小角X光散射在蛋白质及其复合物领域的研究进展

小角X射线散射(SAXS)是能够表征多种形态的样品,解析从原子到几百纳米尺度上微结构的一种重要技术手段.蛋白质复合物正是在这一空间尺度范围表现出了丰富的生理活性.近年来基于分子结构药物设计的飞速发展,极大地促进了SAXS在研究蛋白质复合物中的应用.本文简要介绍了SAXS解析结构的原理、仪器设备和数据采集分析方法.围绕蛋白质复合物的两方面研究热点,溶液中的蛋白构象以及蛋白质复合物中的结构和相互作用解析,对SAXS在该领域的研究进展和一般分析方法进行了综述.     

酶-配体复合物亲和性的计算

描述了一种新的计算酶－配体复合物亲和性的方法．它考虑了酶－配体结合过程中自由能变化的各主要因素，并利用经验公式加以计算、从蛋白质结构数据库中选取了66个酶－配体复合物作为训练集，利用回归分析得出最后的模型．此模型通用于各种类型的酶－配体复合物，计算结果比技准确，预测算合物解离常数的平均偏差小于一个数量级．此方法还可以定量评价配体分子中每个部分对结合过程的贡献大小，可以为先导他合物的优化提供非常直接的信息     

基于可断裂三功能探针的酪氨酸磷酸化蛋白质复合物的规模化分析

在细胞的信号转导过程中,磷酸化酪氨酸（Phosphotyrosine, pTyr）信号在许多通路中起着重要的调控作用。受pTyr信号调控的蛋白质复合物是信号传递早期过程中的关键分子机器,分析该类动态蛋白质复合物具有重要的意义。Photo-pTyr-scaffold是一种结合了化学探针与pTyr识别蛋白质结构域的化学蛋白质组学分析策略,采用该策略已经实现了对表皮生长因子受体（Epidermal growth factor receptor, EGFR）通路相关的pTyr信号蛋白质复合物的规模化分析。但是,该方法无法解析所鉴定复合物的一一对应关系,增加了后续复合物解析的复杂性。本研究在Photo-pTyr-scaffold探针设计的基础上,将可断裂基团引入至其生物素富集端,设计合成了新型的可断裂三功能探针,以实现对富集产物的可控洗脱。基于pTyr信号蛋白质复合物在化学交联后的分子量差异,利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳实现了对洗脱的pTyr蛋白质复合物的分离,进一步将不同分子量区域的蛋白质进行胶内酶解和质谱分析。理论上,发生化学交联的pTyr蛋白质复合物将在高于原蛋白质分子量的区域被...     

单分子水平的酶催化与基因表达研究

近半个多世纪以来生命科学取得了非凡的进展, 从DNA双螺旋结构的提出, 到第一个酶晶体结构的被解析, 都得益于像X射线衍射、核磁共振、质谱这样的物理化学工具的发展. 如今, 在深入细致地定量研究生物活体系统中我们正面临新的挑战, 例如:了解酶及其他大分子复合物在体内是如何实时工作的, 它们在分子数很少时是怎样工作的, 在活细胞中大分子复合物是如何协调工作的, 以及不同的基因在活细胞中分子数很少的情况下是如何实现表达和不表达的等等. 近十多年来, 单分子成像, 超高分辨率显微镜和单分子操纵技术在世界范围内被广泛地运用于生物医学研究, 对生物化学和分子生物学的发展产生着深远的影响, 因为运用这些单分子、超高分辨技术, 使很多如上述的令人感兴趣的生物学问题实现了单分子层面上的研究和理解. 本文拟就近年来相关的物理化学方法特别是单分子方法和技术在生物医学中的应用做一个简要介绍.     

电喷雾质谱的非共价键蛋白质复合物研究

电喷雾质谱(ESI-MS)已经成为检测和研究生物分子弱相互作用,即非共价键作用的一个重要分析手段.ESI-MS除了具有快速、灵敏、专属的特点以外,还有能够直接得出复合物的分子量和化学计量比的优点.本文通过蛋白质与蛋白质、配体、金属离子的非共价复合物的例子阐述了ESI-MS技术的主要特性,综述了ESI-MS在非共价键蛋白质复合物方面的早期和近期应用研究成果.引用文献34篇.     

溶解性可调节的酶载体制备和固定化酶的研究

本文利用自由基沉淀聚合反应,合成了甲基丙烯酸-丙烯酰胺-顺丁烯二酸酐三元共聚物,测定了这些共聚物形成水不溶性的大分子氢键复合物的临界pH值.利用共聚物上的酸酐基团,直接进行了木瓜蛋白酶的固定化,得到了具有液相酶与固相酶两者优点的新型修饰酶。     

交联酶晶体催化剂

交联酶晶体是近年发展起来的新型晶体催化剂，即具有酶的一般特性，催化活性和选择性高，在温和条件下反应等，又具有非均相化学催化剂的操作稳定性高，易回收利用的特点。ＣＬＥＣＳ技术是将酶结晶技术和化学交联技术结合，提高酶抵抗极端条件以及有机溶剂中的稳定性，除了广泛应用于有机合成中，还适合于各种各样的蛋白质及其他领域，如酶传感器，化妆品和洗涤剂等即需要高稳定性和高活性蛋白质的领域。     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

