多肽片段连接方法及肽硫酯和肽醛的合成*

本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法.     

化学裂解结合生物质谱对多肽二硫键的定位

二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键. 当多肽中存在多个半胱氨酸时, 形成的二硫键可能会存在多种配对方式. 快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要. 本文开发了一种基于化学裂解和生物质谱的新方法, 对利那洛肽中3对二硫键进行了精准定位. 通过解析裂解后特异肽段的二级质谱图, 确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6, Cys2-Cys10和Cys5-Cys13. 该方法为二硫键的定位研究提供了新思路.     

蜂毒肽C末端片段的反序肽的抗菌活性和溶血活性

设计并合成了具有不同碱性氨基酸残基数和不同疏水性片段链长的基于Mel(12~26)的系列反序肽类似物.结果表明,反序肽的正电荷和疏水性对于抑菌活性都很重要,N端至少保留3个碱性氨基酸(正电荷>4)和C端的疏水性片段的链长至少为8个氨基酸残基的类似物具有较高的抑菌活性,具有较大的抑菌活性的最小反序肽类似物为具有11个氨基酸残基的RetroMel(13~23).这些反序肽的溶血活性都很小.     

花粉多肽对钙调素的拮抗作用研究

研究了在Ca²⁺存在下荞麦花粉肽及其类似物和丹磺酰标记的钙调素(D-CaM)的相互作用,结果表明,除肽BP-1外,都能与D-CaM结合而形成复合物。利用荧光光谱法测定了这些复合物的解离常数K_d.在所研究的多肽中以BP-13拮抗CaM作用最强,其K_d值为4.6×10^-2μmol/L,抑制钙依赖性磷酸二酯酶活性的IC₅₀为2.2μmol/L.我们还发现,当D-丙氨酸残基取代没有亲和性的BP-1中甘氨酸残基时,其亲和性明显提高。     

尿激酶对纤维蛋白低亲和性的结构基础——Ⅱ.尿激酶A链149—157片段对纤维蛋白促进tPA激活纤溶酶原的抑制作用

本文从低分子量尿激酶(LUK)中分离并纯化了UK 135—157片段,用放射结合分析证明UK 135—157片段和纤维蛋白对tPA的结合呈竞争关系。用酪蛋白降解系统证明此片段可抑制纤维蛋白促进tPA对纤溶酶原的激活。化学合成了UK149—157九肽(R-肽)以及由Asp取代Arg 154和Arg 156的相应九肽(D-肽),发现R-肽对纤维蛋白促进tPA激活纤溶酶原有显著的抑制作用,而D-肽却全无作用。本文结果证明:UK 149—157片段中的正电荷残基在抑制环饼结构域的纤维蛋白结合活性中起重要作用。     

规模化蛋白质生物质谱鉴定中肽段氨基端环化修饰现象

对蛋白质样品制备中引入的氨基酸残基的一种现象--蛋白质酶切肽段氨基端的环化修饰现象的初步研究结果显示,很多以谷氨酰胺(Q)或氨乙酰化修饰的半胱氨酸(CAM_C)残基起始的肽段会发生氨基端的环化修饰,且修饰反应不完全,在同一样本中修饰与非修饰两种状态常同时存在,并且环化修饰后的肽段的反相色谱保留时间发生延迟.在数据库检索时添加环化修饰,可以提高蛋白质的鉴定成功率.本研究结果为大规模的蛋白质质谱数据解析提供了有价值的参考.     

蜂毒肽类似物的合成和生物活性研究

Melittin(GIGAVLKVLTTGLPALISWIKRKRQQ-NH₂)是蜂毒中含26个氨基酸残基的多肽,具有抗菌和溶血等生物活性,是典型的阳离子抗菌肽.本文设计合成了蜂毒肽C端15残基肽片段(GLPALISWIKRKRQQ-NH₂)及其类似物(15残基).研究了Melittin及这些合成肽的抗菌活性、溶血活性、疏水性及二级结构.结果表明,合成的类似物的溶血活性明显降低,抗菌活性基本保留,且与其疏水性相关.类似物中与碱性氨基酸簇(KRKR)距离较远的残基的疏水性对其抗菌活性有较大的贡献.多肽溶血与抗菌机理不同.类似物的抗菌活性和溶血活性与其二级结构(α-螺旋结构)没有明显的相关性.     

