电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白肽链和辅基的相互作用

本文通过牛肝线粒体细胞色素Tb5和它的F35Y突变体蛋白相对分子质量的外标法测定,得到细胞色素Tb5全蛋白的相对分子质量为10077.5脱辅基蛋白的相对分子质量为9461.4F35Y突变体蛋白的相对分子质量为10093.6,它的脱辅基蛋白的相对分子质量为9477.5,不同nozzle电压下的电喷雾质谱结果表明,该电压的大小明显影响蛋白肽链与血红素辅基之间的非共价结合,随着电压的降低,全蛋白谱峰的强度逐渐增大,然而,过低的电压导致了Na^+,K^+离子加合峰相对强度的增加,而不利于谱图分析。同时,考察到细胞色素Tb5在甲醇溶液和酸性溶液中的变性行为,因此选择nozzle电压70V,10%的甲醇水溶液和pH=7为得到全蛋白质谱峰的最佳条件。相同实验条件下得到的野生型CytTb5和F35Y突变体全蛋白的质谱峰相比较,其相对丰度有悬殊的差异,表明F35Y突变体蛋白的血红素结合能力明显低于野生型蛋白。通过解离出的Hemeb的分子离子峰进行解析,证明铁仍以三价离子存在于血红素辅基中。     

电喷雾质谱法研究细胞色素Tb5及其F35Y突变体蛋白 肽链和辅基的相互作用

本文通过牛肝线粒体细胞色素Tb5和它的F35Y突变体蛋白相对分子质量的外标法测定,得到细胞色素Tb5全蛋白的相对分子质量为10077.5脱辅基蛋白的相对分子质量为9461.4F35Y突变体蛋白的相对分子质量为10093.6,它的脱辅基蛋白的相对分子质量为9477.5,不同nozzle电压下的电喷雾质谱结果表明,该电压的大小明显影响蛋白肽链与血红素辅基之间的非共价结合,随着电压的降低,全蛋白谱峰的强度逐渐增大,然而,过低的电压导致了Na^+,K^+离子加合峰相对强度的增加,而不利于谱图分析。同时,考察到细胞色素Tb5在甲醇溶液和酸性溶液中的变性行为,因此选择nozzle电压70V,10%的甲醇水溶液和pH=7为得到全蛋白质谱峰的最佳条件。相同实验条件下得到的野生型CytTb5和F35Y突变体全蛋白的质谱峰相比较,其相对丰度有悬殊的差异,表明F35Y突变体蛋白的血红素结合能力明显低于野生型蛋白。通过解离出的Hemeb的分子离子峰进行解析,证明铁仍以三价离子存在于血红素辅基中。     

神经红蛋白突变体Tyr44Phe的制备、表征和稳定性研究

构建了突变体蛋白Tyr44Phe的基因, 进行了蛋白的表达、分离纯化、谱学表征和稳定性研究. 由电喷雾质谱所得突变体蛋白的分子量与理论值一致; UV-Vis吸收光谱、荧光光谱和圆二色光谱表明, Tyr44Phe的点突变虽没有改变血红素的六配位结构, 但对血红素的构象有所影响. 突变体蛋白的热、酸稳定性研究表明, 定点突变降低了血红素与蛋白肽链之间的结合力, 导致血红素易从疏水腔中脱出, 说明Tyr44对蛋白的结构稳定性起一定的作用.     

鼠脑红蛋白突变体F106L与氰根的结合作用

构建了鼠脑红蛋白(Mouse neuroglobin)的突变体F106L, 以探求近端残基对脑红蛋白血红素口袋结构的贡献. 通过溶液核磁共振方法研究了外来配体氰根离子与NgbF106L蛋白的结合作用, 结果显示, 此结合存在动力学过程, 并且NgbF106LCN 突变蛋白氰根络合物可以可逆地释放氰根离子, 并使原来的第6配体His64(E7)又结合回到血红素铁上. 研究结果揭示, G5(Phe106)残基对脑红蛋白血红素构象而言较为保守; QM/MM结构优化结果表明, 位于G5 和FG5的近端残基对蛋白结构稳定性具有重要作用, 并可调控外来配体与蛋白作用的配位平衡与热动力学性质.     