以蛋白质为基础模拟谷胱甘肽过氧化物酶

硒元素作为一种生命体中必须的微量元素,与人类的健康和疾病息息相关.硒元素主要以硒代半胱氨酸的形式存在于至少25种硒蛋白中,执行着多种生物功能.在这20多种硒蛋白中,谷胱甘肽过氧化物酶(GPx)作为一种主要的抗氧化酶,能够有效地利用谷胱甘肽还原氢过氧化物以防止机体的氧化损伤.这里,我们主要介绍以蛋白质为骨架构筑GPx模拟物的一些策略和方法,以期望于能够更好的理解硒蛋白的生物学性质,甚至开拓更为有效的技术去模拟这种抗氧化酶.     

纳米粒子表面蛋白构象的交联质谱原位蛋白质组表征

采用基于化学交联质谱(Chemical cross-linking mass spectrometry,CXMS)的蛋白质组学方法研究了四氧化三铁(Fe3O4)纳米粒子表面牛血清白蛋白(BSA)的构象变化,考察了纳米粒子的曲率半径和蛋白孵育浓度与Fe_3O_4纳米粒子表面BSA构象的关系。CXMS结合傅里叶变换衰减全反射红外光谱(Attenuated total reflectance Fourier transform infrared spectroscopy,ATR-FTIR)的蛋白质二级结构信息表明,BSA在Fe_3O_4纳米粒子表面存在构象变化和排列取向现象,反映在BSA接触材料后一些位点交联频率的改变。此纳米粒子表面蛋白的CXMS表征方法简单、快速,可提供蛋白在纳米材料上结构变化的化学交联的束缚特征,有望进一步应用于复杂蛋白体系与材料的相互作用研究。     

A Glycosidic-Bond-Based Mass-Spectrometry-Cleavable Cross-linker Enables In Vivo Cross-linking for Protein Complex Analysis

Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful technology to analyze protein complexes. However, the progress of in vivo CXMS studies has been limited by cross-linking biocompatibility and data analysis. Herein, a glycosidic bond-based MS-cleavable cross-linker of trehalose disuccinimidyl ester (TDS) was designed and synthesized, which was fragmented in MS under CID/HCD to simplify the cross-linked peptides into conventional single peptides via selective cleavage between glycosidic and peptide bonds under individual MS collision energy. Consequently, the cross-linking identification accuracy and throughput were significantly enhanced, and the popular MS mode of stepped HCD was allowed. In addition, TDS showed proper cell-penetrating properties while being highly water-soluble, making it non-DMSO dependent during solubilization. Collectively, TDS provides a promising toolkit for CXMS characterization of living systems with high biocompatibility and accuracy.     

A Negative Ion Mass Spectrometry Approach to Identify Cross-Linked Peptides Utilizing Characteristic Disulfide Fragmentations

Chemical cross-linking combined with mass spectrometry (MS) is an analytical tool used to elucidate the topologies of proteins and protein complexes. However, identification of the low abundance cross-linked peptides and modification sites amongst a large quantity of proteolytic fragments remains challenging. In this work, we present a strategy to identify cross-linked peptides by negative ion MS for the first time. This approach is based around the facile cleavages of disulfide bonds in the negative mode, and allows identification of cross-linked products based on their characteristic fragmentations. MS(3) analysis of the cross-linked peptides allows for their sequencing and identification, with residue specific location of cross-linking sites. We demonstrate the applicability of the commercially available cystine based cross-linking reagent dithiobis(succinimidyl) propionate (DSP) and identify cross-linked peptides from ubiquitin. In each instance, the characteristic fragmentation behavior of the cross-linked species is described. The data presented here indicate that this negative ion approach may be a useful tool to characterize the structures of proteins and protein complexes, and provides the basis for the development of high throughput negative ion MS chemical cross-linking strategies.     

Probing the Binding Region of the Single-Stranded DNA-Binding Domain of Rat DNA Polymerase β Using Nanosecond-Pulse Laser-Induced Cross-Linking and Mass Spectrometry

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase β (pol β-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorp-tion/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol β-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.     

Unique Fragmentation of Singly Charged DEST Cross-Linked Peptides

It has previously been shown that when cross-linking reagent diethyl suberthioimidate (DEST) reacts with primary amines of proteins to yield amidinated residues, the primary amines retain their high basicity, and cross-linked species can be enriched by strong cation exchange. It is now demonstrated that collisional activation of singly-charged DEST cross-linked peptide ions leads to preferential cleavage at the cross-linked sites. The resulting product ions facilitate the detection and identification of cross-linked peptides.     

Characterization of a tryptic digest by high-performance displacement chromatography and mass spectrometry.

