家蝇幼虫抗菌肽MDL-1的荧光特性分析

本文研究了家蝇幼虫抗菌肽MDL-1的荧光光谱和淬灭剂对内源性荧光的影响。家蝇幼虫抗菌肽MDL-1在激发波长280 nm时,其荧光光谱为酪氨酸（Tyr）残基和色氨酸（Trp）残基共同提供。结果表明,KI不能淬灭抗菌肽MDL-1的Trp残基的荧光,而丙烯酰胺（Acr）能淬灭几乎所有的Trp残基的荧光（f-0．92）;这说明,Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部。     

尖吻蝮蛇蛇毒出血毒素Ⅲ的荧光光谱

皖南尖吻蝮蛇蛇毒经分离和纯化得到出血毒素Ⅲ(AaH Ⅲ),质谱测定分子量为23,284.4±0.1。实验表明,Trp残基较大程度位于AaH Ⅲ分子的疏水区。十二烷基磺酸钠(SDS)和盐酸胍的加入使AaH Ⅲ的分子构象发生变化,导致分子内色氨酸(Trp)残基的荧光淬灭。盐酸胍的加入使Trp残基的荧光发射峰位明显红移,表明位于AaH Ⅲ分子较疏水环境的Trp残基相对外露。我们还就酸度、EDTA和金属离子对AaH Ⅲ分子内Trp残基荧光光谱的影响进行了研究和讨论。     

光谱法与分子对接法研究山柰酚对牛血清白蛋白二级结构的影响

采用分子对接技术和同步荧光光谱法、红边激发荧光位移法(REES法)及圆二色谱法(CD)共同研究了山柰酚与牛血清白蛋白(BSA)在pH7.40的缓冲溶液中的相互作用。分子对接的结果表明,山柰酚的B环插入到BSA的ⅡA结构域中的疏水腔内,与色氨酸残基(Trp212)的距离为12.96,维系药物与蛋白质的主要作用力为疏水作用。通过荧光光谱法测得二者之间相互作用力主要为疏水性相互作用,结合位点为1,与分子模拟结果一致。同步荧光光谱及REES法的研究表明,发生相互作用的过程中BSA的色氨酸残基处于运动受限的微环境中,而适当增加山柰酚的浓度能够改变色氨酸微环境的流动性,进而对BSA的构象产生一定影响;同时,圆二色谱的定量计算结果也表明,一定浓度的山柰酚与BSA的相互作用引起了α-螺旋含量的显著降低,从11.91%降低到1.67%,对BSA的二级结构产生一定影响。     

黄芩苷与人血清白蛋白的相互作用研究

利用紫外光谱、荧光光谱、傅立叶红外谱、圆二色谱及分子模型等技术,在生理pH条件下,研究了黄芩苷与人血清白蛋白(HSA)的相互作用,并计算了其结合常数和热力学参数.分子模型研究表明,黄芩苷与HSA在亚结构域ⅡA结合,二者间的作用主要为静电作用和疏水作用,与荧光光谱结果基本一致.红外光谱和圆二色谱显示黄芩苷与HSA结合后未...     

不同介质中蜂毒素聚集/解聚集的超分子调控及相关机理

利用荧光光谱和圆二色谱等技术手段研究了蜂毒素在2-羟丙基环糊精、氯化钠或 DOPC 调控下的聚集/解聚集过程、相关机理以及不同介质中α-helix的含量. 研究结果表明, 蜂毒素在不同介质中的聚集能力、构象以及和介质分子相互作用力均存在很大差别.     

溶液中溶菌酶的荧光光谱研究

本文采用荧光分析法(荧光光谱、荧光偏振度)结合内源荧光探针色氨酸残基对溶菌酶及其在不同条件下的构象变化进行了研究。结果表明,利用荧光光谱可对溶菌酶溶液构象进行有效的分析,能够较直观地表征色氨酸残基在溶菌酶分子中的微环境及其在不同条件下构象的变化,得出了一些较有价值的结果。     

