胞内的大分子拥挤环境

所有的细胞中都存在着大量的蛋白质、核酸、多糖等各种生物大分子,它们大约占用细胞容积的20%～30%,总浓度高达80～200 g/L,因此任何一种大分子都处于一个充满其他大分子的拥挤环境中. 对源于排斥容积效应的拥挤理论分析表明它对所有大分子之间的反应在热力学和动力学上都有很大的影响. 可是以往人们在体外研究生物大分子的性质和相互作用时几乎都忽略了这样一个细胞大分子拥挤的实际环境. 最近几年建议把大分子拥挤与pH、离子强度和溶液组成等一样作为常规因素来研究生物大分子的呼声很高,在体系中添加大分子拥挤试剂以在体外模拟细胞内环境研究蛋白质折叠已有一些实验报道.     

生物大分子溶液三维结构及分子间相互作用的波谱研究

生物大分子溶液三维结构及分子间相互作用的波谱研究吴厚铭（中国科学院上海有机化学研究所生命有机化学国家重点实验室２０００３２）生物活性分子的溶液空间结构及分子间作用（如蛋白质与核酸,信息分子,各种调节剂,拮抗剂,以及核酸与药物分子等相互作用）的研究是发...     

超离心法及其在放射损伤研究中的应用

超离心机是研究高分子化合物如生物大分子蛋白质、核酸和病毒等的一个重要工具。在分析方面主要用以测定这些生物大分子的沉降系数与分子量。在制备方面可用以分离这些生物大分子和有关的亚细胞成分。在研究电离辐射对细胞原发损伤的规律时,观察射线对生物大分子的损伤和亚细胞成分的破坏,及其对生化功能的影响等问题,超离     

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA^val, and yeast tRNA^phe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.     

Specific radiation damage can be used to solve macromolecular crystal structures

The use of third generation synchrotron sources has led to renewed concern about the effect of ionizing radiation on crystalline biological samples. In general, the problem is seen as one to be avoided. However, in this paper, it is shown that, far from being a hindrance to successful structure determination, radiation damage provides an opportunity for phasing macromolecular structures. This is successfully demonstrated for both a protein and an oligonucleotide, by way of which complete models were built automatically. The possibility that, through the exploitation of radiation damage, the phase problem could become less of a barrier to macromolecular crystal structure determination is discussed.     

How does radiation damage in protein crystals depend on X-ray dose?

Is radiation damage to cryopreserved protein crystals strictly proportional to accumulated dose at the high-flux density of beams from undulators at third-generation synchrotron sources? The answer is "yes," for overall damage to several different kinds of protein crystals at flux densities up to 10(15) ph/sec/mm(2) (APS beamline 19-ID). We find that, at 12 keV (1 A wavelength), about ten absorbed photons are sufficient to "kill" a unit cell. As this corresponds to about one elastically scattered photon, each unit cell can contribute only about one photon to total Bragg diffraction. The smallest crystal that can yield a full data set to 3.5 A resolution has a diameter of about 20 microm (100 A unit cell).     

Time-resolved methods in biophysics. 6. Time-resolved Laue crystallography as a tool to investigate photo-activated protein dynamics.

When polychromatic X-rays are shined onto crystalline material, they generate a Laue diffraction pattern. At third generation synchrotron radiation sources, a single X-ray pulse of approximately 100 ps duration is enough to produce interpretable Laue data from biomolecular crystals. Thus, by initiating biological turnover in a crystalline protein, structural changes along the reaction pathway may be filmed by ultra-fast Laue diffraction. Using laser-light as a trigger, transient species in photosensitive macromolecules can be captured at near atomic resolution with sub-nanosecond time-resolution. Such pump-probe Laue experiments have now reached an outstanding level of sophistication and have found a domain of excellence in the investigation of light-sensitive proteins undergoing cyclic photo-reactions and producing stiff crystals. The main theoretical concepts of Laue diffraction and the challenges associated with time-resolved experiments on biological crystals are recalled. The recent advances in the design of experiments are presented in terms of instrumental choices, data collection strategy and data processing, and some of the inherent difficulties of the method are highlighted. The discussion is based on the example of myoglobin, a protein that has traversed the whole history of pump-probe Laue diffraction, and for which a massive amount of data have provided considerable insight into the understanding of protein dynamics.     

Direct comparison of the crystal and solution structure of xylanase from Trichoderma longibrachiatum

The small angle X-ray scattering (SAXS) data of xylanase XYNII (endo-1,4-beta-xylan xylanohydrolase EC 3.2.1.8) from Trichoderma longibrachiatum, an enzyme catalysing the reaction of accidental hydrolysis of beta-1,4-D-xylosidic linkages of xylan, were recorded for protein solution using synchrotron radiation. The experimental data were compared with those of theoretical scattering calculated on the basis of the known crystal structure. The radius of gyration measured by SAXS (RG = 1.7 nm) was about 3.5% larger and the maximum dimension in the distance distribution function about 5 % larger than the corresponding values calculated on the basis of the crystal structure.     

Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubiginosus

Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (R(G)=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.     

Solution studies and structural model of the extracellular domain of the human amyloid precursor protein

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimer's disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.     

Small angle X-ray scattering as a complementary tool for high-throughput structural studies

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples. Small angle X-ray scattering (SAXS) is a solution technique that provides data on the size and multiple conformations of a sample, and can be used to reconstruct a low-resolution molecular envelope of a macromolecule. In this study, SAXS has been used in a high-throughput manner on a subset of 28 proteins, where structural information is available from crystallographic and/or NMR techniques. These crystallographic and NMR structures were used to validate the accuracy of molecular envelopes reconstructed from SAXS data on a statistical level, to compare and highlight complementary structural information that SAXS provides, and to leverage biological information derived by crystallographers and spectroscopists from their structures. All the ab initio molecular envelopes calculated from the SAXS data agree well with the available structural information. SAXS is a powerful albeit low-resolution technique that can provide additional structural information in a high-throughput and complementary manner to improve the functional interpretation of high-resolution structures.     

X-ray fluorescence analysis using synchrotron radiation at the photon factory

X-ray fluorescence experiments at the Photon Factory in Japan are described. An energy-dispersive X-ray fluorescence system was combined with various excitation modes, i.e., a continuum and a monochromatic excitation, which consist of a crystal monochromator or a wide band pass monochromator. These excitation modes provide a wide range of band width and photon flux of excitation beams. Minimum detection limits obtained for a thin sample were less than 0.1 ppm and 0.1 pg when there was no line interference. Advantages of using monochromatic excitation are discussed, with emphasis on the possibility of chemical state analysis. Grazing incidence X-ray fluorescence is a technique very appropriate to synchrotron radiation characteristics. Near-surface analysis and trace analysis of solution samples placed on a total reflection support were made. Future plans are discussed, including microbeam analysis, tomography, X-ray excited optical fluorescence, and applications of insertion devices (undulator and wiggler).