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。     

多肽对钙调素亲和力的预测

根据前文提出的钙调素可结合多肽的钙调素结合部位的模型,并综合钙调素可结合多肽的报道,提出一种简单方法来预测多肽的钙调素结合部位和其复合物的解离常数,研究了多肽的疏水性、形成α螺旋结构的倾向、碱性等因素对解离常数的影响.为了进一步检验模型和预测方法,设计合成了模型肽,合成的模型肽与钙调素的复合物的解离常数的实测值与预测值相符.     

多肽与花粉钙调素的相互作用及对细胞功能的影响

研究了在Ca²⁺存在下,丹磺酰标记的花粉钙调素(D-pCaM)与合成的多肽相互作用的规律.研究结果表明,被研究的绝大多数多肽能与D-pCaM结合而形成复合物,使D-pCaM的荧光光谱发生变化.用荧光法测定了这些复合物的解离常数K_d,其中肽BP-13的Kd值为4.0nmol/L,是与pCaM结合能力最强的一个多肽.除证明了多肽的疏水性,预测二级结构形成倾向和链长对其与pCaM结合能力有影响外,还发现用D-丙氨酸取代肽链中的Gly或L-丙氨酸后,可改变肽对pCaM的亲和性,这为提高多肽与钙调素的亲和力提供了一条改进的途径.我们的研究还可以定性地说明不同来源CaM性质相似又有差异,反映了它们的高度保守性和变异性.同时还展示了所研究的多肽对细胞信息传导过程的影响,为研究CaM对细胞功能调节的作用机制和建立pCaM拮抗肽的活性筛选模型提供了思路.     

多肽及蛋白质的化学合成研究

多肽与蛋白质作为生物体内的活性物质和生命活动的物质基础,在信号传递、能量利用、免疫应答等基础生理过程发挥着至关重要的作用,并与多种疾病的发生密切相关。获得一定数量高纯度的多肽和蛋白质是研究其结构、生物学功能以及开发相关药物的重要前提。天然多肽与蛋白质的来源主要有动植物的组织器官、微生物的次级代谢产物等。目前,自然提取、重组技术和化学合成是多肽与蛋白质的主要获得途径。相较于从天然产物中提取分离和基因重组表达,化学合成能够方便地在多肽与蛋白质的任意位点引入非天然氨基酸或特定类型的翻译后修饰基团,如糖基化、磷酸化、荧光团及光交联反应基团等,极大地促进了多肽与蛋白质在基础医学及生物医药研究领域的应用发展。本综述全面介绍了多肽与蛋白质的各种化学合成研究策略,并讨论了这些策略的基本原理、优缺点及应用价值,旨在为多肽及蛋白质的合成研究提供参考。     

多肽IS4溶液构象的进一步NMR研究

利用二维核磁共振技术对多肽IS4中除Ser-1外的所有残基质子进行了指认.积分NOESY谱中的相关峰可得到距离约束.二面角的约束来自偶合常数³J_NHα.由慢交换质子可得到氢键的约束.在进行距离几何程序计算时利用这些约束可得到一组构象,用能量最优化程序优化后的结果表明,多肽IS4在CF₃CD₂OH中的构象为α-螺旋.     

光谱法研究钙调素与蜂毒肽片断及其类似物的相互作用

设计合成了蜂毒肽片断及其类似物:Mel15,Mel15(8F)和Mel15(7P),这些多肽与钙调素有很强的结合力,而且链段很短,因此它们可作为钙调素可结合蛋白质的结合部位的模型.本文采用光谱法研究了它们与钙调素的相互作用.荧光发射光谱法结果表明,多肽Mel15在与钙调素相互作用时,肽链中的Trp基团的微环境变得更加疏水,说明Mel15中的Trp残基可能与钙调素的疏水性表面靠近.紫外差谱测试表明,只有当钙调素分子结合2个Ca~(2+)后,才可以与多肽Mel15(8F)结合.圆二色谱法研究表明,多肽与钙调素结合后多肽分子和钙调素分子的α-螺旋结构的含量都被诱导而增加,结合力越大,则越多的残基被诱导形成α-螺旋结构.     

寡肽Asterin B和C溶液构象的NMR研究 Ⅱ. NMR及分子动力学模拟

利用NMR和分子动力学方法研究了寡肽Asterin B和C的溶液构象.结果表明,Asterin B在溶液中形成了某种非氢键的转角结构,并由残基间的疏水相互作用使整个分子具有两亲性,这种结构特征可能和其生物活性有关.并进一步讨论了这种结构的形成在蛋白质卷曲的起始过程中的意义.而Asterin C在溶液中柔性较大,存在多种构象的平均.     

新多肽配体GE11与表皮生长因子受体的结合能力分析

应用亲和毛细管电泳（ACE）分析方法,对表皮生长因子受体（EGFR）和新多肽配体GE11之间的结合能力进行分析。结果表明,EGFR与多肽配体GE11之间存在特异性相互作用,考察EGFR在不同浓度GE11溶液中的迁移情况,采用非线性、双倒数、Y-倒数和X-倒数4个数据处理方法得到较好的数据拟合,并测得结合常数。该文为筛选多肽配体以及测定受体与多肽配体之间的结合常数提供了简便的方法,将有力推动肿瘤靶向药物输送的研究。     

缓激肽多肽片段间非共价作用的质谱研究

以缓激肽(R¹P²P³G⁴F⁵S⁶P⁷F⁸R⁹)分子作为研究模型, 用电喷雾质谱研究缓激肽分子碎片片段之间的非共价相互作用, 探讨了影响气相多肽分子构象稳定的氢键作用. 合成了与缓激肽分子在位置1断裂形成的碎片一致的RPPGFS和PFR多肽序列, 与在位置2断裂形成碎片一致的RPPGF和SPFR多肽, 以及N端或者C端去掉精氨酸的相应碎片多肽. 实验结果表明, 上述两个断裂位置产生的碎片多肽分别进行反应后, 都能发生非共价作用. 在断裂方式1下, PFR多肽在去掉C端的精氨酸R后, 与其他大多数多肽不发生非共价结合, 表明PFR中的R在缓激肽气相分子的构象中发挥重要的作用. 而在断裂方式2下, 去掉N端或者C端精氨酸的多肽之间都存在非共价结合, 即C端带有丝氨酸的SPF或SPFR多肽碎片仍然可以与N端碎片发生氢键结合, 表明丝氨酸很可能处于转角的位置. 通过对碰撞诱导解离(CID)的碰撞能量分析, 发现多肽RPPGFS和PFR, 以及多肽RPPGF和SPFR之间氢键结合较强, 而同时去掉N端和C端精氨酸得到的多肽之间的氢键结合较弱. 质谱滴定法定量测得的RPPGFS和PFR的结合常数为3.53×10³, 与RPPGF和SPFR的结合常数(3.16×10³)相接近,它们均大于去除精氨酸的PPGF和SPF的结合常数(1.25×10³). 质谱滴定实验结果进一步确认了碰撞诱导解离的分析结果, 表明缓激肽分子两端的精氨酸之间的氢键作用是气相缓激肽分子构象稳定的重要因素之一.     

Ranking Peptide Binders by Affinity with AlphaFold**

AlphaFold has revolutionized structural biology by predicting highly accurate structures of proteins and their complexes with peptides and other proteins. However, for protein-peptide systems, we are also interested in identifying the highest affinity binder among a set of candidate peptides. We present a novel competitive binding assay using AlphaFold to predict structures of the receptor in the presence of two peptides. For systems in which the individual structures of the peptides are well predicted, the assay captures the higher affinity binder in the bound state, and the other peptide in the unbound form with statistical significance. We test the application on six protein receptors for which we have experimental binding affinities to several peptides. We find that the assay is best suited for identifying medium to strong peptide binders that adopt stable secondary structures upon binding.     