莲藕多酚氧化酶与底物或抑制剂相互作用的光谱学研究

本文研究了纯化的莲藕多酚氧化酶(PPO)与底物和抑制剂相互作用时的二级结构变化。园二色谱分析表明莲藕PPO主要含有α-螺旋和β-折叠结构。与抑制剂作用后,莲藕PPO活性显著降低,同时伴随其二级结构中α-螺旋结构明显减少,表明莲藕PPO的活性中心可能位于α-螺旋结构中。荧光分析表明,莲藕PPO与邻苯三酚(pyrogallic acid,PA)作用后,酪氨酸残基荧光强度略有降低,λmax位移不明显,色氨酸残基荧光强度略有降低,λmax红移2 nm;而与莲藕多酚(Lotus root polyphenol,LRP)作用后,莲藕PPO分子中酪氨酸(Tyr)和色氨酸(Trp)残基荧光强度显著提高,且其最大发射波长分别蓝移6nm和红移5nm。当加入异Vc钠后,Tyr和Trp残基最大发射峰显著红移,说明Trp和Tyr残基位于一定的疏水环境对维持PPO催化活性的优势构象至关重要。     

铜离子对水溶液中再生丝素蛋白构象转变的诱导作用

综合外加疏水性荧光探针ANS和荧光猝灭等多种荧光分析手段以及远紫外圆二色(far-UVCD)实验,发现在再生丝素蛋白稀溶液中,Cu(Ⅱ)通过与丝素蛋白疏水区域的直接络合从而诱导蛋白质分子发生构象转变,这一过程与乙醇诱导下的丝素蛋白变性有着相当大的区别.就络合位点而言,除了普遍认为的组氨酸(His)残基外,丝素蛋白分子中的色氨酸(Trp)残基是Cu(Ⅱ)的另一个有效络合点,并且这种络合在一定条件下是可逆的.     

咪唑型离子液体与牛血清蛋白的缔合特性

通过紫外-可见光谱、荧光光谱、同步荧光光谱、圆二色谱、衰减全反射红外光谱、负染-透射电镜、等温滴定微量热等实验方法系统地探讨了咪唑型离子液体与牛血清蛋白(BSA)的缔合特性.结果发现,离子液体[Bmim]Cl的加入使得BSA的紫外吸收强度增加,同时也会导致其荧光猝灭,并且这种猝灭是静态猝灭.同步荧光的研究结果表明,[Bmim]Cl分子可与蛋白质中接近色氨酸残基的区域发生相互作用,使蛋白质的构象和内部的疏水结构发生改变;负染色法透射电镜直观地显示了加入离子液体后形成的蛋白质-离子液体复合物结构逐渐变大;圆二色谱和衰减全反射红外光谱表明:在离子液体与BSA缔合过程中,离子液体的加入使得BSA二级结构中的α-螺旋和β-折叠的含量降低,从而引起蛋白质二级结构的变化;表面张力法和等温滴定微量热法进一步证实上述缔合作用为静电作用和疏水作用共同作用的结果,但离子液体的烷基链与BSA疏水内腔之间的疏水作用是离子液体与BSA缔合的主要驱动力.     

一种中国林蛙抗菌肽的光谱研究

从中国林蛙皮中纯化得到了一种抗多种临床多重耐药菌的抗菌肽(RTCⅠ), 初步氨基酸组成分析结果表明其不含碱性氨基酸, 此抗菌肽在276.5和356.5 nm波长光的激发下发射出448 nm的荧光, 利用傅里叶变换红外光谱、拉曼光谱、电子吸收光谱及荧光光谱等技术研究了此特定荧光产生的结构依据. 此抗菌肽的主要组成是Tyr, Asn(Asp)和Glu(Gln), 抗菌肽特殊的荧光光谱和电子吸收谱与Tyr的酚羟基和Asn侧链的强氢键有关. 这一特殊的荧光(448 nm)及圆二色谱(259, 263和267 nm)信号为进一步在分子水平上研究此抗菌肽的抗菌机理提供了依据.     