蛋白质表面模块划分及其在结合位点预测中的应用

蛋白质-蛋白质复合物的结合位点预测是计算分子生物学的一个难题. 本文对蛋白质-蛋白质复合物数据集Benchmark 3.0 中的双链蛋白质复合物进行了研究, 计算了单体的残基溶剂可接近表面积和残基间的接触面积, 并据此提出了蛋白质表面模块划分方法. 发现模块的溶剂可接近表面积与其内部接触面积的乘积(PSAIA)值能够提供结合位点的信息. 在78 个双链蛋白质复合物中, 有74 个体系其受体或配体上具有最大或次大PSAIA值的模块是界面模块. 将该方法获得的结合位点信息应用在CAPRI竞赛Target 39 的复合物结构预测中取得了较好的结果. 本文提出的基于模块的蛋白质结合位点预测方法不同于以残基为基础且仅考虑表面残基的传统预测方法, 为蛋白质-蛋白质复合物结合位点预测提供了新思路.     

蛋白质的化学全合成

含有非天然氨基酸的蛋白质（如翻译后修饰蛋白质、修饰有探针分子的蛋白质等）是化学生物学中重要的生理活性分子。这些分子难以通过生物表达来获取，而必须使用化学方法来合成。半胱氨酸肽片段连接方法是目前应用于蛋白质化学全合成中的一种重要方法，该方法能够在温和的水溶液中高效地实现肽片段的连接，从而生成天然或者非天然的蛋白质。本文系统地综述了半胱氨酸肽片段连接方法的基本原理，详细讨论了近年来人们对该方法的一些重要改进。最后又介绍了该方法在几类重要的蛋白质分子合成中的代表性应用。     

SARS病毒E蛋白的计算机模拟的初步研究

以粗粒化的多肽链模型进行了SARS病毒包膜中E蛋白的计算机模拟,描述了该蛋白质空间构象的概貌.首先扩展了多肽链的HP模型,使之能够用于研究在水或脂环境下蛋白质折叠的行为,并且考虑了全部氨基酸残基疏水相互作用能的差异.相关格子链的MonteCarlo模拟显示了很高的计算效率.模拟再现了蛋白质的coil-globule转变,验证了蛋白质序列分布的重要性.结果表明,在水环境中,E蛋白质空间结构由紧致的疏水内核和部分向外延伸的亲水片段组成;在脂环境中,中部疏水片段会成为向外延伸的环,而当两侧紧致的亲水片段分开时,则形成桥.     

Efficient estimation of binding free energies between peptides and an MHC class II molecule using coarse‐grained molecular dynamics simulations with a weighted histogram analysis method

We estimate the binding free energy between peptides and an MHC class II molecule using molecular dynamics (MD) simulations with the weighted histogram analysis method (WHAM). We show that, owing to its more thorough sampling in the available computational time, the binding free energy obtained by pulling the whole peptide using a coarse‐grained (CG) force field (MARTINI) is less prone to significant error induced by inadequate‐sampling than using an atomistic force field (AMBER). We further demonstrate that using CG MD to pull 3–4 residue peptide segments while leaving the remaining peptide segments in the binding groove and adding up the binding free energies of all peptide segments gives robust binding free energy estimations, which are in good agreement with the experimentally measured binding affinities for the peptide sequences studied. Our approach thus provides a promising and computationally efficient way to rapidly and reliably estimate the binding free energy between an arbitrary peptide and an MHC class II molecule. © 2017 Wiley Periodicals, Inc.     

Aromatic Radical Anions in Neat Aromatic Hydrocarbons as Solvents. Direct evidence of through space spin-density transfer to the ligand of the counter ion

Contact ion pairs of aromatic radical anions, with a crown ether complex of potassium as cation in a neat aromatic hydrocarbon, can be obtained by reducing the aromatic hydrocarbon in which a small amount of crown ether is dissolved. The unpaired electron stays attached to one aromatic molecule during a time interval which is long on the ESR. time scale. The radicals are stabilized by ion-pair formation in the low polarity solvent. As a consequence of this stabilization, radicals of compounds with low electron affinities, e.g. mesitylene, can be prepared. Mesitylene, m-xylene, and toluene show additional hyperfine splitting in the ESR. spectra of their anion radical pairs of the order of 18 μT. The proton ENDOR. spectra have signals at the corresponding frequencies, indicating a hyperfine coupling with protons of the crown ether ligand. Using mixtures of two aromatic compounds, their relative electron affinities can be determined by studying the temperature dependence of the radical concentrations.     

Specific Purine‐Purine Base Pairing in Linear Alanyl‐Peptide Nucleic Acids

Purine‐purine base pairing with guanine, isoguanine, 2,6‐diaminopurine, and xanthine is investigated within the topology of alanyl‐PNA. Alanyl‐PNA is based on a regular peptide backbone with alternating configuration of the amino acids. The nucleobases are covalently linked as side chains. Their distance in peptides with β‐sheet conformation is similar to the favored base‐pair stacking distance. Therefore, alanyl‐PNA provides self‐pairing linear double‐strands. The linear double‐strand topology does not restrict base‐pair size and geometry. The favored base pairs are formed mostly dependent on recognition by H‐bonding. The synthesis of the nucleo‐amino acids with unnatural nucleobases and their oligomerization is described. Hexamers and a tetramer based on 2,6‐diaminopurine‐xanthine and guanine‐isoguanine base pairs were observed with very high stabilities. For xanthine‐xanthine self‐pairing, an unusual tridentate reverse Watson‐Crick pairing mode is indicated, that is only possible with xanthines pairing in different tautomeric forms. To investigate the nature of xanthine‐xanthine base pairs in more detail, quantum‐chemical calculations were performed. They establish the easier tautomerization of xanthine compared to uracil and indicate that, in the AO basis‐set limit, the tridentate pairing mode with mixed tautomers is favored.     

DFT investigation of dehalogenation of adenine-halouracil base pairs upon low-energy electron attachment

The energetics of the dehalogenation of adenine-halouracil base pairs (A5XU), upon attachment of low-energy electrons, was investigated by use of density functional theory. These results are compared to those of single halouracils reported previously [J. Phys. Chem. A 2002, 106, 11248-11253]. Using the B3LYP functionals it was found that the gas phase adiabatic electron affinities (EA) of halogenated base pairs (A5BrU 0.59, A5ClU 0.56, A5FU 0.47 eV) are higher than that of AU (0.32 eV) and are slightly higher or comparable to the other DNA abundant base pair, guanine-cytosine (0.49 eV). Base pairing with adenine slightly decreases the EA of the halouracils, in contrast to the substantial increase in EA on base pairing of natural bases; as a result, the probability of electron capture by halouracils when in double-stranded DNA is suggested to be substantially reduced relative to that in single-stranded DNA. Even though the activation barriers for dehalogenation are small for both BrU-A and ClU-A, only the former has negative values of both DeltaH (-0.95 kcal/mol) and DeltaG (-1.52), while the latter has negative DeltaG (-0.28) but positive DeltaH (1.27). Infinite separations into halogen anions plus the remaining A-U-5-yl neutral radical are energetically unfavorable owing to sizable halide ion, radical interactions as reported earlier for non base paired halouracils. It is found that base pairing does not change the reactive nature of the uracil-5-yl radical. The results suggest that the radiosensitization properties of halouracils should be less effective in double-stranded DNA than in single-stranded DNA.     

Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies

The base pairing patterns in RNA structures are more versatile and completely different as compared to DNA. We present here results of ab-initio studies of structures and interaction energies of eight selected RNA base pairs reported in literature. Interaction energies, including BSSE correction, of hydrogen added crystal geometries of base pairs have been calculated at the HF/6-31G** level. The structures and interaction energies of the base pairs in the crystal geometry are compared with those obtained after optimization of the base pairs. We find that the base pairs become more planar on full optimization. No change in the hydrogen bonding pattern is seen. It is expected that the inclusion of appropriate considerations of many of these aspects of RNA base pairing would significantly improve the accuracy of RNA secondary structure prediction.     

Hydrophobic, Non-Hydrogen-Bonding Bases and Base Pairs in DNA

We report the properties of hydrophobic isosteres of pyrimidines and purines in synthetic DNA duplexes. Phenyl nucleosides 1 and 2 are nonpolar isosteres of the natural thymidine nucleoside, and indole nucleoside 3 is an analog of the complementary purine 2-aminodeoxyadenosine. The nucleosides were incorporated into synthetic oligodeoxynucleotides and were paired against each other and against the natural bases. Thermal denaturation experiments were used to measure the stabilities of the duplexes at neutral pH. It is found that the hydrophobic base analogs are nonselective in pairing with the four natural bases but selective for pairing with each other rather than with the natural bases. For example, compound 2 selectively pairs with itself rather than with A, T, G, or C; the magnitude of this selectivity is found to be 6.5-9.3 °C in Tm or 1.5-1.8 kcal/mol in free energy (25 °C). All possible hydrophobic pairing combinations of 1, 2, and 3 were examined. Results show that the pairing affinity depends on the nature of the pairs and on position in the duplex. The highest affinity pairs are found to be the 1-1 and 2-2 self-pairs and the 1-2 heteropair. The best stabilization occurs when the pairs are placed at the ends of duplexes rather than internally; the internal pairs may be destabilized by imperfect steric mimicry which leads to non-ideal duplex structure. In some cases the hydrophobic pairs are significantly stabilizing to the DNA duplex; for example, when situated at the end of a duplex, the 1-1 pair is more stabilizing than a T-A pair. When situated internally, the affinity of the 1-1 pair is the same as, or slightly better than, the analogous T-T mismatch pair, which is known to have two hydrogen bonds. The studies raise the possibility that hydrogen bonds may not always be required for the formation of stable duplex DNA-like structure. In addition, the results point out the importance of solvation and desolvation in natural base pairing, and lend new support to the idea that hydrogen bonds in DNA may be more important for specificity of pairing than for affinity. Finally, the study raises the possibility of using these or related base pairs to expand the genetic code beyond the natural A-T and G-C pairs.     

无规共聚物熔体结晶的计算机模拟

1956年Flory基于Onsager的思想提出了高分子有序化的"堆积原理",即由于链的非柔顺性导致链构象的空间各向异性,只有有序化排列才能在有限的空间中放入大量的各向异性分子而不必改变其构象,由此发展的平均场格子理论被向列型有序的MonteCarlo模拟所证明。     

The sodium ion affinities of simple Di-, Tri-, and tetrapeptides

The sodium ion affinities (binding energies) of nineteen peptides containing 2-4 residues have been determined by experimental and computational approaches. Na(+)-bound heterodimers with amino acid and peptide ligands (Pep(1), Pep(2)) were produced by electrospray ionization. The dissociations of these Pep(1)-Na(+)-Pep(2) ions to Pep(1)-Na(+) and Pep(2)-Na(+) were examined by collisionally activated dissociation to construct a ladder of relative affinities via the kinetic method. The accuracy of this ladder was subsequently ascertained by experiments using several excitation energies for four peptide pairs. The relative scale was converted to absolute affinities by anchoring the relative values to the known Na(+) affinity of GlyGly. The Na(+) affinities of AlaAla, HisGly, GlyHis, GlyGlyGly, AlaAlaAla, GlyGlyGlyGly, and AlaAlaAlaAla were also calculated at the MP2(full)/6-311 + G(2d,2p) level of ab initio theory using geometries that were optimized at the MP2(full)/6-31G(d) level for AlaAla or HF/6-31G(d) level for the other peptides; the resulting values agree well with experimental Na(+) affinities. Increasing the peptide size is found to dramatically augment the Na(+) binding energy. The calculations show that in nearly all cases, all available carbonyl oxygens are sodium binding sites in the most stable structures. Whenever side chains are available, as in HisGly and GlyHis, specific additional binding sites are provided to the cation. Oligoglycines and oligoalanines have similar binding modes for the di- and tripeptides, but differ significantly for the tetrapeptides: while the lowest energy structure of GlyGlyGlyGly-Na(+) has the peptide folded around the ion with all four carbonyl oxygens in close contact with Na(+), that of AlaAlaAlaAla-Na(+) involves a pseudo-cyclic peptide in which the C and N termini interact via hydrogen bonding, while Na(+) sits on top of the oxygens of three nearly parallel C=O bonds.     

Quantitative characterization of ion pairing and cluster formation in strong 1:1 electrolytes

Aqueous solutions of 1:1 strong electrolytes are considered to be the prototype for complete ionic dissociation. Nonetheless, clustering of strong 1:1 electrolytes has been widely reported in all atom molecular dynamics simulations, and their presence is indirectly implicated in a diverse range of experimental results. Is there a physical basis for nonidealities such as ion pairing and cluster formation in aqueous solutions of strong 1:1 electrolytes? We attempt to answer this question by direct comparison of results from detailed molecular dynamics simulations to experimentally observed properties of 1:1 electrolytes. We report the analysis of a series of lengthy molecular dynamics simulations of alkali-halide solutions carried out over a wide range of physiologically relevant concentrations using explicit representations of water molecules. We find evidence for pronounced nonideal behavior of ions at all concentrations in the form of ion pairs and clusters which are in rapid equilibrium with dissociated ions. The phenomenology for ion pairing seen in these simulations is congruent with the multistep scheme proposed by Eigen and Tamm based on data from ultrasonic absorption experiments. For a given electrolyte, we show that the dependence of cluster populations on concentration can be described through a single set of equilibrium constants. We assess the accuracy of calculated ion pairing constants by favorable comparison to estimates obtained by Fuoss and co-workers and based on conductometric experiments. Ion pairs and clusters form on length scales where the size of individual water molecules is as important as the hard core radius of ions. Ion pairing results as a balance between the favorable Coulomb interactions and the unfavorable partial desolvation of ions needed to form a pair.