荧光光谱法研究中华蜜蜂化学感受蛋白与特异配基的相互作用

用荧光光谱法研究了中华蜜蜂化学感受蛋白(chemosensory proteins, CSPs)3与其特异性配基N-苯基-1-萘胺(N-Phenyl-1-naphthylamine, 1-NPN)的相互作用关系。研究表明1-NPN能使CSP3在328 nm (λem)处产生猝灭,且猝灭机理为静态猝灭,另外猝灭过程中ΔH0 > 0, ΔS0 > 0,表明二者间的主要作用力为疏水相互作用。依据F?rster非辐射能量转移机制,得到二者的结合距离为9.3 nm,能量转移效率E = 0.054。根据同步荧光技术考察1-NPN对CSP3的构象的影响,表明CSP3荧光主要贡献者--色氨酸残基的最大发射波长略有红移,表明原处于疏水腔中的色氨酸残基由于所处环境的极性增加,而使CSP3构象产生变化。     

神经红蛋白突变体H64V与叠氮离子的作用

将64位组氨酸(His64)突变为缬氨酸(Val),构建了神经红蛋白(Ngb)突变体H64V基因并进行了蛋白的表达纯化,突变使Ngb的血红素Fe由六配位结构变成五配位结构.利用光谱法研究了突变体H64V与叠氮离子(N_3~-)的相互作用.紫外-可见吸收光谱表明,H64V与N_3~-键合后其Soret带吸收峰由406 nm红移至418 nm;荧光光谱表明N_3~-可使H64V的荧光发生静态猝灭.计算得到了H64V与N_3~-键合的平衡解离常数和热力学参数.另外,还研究了不同阴离子对H64V与N_3~-结合的影响,发现阴离子的存在减弱了N_3~-与H64V之间的结合力.     

荧光光谱在研究氯霉素与沙拉沙星间拮抗作用中的应用

氯霉素(CHL)和沙拉沙星(SLFX)均能够猝灭牛血清白蛋白(BSA)的荧光. 当两种药物共存时使BSA荧光进一步猝灭, 据此利用荧光光谱法研究了氟喹诺酮类药物SLFX与CHL间相互作用. 结果表明: 两种药物间存在拮抗作用, 使药物与蛋白的结合稳定性增加, 致使能够转运到作用部位产生药理效应的游离型药物含量减少, 造成药效降低; 药物对蛋白荧光的猝灭属于静态猝灭; 药物与蛋白结合位点数约为1. 根据Forster非辐射能量转移理论, 确定了药物与蛋白之间的结合距离r, 药物间拮抗作用的存在使r值降低, 结合距离减小. 同步荧光光谱研究表明, 药物间的拮抗作用对蛋白质构象产生影响, 使蛋白质分子伸展, 疏水性降低.     

环丙沙星与牛血清白蛋白的结合反应

以光谱技术与微量热技术相结合研究了水溶液中牛血清白蛋白与环丙沙星分子间结合作用的机制. 荧光猝灭法测得该反应的结合常数K=8.39×104 L•mol－1,结合位点数n =1.18;依据Fōrster非辐射能量转移机制,得到授体 受体间的结合距离(r=2.46 nm)和能量转移效率(E=0.33).微量热法测得反应的焓变 ΔrHm≈0;牛血清白蛋白与环丙沙星分子间有较强的结合作用,且以疏水作用为主.     

W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.     

p53突变体Y220C小分子稳定剂的虚拟筛选

为了获得p53突变体的稳定剂,依次利用利宾斯基五原则,通过2次分子对接和全原子分子动力学(MD)模拟从Drug Bank 4.0数据库中筛选获得了潜在的稳定剂他克林.利用MD模拟进一步验证他克林和目标蛋白质之间的亲和作用.结果表明,他克林能够紧密结合到Y220C突变所形成的疏水空腔之中;他克林和目标蛋白质之间的主要作用力为疏水和静电相互作用,其中疏水相互作用占主导地位.此外,他克林分别与目标蛋白质的残基Leu145,Val147和Asp228形成3个氢键.基于MD模拟轨迹分析了他克林与p53CY220C的结合过程.由硫黄素T荧光光谱进一步证明他克林能够提高p53C-Y220C突变体的稳定性.     

Study on the Gas Phase Stability of Heme-binding Pocket in Cytochrome Tb_5 and Its Mutants by Electrospray Mass Spectrometry

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Action spectra for photogravitropism of Phycomyces wild type and three behavioral mutants (L150, L152, and L154)

We have measured fluence rate-response curves and action spectra for photogravitropism in Phycomyces wild type and in three recently isolated mutants with elevated phototropic thresholds. The action spectra were determined from least-squares fits of a sigmoidal function to the fluence rate-response data for each wavelength. The action spectrum for wild type has maxima near 383, 413, 452, and 490 nm and minima near 397, 425, and 469 nm. This photogravitropism action spectrum is very similar to the Phycomyces phototropic balance action spectrum between 413 but has significantly higher effectiveness below 400 nm and above 490 nm. These differences may be caused by dichroic effects of the oriented receptor pigment and/or by multiple receptor pigments. The action spectra of the three mutants differ significantly from one another and from that of wild type. Relative to the wild type spectrum, all three mutants exhibit a suppression in effectiveness near 425 nm, which is near the transmission peak of the broadband blue filter used to isolate the mutants.     