High-performance displacement chromatography (HPDC) provides a means of increasing the capacity of a chromatographic column, while maintaining the resolution afforded by high-performance liquid chromatographic (HPLC) instruments. The high capacity and high resolution of HPDC can be exploited in tryptic mapping to facilitate the characterization of a protein preparation. In this manner, minor constituents of the mixture, which may be difficult to isolate by conventional chromatographic methods, can be obtained in sufficient amounts to permit chemical characterization by established techniques. The isolation by HPDC of peptides obtained by digestion of recombinant human growth hormone (rhGH) and the subsequent characterization of the peptides are described. The identification of certain of these peptides revealed information on the specificity of trypsin for the substrate, rhGH, and for autolysis. Fractions from the HPDC tryptic map were collected and analyzed by electrospray ionization mass spectrometry (ESI-MS) either directly or following further separation by gradient elution HPLC. Fragment ions observed in the ESI mass spectra facilitated identification of peptides obtained by HPDC tryptic mapping.     

Probing three-dimensional structure of bovine serum albumin by chemical cross-linking and mass spectrometry

Serum albumin is the principal transporter of fatty acids that are otherwise insoluble in circulating plasma. While the crystal structure of human serum albumin (HSA) as well as its binding with fatty acids has been characterized, the three dimensional structure of bovine serum albumin (BSA) has not been determined although both albumins share 76% sequence homology. In this study we used mass spectrometry coupled with chemical cross-linking, to probe the tertiary structure of BSA. BSA was modified with lysine specific cross-linkers, bis(sulfosuccinimidyl) suberate (BS(3)), disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), digested with trypsin and analyzed by tandem mass spectrometry. With O-18 labeling during the digestion, through-space cross-linked peptides were readily identified in mass spectra by a characteristic 8 Da shift. From the cross-linked peptides identified in this study, we found that 12 pairs of lysine residues were separated within 20 A, while 5 pairs were spaced between 20 and 24 A. The spatial distance constraints generated from five K-K pairs in BSA were consistent with the corresponding distance obtained from the crystal structure of HSA, although only six equivalent K-K pairs could be compared. According to our data, the distance between K235 of IIA and K374 of IIB domain in BSA was farther by 7-11 A than that expected from the crystal structure of HSA, suggesting structural differences between BSA and HSA in this region. The distance constraints obtained for lysine residues using various cross-linkers should be valuable in assisting the determination of the 3-D structure of BSA.     

Mapping protein interfaces by a trifunctional cross-linker combined with MALDI-TOF and ESI-FTICR mass spectrometry

Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.     

Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.     

Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry

An analytical strategy for the analysis of antigen epitopes by chemical cross-linking and mass spectrometry is demonstrated. The information of antigen peptides involved in the binding to an antibody can be obtained by monitoring the antigen peptides modified by a partially hydrolyzed cross-linker in the absence and in the presence of an antibody. This approach was shown to be efficient for characterization of the epitope on bovine prion protein bPrP(25-241) specifically recognized by a monoclonal antibody, 3E7 (mAb3E7), with only a small amount of sample (200 picomoles) needed. After cross-linking of the specific immuno complex, a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer equipped with an ion conversion dynode (ICD) high-mass detector was used to optimize the amount of cross-linked complex formed at 202 kDa before proteolytic digestion. To identify the cross-linked peptides after proteolysis without ambiguity, isotope-labeled cross-linkers, disuccinimidyl suberate (DSS-d0/d12) and disuccinimidyl glutarate (DSG-d0/d6), together with high-resolution Fourier transform ion-cyclotron resonance mass spectrometry (FTICR-MS) were used. As a result, a complete fading of the peak intensities corresponding to the peptides representing the epitope was observed when bPrP/mAb3E7 complexes were formed.     

Mass spectrometric identification of formaldehyde-induced peptide modifications under in vivo protein cross-linking conditions

Formaldehyde cross-linking of proteins is emerging as a novel approach to study protein-protein interactions in living cells. It has been shown to be compatible with standard techniques used in functional proteomics such as affinity-based protein enrichment, enzymatic digestion, and mass spectrometric protein identification. So far, the lack of knowledge on formaldehyde-induced protein modifications and suitable mass spectrometric methods for their targeted detection has impeded the identification of the different types of cross-linked peptides in these samples. In particular, it has remained unclear whether in vitro studies that identified a multitude of amino acid residues reacting with formaldehyde over the course of several days are suitable substitutes for the much shorter reaction times of 10-20 min used in cross-linking experiments in living cells. The current study on model peptides identifies amino-termini as well as lysine, tryptophan, and cysteine side chains, i.e. a small subset of those modified after several days, as the major reactive sites under such conditions, and suggests relative position in the peptide sequence as well as sequence microenvironment to be important factors that govern reactivity. Using MALDI-MS, mass increases of 12 Da on amino groups and 30 Da on cysteines were detected as the major reaction products, while peptide fragment ion analysis by tandem mass spectrometry was used to localize the actual modification sites on a peptide. Non-specific cross-linking was absent, and could only be detected with low yield at elevated peptide concentrations. The detailed knowledge on the constraints and products of the formaldehyde reaction with peptides after short incubation times presented in this study is expected to facilitate the targeted mass spectrometric analysis of proteins after in vivo formaldehyde cross-linking.