Design of Ligands for Affinity Purification of G-CSF Based on Peptide Ligands Derived from a Peptide Library

Introduction　　 Affinity chromatography is of great interest in pharmaceutical industry as it is simple,fast and is of a high efficiency to purify proteins from a complicated mixture to homogenousones in a single step. The most common affinity ligands are monoclonal antibodies,smallmolecules such as biotin and those specific to the bio-function of the protein of interest,suchas substrates and inhibitors. Although m Ab is less limited by the bioactivity of proteins,itspreparation is a complex …     

二维核磁共振波谱和分子力学方法计算香茶菜醛溶液中的三维空间结构

采用二维核磁共振波谱完全归属了天然有机化合物香茶菜醛的~1H和~(13)C NMR化学位移,测定了部分质子的偶合常数,并用孤立自旋对近似方法定量处理香茶菜醛的相敏NOESY谱,根据1/r_(ij)~8∝A_(ij)计算出相应的质子间距离.其溶液中的三维空间结构采用以NMR结构参数为初始数据的WUPH计算程序和分子力学能量优化(MM2)完成计算.计算结果表明,得到的香茶菜醛在溶液中的三维空间结构反映了该化合物在溶液中的真实空间结构.     

孤啡肽OFQ的溶液构象研究

应用近代NMR波谱技术对孤啡肽OFQ及其片段OFQ_(8-17)的溶液结构进行了比较 研究，测定了孤啡肽OFQ和片段OFQ_(8-17)在H_2O和TEF/H_2O两种溶剂条件下的2D －DQF－COSY，2D－TOCSY，2D－NOSESY(ROESY)谱，完成了它们氨基酸残基的处旋 系统识别和序列特异性归属，获得分子中质子化学位移的完全归属，根据NOE特征 和主链偶合常数等结构参数确定了它们的特征二级结构，以NOE提供的距离约束， 进行何，分子力学和分子动力学模拟，得到了孤啡肽OFQ在不同溶剂条件下的溶液 结构。在H_2O溶济中，孤啡肽OFQ在G3－F4－T5－G6处形成一复合型转角结构。在 TFE/H_2O溶剂中，OFQ整个分子形成两新性α－螺旋结构。而孤啡肽片OFO_(8-17) 在这两种环境中均为伸展的无规结构。孤啡肽OFQ在类似膜环境(TFE/H_2O)下的α 螺旋结构可能为其活性构象。     

蛋白质全新设计：八残基序列形成发夹结构的圆二色谱

β-发夹是天然蛋白质中丰富的二级结构单元之一，在蛋白折叠和功能方面扮演着重要角色.文章报导了二条多肽序列（LTVd-PGLTV，n7和 LTVGDDTV， n5）的设计、合成和园二色谱研究结果.结果显示，n5在198 nm附近呈现负峰，表现为非规整结构特征；相反，n7表现为典型的发夹结构特征，在218 nm附近呈负峰,196 nm附近呈正峰，为β-转角与β-折叠的共同贡献.初步研究表明，β-转角、序列关系和氨基酸形成β-折叠结构倾向性是β-发夹结构形成和稳定的决定性因素.     

Investigation of microstructure of glycidyl methacrylate/α-methyl styrene copolymers by 1D- and 2D-NMR spectroscopy

The copolymers of glycidyl methacrylate/α-methyl styrene were synthesized. The quantitative ¹³C{¹H}-NMR spectroscopy was used to determine the copolymer compositions. The distortionless enhancement by polarization transfer, ¹³C-¹H heteronuclear single quantum coherence and total correlation spectroscopy were used for the complete spectral assignment of ¹³C{¹H}- and ¹H-NMR spectra.     

2H QUOSY 2D-NMR Experiments in Weakly Aligning Systems: From the Conventional to the Ultrafast Approach

We describe three anisotropic ultrafast (UF) QUadrupolar Ordered SpectroscopY (QUOSY) 2D-NMR experiments (referred to as ADUF 2D NMR spectroscopy) designed for recording the ²H homonuclear 2D spectra of weakly aligned (deuterated) solutes in sub-second experiment times. These new ADUF 2D experiments derive from the Q-COSY, Q-resolved and Q-DQ 2D pulse sequences (J. Am. Chem. Soc. 1999 , 121, 5249) and allow the correlation between the two components of each quadrupolar doublet, and then their assignment on the basis of ²H chemical shifts. The UF 2D pulse sequences are analyzed by using the Cartesian spin-operator formalism for spin I=1 nuclei with a small quadrupolar moment. The optimal experimental/practical conditions as well as the resolution, sensitivity and quantification issues of these ADUF 2D experiments are discussed on comparison to their conventional 2D counterparts and their analytical potentialities. Illustrative ADUF 2D experiments using deuterated achiral/prochiral/chiral solutes in poly-γ-benzyl-L-glutamate based chiral liquid crystals are presented, as well as the first examples of natural abundance deuterium (ANADUF) 2D spectrum using 14.1 T magnetic field and a basic gradient unit (53 G.cm⁻¹) in oriented solvents.     

亲和层析结合生物质谱技术分析鉴定猕猴血清中的重组人内皮抑制素

建立了将固相亲和层析与生物质谱技术相结合 ,分离提取鉴定给药猕猴血清中含有 6×His序列的重组人内皮抑制素的方法。用固相免疫亲和层析和金属螯合层析两种方法分别提取并富集血清中的重组药物 ,采用基质辅助激光解吸 电离飞行时间质谱的直接分析和肽质量指纹谱分析与数据库检索 ,分别对两种亲和提取方法得到的药物蛋白前体collagenXVIII[Homosapiens](IDofSWISS -PROT TrEMBL :Q8WXI5 )进行了鉴定     

Affinity capillary electrophoresis employed for determination of stability constants of antamanide complexes with univalent and divalent cations in methanol

Affinity capillary electrophoresis (ACE) and pressure‐assisted ACE were employed to study the noncovalent molecular interactions of antamanide (AA), cyclic decapeptide from the deadly poisonous fungus Amanita phalloides, with univalent (Li⁺, Na⁺, K⁺, and NH₄⁺) and divalent (Mg²⁺ and Ca²⁺) cations in methanol. The strength of these interactions was quantified by the apparent stability constants of the appropriate AA‐cation complexes. The stability constants were calculated using the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of AA on the concentration of the above ions in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, pH_MeOH 7.8, containing 0–50 mM concentrations of the above ions added in the form of chlorides). Prior to stability constant calculation, the AA effective mobilities measured at actual temperature inside the capillary and at variable ionic strength of the BGEs were corrected to the values corresponding to the reference temperature of 25°C and to the constant ionic strength of 10 mM. From the above ions, sodium cation interacted with AA moderately strong with the stability constant 362 ± 16 L/mol. K⁺, Mg²⁺, and Ca²⁺ cations formed with AA weak complexes with stability constants in the range 37–31 L/mol decreasing in the order K⁺ > Ca²⁺ > Mg²⁺. No interactions were observed between AA and small Li⁺ and large NH₄⁺ cations.     

Theoretical Study Of β2,3‐Peptide Models

Conformational features of α,β‐disubstituted β^2,3‐dipeptide models have been studied with quantum mechanics method. Geometries were optimized with the HF/6‐31G** method, and energies were evaluated with the B3LYP/6‐31G** method. Solvent effect was evaluated with the SCIPCM method. For (2S,3S)‐β^2,3‐dipeptide model 1 , a six‐membered‐ring hydrogen bonded structure is most stable. However, the conformation corresponding to the formation of the 14‐helix is only about 1.7 kcal/mol less stable in methanol solution, indicating that the 14‐helix is favored if a (2S,3S)‐β^2,3‐polypeptide contains more than 5 residues. On the other hand, the conformation corresponding to the formation of β‐sheet is most stable for (2R,3S)‐β^2,3‐dipeptide model 2 , suggesting that this type of β‐peptides is intrinsically favored for the formation of β‐sheet secondary structure.