Structure-activity relationships of GHRP-6 azapeptide ligands of the CD36 scavenger receptor by solid-phase submonomer azapeptide synthesis

The cluster of differentiation 36 (CD36) class B scavenger receptor binds a variety of biologically endogenous ligands in addition to synthetic peptides (i.e., growth hormone-releasing peptides, GHRPs), which modulate biological function related to anti-angiogenic and anti-atherosclerotic activities. Affinity labeling had previously shown that GHRP-6 analogues such as hexarelin, [2-Me-W(2)]GHRP-6 (1), bind to the lysine-rich domain of the CD36 receptor. Moreover, the azapeptide analogue [aza-F(4)]GHRP-6, 2, exhibited a characteristic β-turn conformation as described by CD and NMR spectroscopy and a slightly higher CD36 binding affinity relative to hexarelin (1.34 and 2.37 μM, respectively), suggesting receptor binding was mediated by the conformation and the aromatic residues of these peptide sequences. Ligand-receptor binding interactions were thus explored using azapeptides to examine influences of side-chain diversity and backbone conformation. In particular, considering that aromatic cation interactions may contribute to binding affinity, we have explored the potential of introducing salt bridges to furnish GHRP-6 azapeptide ligands of the CD36 receptor. Fifteen aza-glutamic acid analogues related to 2 were prepared by submonomer solid-phase synthesis. The azapeptide side chains were installed by novel approaches featuring alkylation of resin-bound semicarbazone with Michael acceptors and activated allylic acetates in the presence of phosphazene base (BTPP). Moreover, certain Michael adducts underwent intramolecular cyclization during semicarbazone deprotection, leading to novel pyrrazoline and aza-pyroglutamate N-terminal residues. Structural studies indicated that contingent on sequence the [aza-Glu]GHRP-6 analogues exhibited CD spectra characteristic of random coil, polyproline type II and β-turn secondary structures in aqueous media. In covalent competition binding studies with the GHRP-6 prototype hexarelin bearing a radiotracer, certain [aza-Glu]GHRP-6 azapeptides retained relatively high (2-27 μM) affinity for the CD36 scavenger receptor.     

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。     

Two-dimensional fluorescence correlation spectroscopy IV: resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching.     

A 10-A spectroscopic ruler applied to short polyprolines

Fluorescence resonance energy transfer (FRET) from the amino acid tryptophan (Trp) as donor and a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as acceptor in peptides of the general structure Trp-(Pro)n-Dbo-NH2 (n = 1-6) was investigated by steady-state and time-resolved fluorescence, CD, and NMR spectroscopy as well as by molecular dynamics (MD) simulations (GROMOS96 force field). The Trp/Dbo FRET pair is characterized by a very short F?rster radius (R0 ca. 9 A), which allowed distance determinations in such short peptides. Water and propylene glycol were investigated as solvents. The peptides were designed to show an early nucleation of the poly(Pro)II (PPII) secondary helix structure for n > or = 2, which was confirmed by their CD spectra. The shortest peptide (n = 1) adopts preferentially the trans conformation about the Trp-Pro bond, as confirmed by NMR spectra. The FRET efficiencies ranged 2-72% and were found to depend sensitively on the peptide length, i.e., the number of intervening proline residues. The analysis of the FRET data at different levels of theory (assuming either a fixed distance or distance distributions according to a wormlike chain or Gaussian model) afforded donor-acceptor distances between ca. 8 A (n = 1) and ca. 16 A (n = 6) in water, which were found to be similar or slightly higher in propylene glycol. The distances afforded by the Trp/Dbo FRET pair were found to be reasonable in comparison to literature data, expectations from the PPII helix structure, and the results from MD simulations. The persistence lengths for the longer peptides were found to lie at 30-70 A in water and 220 +/- 40 A in propylene glycol, suggesting a more rigid PPII helical structure in propylene glycol. A detailed comparison with literature data on FRET in polyprolines demonstrates that the donor-acceptor distances extracted by FRET are correlated with the F?rster radii of the employed FRET pairs. This demonstrates the limitations of using FRET as a spectroscopic ruler for short polyprolines, which is presumably due to the breakdown of the point dipole approximation in F?rster theory, when the size of the chromophores becomes comparable or larger than the distances under investigation.     

手性二茂铁色氨酸化合物[Fc-(CO-L-Trp-OMe)2]的合成和结构表征

由色氨酸甲酯和1,1′-二茂铁二羧酸合成了化合物1[Fc-(CO-L-Trp-OMe)₂],并对化合物1在固体和溶液中的结构进行了表征。单晶结构表明,该化合物通过2个分子内氢键,形成了规则的手性构象。通过分子间的氢键,形成了二维网状结构。对化合物1的乙腈溶液进行了CD谱的测定,结果表明,该化合物在溶液中呈现P螺旋构象。¹H NMR谱和FTIR谱也证实了该化合物在固体和溶液状态中都形成了含分子内氢键的构象。     

Expected and unexpected results from combined beta-hairpin design elements

A model beta-hairpin dodecapeptide [EFGWVpGKWTIK] was designed by including a favorable D-ProGly Type II' beta-turn sequence and a Trp-zip interaction, while also incorporating a beta-strand unfavorable glycine residue in the N-terminal strand. This peptide is highly folded and monomeric in aqueous solution as determined by combined analysis with circular dichroism and 1H NMR spectroscopy. A peptide representing the folded conformation of the model beta-hairpin [cyclic(EFGWVpGKWTIKpG)] and a linear peptide representing the unfolded conformation [EFGWVPGKWTIK] yield unexpected relative deviations between the CD and 1H NMR spectroscopic results that are attributed to variations in the packing interactions of the aromatic side chains. Mutational analysis of the model beta-hairpin indicates that the Trp-zip interaction favors folding and stability relative to an alternate hydrophobic cluster between Trp and Tyr residues [EFGYVpGKWTIK]. The significance of select diagonal interactions in the model beta-hairpin was tested by rearranging the cross-strand hydrophobic interactions to provide a folded peptide [EWFGIpGKTYWK] displaying evidence of an unusual backbone conformation at the hydrophobic cluster. This unusual conformation does not appear to be a result of the glycine residue in the beta-strand, as replacement with a serine results in a peptide [EWFSIpGKTYWK] with a similar and seemingly characteristic CD spectrum. However, an alternate arrangement of hydrophobic residues with a Trp-zip interaction in a similar position to the parent beta-hairpin [EGFWVpGKWITK] results in a folded beta-hairpin conformation. The differences between side chain packing of these peptides precludes meaningful thermodynamic analysis and illustrates the caution necessary when interpreting beta-hairpin folding thermodynamics that are driven, at least in part, by aromatic cross strand interactions.     

铱(IV)离子与人血丙种球蛋白的作用研究

在0.1 mol•L^－1醋酸-醋酸钠(pH 5.0)体系中, 采用紫外吸收光谱、荧光光谱及同步荧光光谱法研究了人血丙种球蛋白(gamma seroglobulinum humanum, 简称GSH)与铱(IV)离子的相互作用. 结果表明, Ir(IV)离子使人血丙种球蛋白的构象发生了改变, α-螺旋含量减少, 并且用同步荧光光谱发现Ir(IV)离子与人血丙种球蛋白的作用位点更接近于色氨酸, 从而使色氨酸残基的疏水性略有减小. 荧光光谱结果表明Ir(IV)对人血丙种球蛋白内源荧光(342 nm)产生了较强的荧光猝灭作用, 根据不同温度下Ir(IV)对人血丙种球蛋白的荧光猝灭作用, 证明了这种荧光猝灭为静态猝灭机制, 计算了其结合常数和结合位点数, 从而得出了静电作用力为其主要的作用力.     

Study on specific interaction of new ferrocene-substituted carborane conjugates with hemoglobin protein

The interactions between the new organometallic complexes, ferrocene-substituted dithio-o-carborane conjugates (denoted as FcSB1, FcSB2 and FcSBCO) and hemoglobin (Hb) are investigated by electrochemistry, fluorescence and UV-vis absorption spectroscopy. The results demonstrate that FcSB1, FcSB2 and FcSBCO can bind to the heme iron center through the replacement of the weakly bound H2O/O2 in the distal heme pocket of Hb by their sulfur donor atoms, inducing the allosteric change from the R state (oxygenated conformation, relax) to T state (deoxygenated conformation, tense). The binding affinity is in the order of FcSBCO>FcSB2>FcSB1. Moreover, the fluorescence study illustrates that the three ferrocene-carborane conjugates differently affect the quarterly and tertiary structures as well as the polarity in the surrounding of the Trp and Tyr residues in Hb. Typically, FcSB2 mainly induces alterations of the microenvironment around the β37Trp residue which is located on the α1β2 interface of Hb. Such distinct influences are attributed to the structural features of FcSB1, FcSB2 and FcSBCO containing hydrophobic ferrocenyl and carboranyl units as well as C=O group. Screening the protein-binding behavior can signify the potential bioactivity of such molecules and may be helpful in the future development of promising multifunctional metallodrugs.