Array of aromatic amino acid side chains located near the chromophore of photoactive yellow protein

The role of the array of aromatic amino acid side chains located close to the chromophore binding loop of photoactive yellow protein (PYP) was studied using the alanine-substitution mutagenesis. Phe92, Tyr94, Phe96 and Tyr98 were replaced with alanine (F92A, Y94A, F96A and Y98A, respectively), then these mutants were characterized by UV-visible absorption spectra, circular dichroism (CD) spectra, thermal stability and photocycle kinetics. Absorption maxima of F92A, Y94A, F96A and Y98A were 444, 442, 439 and 447 nm, respectively, different to wild type (WT) at 446 nm. Far-UV CD spectra of mutants other than F92A were different from WT, indicating that Tyr94, Phe96 and Tyr98 maintain the native secondary structure of PYP. Mid-point temperatures of thermal denaturation of F92A, Y94A and F96A, estimated by the CD signal at 222 nm, were 5-10 degrees C lower than WT. Time constants of the photocycle estimated by flash-induced absorbance change were 0.36 s for WT and 1.4 s for Y98A, however, 100, 30 and 3000 times slower than WT for F92A, Y94A and F96A, respectively. Tyr98 is located in the loop region, whereas Phe92, Tyr94 and Phe96 are incorporated in the beta4 strand, showing that aromatic amino acid residues in the beta-sheet regulate the absorption spectrum, thermal stability and photocycle of PYP. Aromatic rings of Phe92, Tyr94 and Phe96 lie nearly perpendicular to the aromatic ring of Phe75 or chromophore. Possible weak hydrogen bonds between the aromatic ring hydrogen and pi-electrons of these residues are discussed.     

Structural Identification of Spectroscopic Substates in Neuroglobin

The structural origins of infrared absorptions of photodissociated CO in murine neuroglobin (Ngb) are determined by combining Fourier transform infrared (FTIR) spectroscopy and molecular dynamics (MD) simulations. Such an approach allows to identify and characterize both the different conformations of the Ngb active site and the transient ligand docking sites. To capture the influence of the protein environment on the spectroscopy and dynamics, experiments and simulations are carried out for the wild type protein and its F28L and F28W mutants. It is found that a voluminous side chain at position 28 divides site B into two subsites, B’ and B”. At low temperatures, CO in wt Ngb only migrates to site B’ from where it can rebind, and B” is not populated. The spectra of CO in site B’ for wt Ngb from simulations and experiments are very similar in spectral shift and shape. They both show doublets, red‐shifted with respect to gas‐phase CO and split by≈8 cm^?1. The FTIR spectra of the F28L mutant show additional bands which are also found in the simulations and can be attributed to CO located in substate B”. The different bands are mainly related to different orientations of the His64 side chain with respect to the CO ligand. Large red‐shifts arise from strong interactions between the Histidine? NH and the CO oxygen. After dissociation from the heme iron, the CO ligand visits multiple docking sites. The locations of the primary docking site B and a secondary site C, which corresponds to the Mb Xe4 cavity, could be identified unambiguously. Finally, by comparing experiment and simulations it is also possible to identify protonation of its ε position (His_ε64 NgbCO) as the preferred heme‐bound conformation in the wild type protein with a signal at 1935 cm^?1.     

Y443F mutation in the substrate-binding domain of extracellular PHB depolymerase enhances its PHB adsorption and disruption abilities

Extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ_RpiT1) from Ralstonia pickettii T1 adsorbs to the PHB surface via its substrate-binding domain (SBD) and cleaves the PHB chain using its catalytic domain. Our previous study (Biomacromolecules 2010; 11: 113-119) has suggested that the hydrophobic interaction between the amino acid residues at positions 441, 443, and 445 in the SBD and the PHB surface plays a crucial role in facilitating the association phase of the enzyme adsorption process. In the present study, in order to improve PhaZ_RpiT1 for effective PHB degradation, we targeted Tyr at position 443 for substitution with a more highly hydrophobic amino acid residue because its hydrophobicity shows medium to high degree compared to those of general naturally occurring amino acid residues. We designed a mutant enzyme with an amino acid substitution at this position, taking the following factors into consideration: (1) to achieve higher hydrophobicity than the original residue, (2) to retain the β-sheet structure, and (3) to change as little as possible the volume of the amino acid residue after the substitution. As a result, the substitution of Tyr443 with Phe (Y443F) was considered to be appropriate. The purified Y443F enzyme showed identical CD spectrum and hydrolysis activity for a water-soluble substrate with the wild type, indicating that the mutation had no influence on the structure and the ester bond cleavage activity. In contrast, the Y443F enzyme had higher PHB degradation activity than the wild type. Kinetic analysis of PHB degradation suggests that this amino acid substitution promoted not only the adsorption of the mutant enzyme to PHB, but also the disruption of the PHB surface to enhance the hydrolysis of the PHB polymer chain.     

Influences of the Hydrophobicity of the Heme－binding Pocket on the Properties and Functions of Cytochrome b5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b5 are located at the edge of the heme-binding pocket. The solvent accessible areas of the “pocket inte-rior“ of the four mutants and the wild-type cytochrome b5 have been calculated based on their crystal structures at high resolu-tion. The change in the hydrophobicity of the heme-binding pocket resulting from the mutation can be quantitatively de-scribed using the difference of the solvent accessible area of the “pocket interior“ of each mutant from that of the wild-type cy-tochrome b5. The influences of the hydrophobicity of the heme-binding pocket on the protein stability and redox potential are discussed.     

Synthesis and cofluorescence of Eu(Y) complexes with salicylic acid and o-phenanthroline

A series of dinuclear complexes of salicylic acid (HSal) and o-phenanthroline (Phen) with different molar ratios of Eu3+ to Y3+ have been synthesized. Their compositions are Eu(x)Y(1-x) (Sal)3(Phen) (x = 0 to approximately 1). Their UV spectra, IR spectra, and fluorescence spectra were studied. The UV spectra of the complexes reflect essentially absorption of the ligands for the fact that no obvious change of wavelength and band shape is found between the spectra of the complexes and that of the ligands except slight red shift. The IR absorption spectra indicate that salicylic acid is coordinated to the rare earth ions and chemical bonds are formed between rare earth ions and nitrogen atoms of o-phenanthroline. The fluorescence spectra of the complexes indicate that the fluorescence emission intensity of europium ion was enhanced by the addition of Y3+, which is referred to as cofluorescence. These facts show that not only the ligands but also the yttrium complex can transfer the absorbed energy to Eu3+ ion in the complexes. Formation of polynuclear complexes appears to be responsible for cofluorescence.     

Use of oxidation-state differences and molecular orbitals to interpret bonding in the series ONXYZ (X, Y, Z=H, F, Cl), HNNX3, HNNX2Y, and HNNXY2 (X, Y=H, F) and OCX3-, OCX2Y-, and OCXY2- (X, Y=H, F)

Bonding in the series ONXYZ (X, Y, Z=H, F, Cl), HNNX3, HNNX2Y, HNNXY2 (X, Y=H, F), and OCX3-, OCX2Y-, OCXY2- (X, Y=H, F) shows evidence of a significant ionic contribution modifying the underlying covalent bonding. Increased ionic character can be correlated with oxidation-state differences between the bound atoms and is expressed in terms of shorter bond lengths. All members of the series, with the exception of ONH3, HNNH3, and OCH3-, possess a multiple O-N, N-N, or C-O bond modified by the ionic character of that bond. The O-N, N-N, and O-C single bonds in ONH3, HNNH3, and OCH3-, respectively, show some variation in length relative to "typical" single bonds of these types due to differences in ionic character. The two highest-occupied molecular orbitals in the ONXYZ or OCXYZ- (X, Y, Z=H, F) series which are piNO or piCO (when X=Y=Z=H) exhibit a distinct shift in their nodal plane as hydrogen is replaced by fluorine. The nodal plane moves from a location between the oxygen and the nitrogen or carbon to between the nitrogen or carbon and the fluorines impacting on the nature and length of the bonds joining these atoms. The pattern of N-F and C-F bond lengths in the series, ONH3-ONF3 and OCH3--OCF3-, respectively, lends support to the idea of resonance structures of the form ONXY+ F- or OCXY F- (where X, Y=H, F).     

The CO Photodissociation and Recombination Dynamics of the W172Y/F282T Ligand Channel Mutant of Rhodobacter sphaeroides aa3 Cytochrome c Oxidase

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa₃) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa₃ were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba₃ cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild‐type Rs aa₃ and the W172Y/F282T mutant were investigated using time‐resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild‐type enzyme (~23 ms). Classical dynamic simulations of the wild‐type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a₃ ring‐D propionate toward Cu_B and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant.