N-溴代琥珀酰亚胺修饰菌紫质中色氨酸的光谱学研究

采用紫外-可见吸收光谱和荧光光谱方法研究了菌紫质（Bacteriorhodopsin,bR）中的8个色氨酸（Tryptophan,Trp）残基在被N-溴代琥珀酰亚胺（N-bromosuccinimide,NBS）修饰过程中的残基数目及对应的光谱变化。研究结果显示：随着NBS／bR摩尔比例增加逐渐被修饰的Trp残基有4个左右,如果NBS过量,则Trp残基的修饰个数最终可迭6～7个;伴随化学修饰出现Trp残基特征荧光峰值下降及峰位蓝移。研究结果揭示了bR中Trp残基可能的三种结构分布,对于进一步弄清bR中Trp-视黄醛（Retinal）偶联能量传递、单独Trp残基的荧光寿命和Tm残基在膜蛋白结构和功能中的作用具有积极而重要的意义。     

色氨酸残基在内切葡聚糖酶分子中的作用

内切葡聚糖酶的化学修饰研究表明：色氨酸残基可能位于活性位点,与底物结合有关．荧光光谱测定指出该酶的荧光几乎都来自色氨酸残基,酶分子中色氨酸微环境对ｐＨ变化非常敏感,降低ｐＨ导致了酶分子构象发生了较大变化,配基结合使酶分子色氨酸微环境产生了改变,引发了与ｐＨ诱导不同的构象变化．     

硒与红细胞血影收缩蛋白(Spectrin)作用导致构象变化

从人红细胞膜提取血影收缩蛋白(Spectrin),研究不同浓度Na_2SeO_3与其作用后的构象变化.用N—[3—芘]—马来酰胺(N-[3-P]M)作荧光探针标记Spectrin,经SDS处理后,其荧光强度随硒的浓度增加而逐步降低.但未经SDS处理的样品,加入0.2—1.0ppm的Na_2SeO_3后反而使荧光强度有所增加.Spectrin经硒作用与未经作用相比较在色氨酸内源荧光、丹磺酰氯(DNS-Cl)标记后(DNS-Spectrin)的荧光光谱以及色氨酸残基与DNS基团间的能量转移实验结果均有明显的差别.这反映Spectrin的巯基经与硒作用后会导致构象的变化.     

兔肌醛缩酶中色氨酸残基的分布及其作用

在不同条件下,用NBS修饰兔肌醛缩酶的色氨酸残基。pH4.0时测定到全酶分子的总色氨酸残基数为12,用邹氏图解法求得其中2个色氨酸残基为表现活性所必需。而在pH7.5条件下,仅鉴定出2个色氨酸残基。这些实验表明此2个色氨酸残基很可能就位于分子表面。此外,紫外光谱和萤光光谱指出,pH4.0时,NBS引起酶构型的较大的变化,而在pH7.5时仅引起较轻微变化。这些结果认为:醛缩酶的四个亚基对整个分子构象的贡献是不完全相同的,同时醛缩酶整个分子也是不对称的。     

兔肌醛缩酶中色氨酸残基的分布及其作用

在不同条件下,用NBS修饰兔肌醛缩酶的色氮酸残基。pH4.0时测定到全酶分子的总色氨酸残基数为12,用邹氏图解法求得其中2个色氨酸残基为表现活性所必需。而在pH7.5条件下,仅鉴定出2个色氨酸残基。这些实验表明此2个色氨酸残基很可能就位于分子表面。此外,紫外光谱和萤光光谱指出,pH4.0时,NBS 引起酶构型的较大的变化,而在pH7.5时仅引起较轻微变化。这些结果认为:醛缩酶的四个亚基对整个分子构象的贡献是不完全相同的,同时醛缩酶整个分子也是不对称的。     

酰化菌紫质的动力学光谱及光电特性研究

用人工双分子膜(BLM)技术及动力学光谱研究了赖氨酸残基在紫膜的结构和功能中所起的作用.酰化基团与菌紫质(bR)分子中的赖氨酸残基的ε氨基作用,使光照后bR的跨膜质子迁移信号及膜的充放电速度变慢,光循环中间产物M412的产量下降,半衰期延长.但UV/Vis吸收光谱表明酰化并未破坏bR中视黄醛的构象环境.在高pH或高盐浓度下,酰化的影响降低.这些结果表明:赖氨酸残基并不是泵出质子的提供者,没有直接参与质子的跨膜输运,而是通过表面电位来影响bR的质子泵功能.     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。     

葡萄糖淀粉酶色氨酸残基及底物结合位点的研究

 本文用N-溴代琥珀酰亚胺(NBS)对葡萄糖淀粉酶进行特异性修饰,当酶分子表面有3个色氨酸残基被修饰后,酶活力完全丧失。用邹氏图解法测得酶活性中心有一个色氨酸残基是必需的。如果在酶液中加入不同的底物再用NBS氧化,用荧光发射和荧光猝灭光谱检测表明,底物对酶分子有不同程度的保护作用。在被测试的三种底物中,这种保护能力依为糊精>淀粉>麦芽糖。     

人肌和兔肌肌酸激酶在低pH条件下再折叠的研究

利用蛋白质内源荧光和远紫外ＣＤ光谱研究了在低ｐＨ条件下人肌肌酸激酶和兔肌肌酸激酶的构象状态。结果表明,在低离子强度下,随着酸的加入人肌和兔肌肌酸激酶去折叠,至ｐＨ２．０附近几乎都达到充分去折叠。它们的色氨酸荧光发射峰位红移至３５０ｎｍ,这表明了内埋的色氨酸残基已经完全暴露到极性溶剂之中,它们的远紫外ＣＤ光谱表明二级结构也遭破坏,但是仍保留有相当部分的二级结构。而且在高离子强度低ｐＨ条件下由Ｇｏｔｏ等人首先发现的中间体构象状态（融球结构状态）在我们的实验中也被观察到了。它具有与天然酶类似的二级结构。色氨酸荧光发射光谱表明与天然酶类似,它的最大发射峰位也为３３５ｎｍ表明了蛋白质已经完全折叠,然而其荧光强度远低于天然酶,表明这种结构状态具有较大运动性而导致荧光的动态淬灭。上述结果支持了Ｇｏｔｏ等人的发现,说明了融球中间体结构可能是蛋白质折叠过程需经历的一个中间态。     

铝与钙调蛋白相互作用及其作用位点的研究

用500MHzNMR研究了铝与钙调蛋白的相互作用,主要研究了铝对钙调蛋白中芳香氨基酸残基(Tyr,His,Phe)构象变化的影响。实验结果表明,铝在钙饱和的钙调蛋白上存在着特异性的结合位点,结合位点数目至少为两个,第一结合位点可能位于钙调蛋白的N端结构域,第二结合位点靠近Ca~(2+)的Ⅲ结合域。Al~(3+)结合引起脱钙的钙调蛋白的构象变化不同于与Ca~(3+)结合引起的构象变化。Al~(3+)在CaM上的结合位点与Ca~(2+)的并不相同。柠檬酸等有机酸对铝的毒性有保护作用,这种保护作用是由于柠檬酸分子对铝的络合。     

Time resolved fluorescence and phosphorescence properties of the individual tryptophan residues of barnase: evidence for protein-protein interactions.

Steady-state and time-resolved fluorescence, as well as phosphorescence measurements, were used to resolve the luminescence properties of the three individual tryptophan residues of barnase. Assignment of the fluorescence properties was performed using single-tryptophan-containing mutants and the results were compared with the information available from the study of wild-type and two-tryptophan-containing mutants (Willaert, Lowenthal, Sancho, Froeyen, Fersht, Engelborghs, Biochemistry 1992;31:711-716). The fluorescence and the phosphorescence emission of wild-type barnase is dominated by Trp35, although Trp71 has the strongest intrinsic fluorescence when present alone. Fluorescence emission of these two tryptophan residues is blue-shifted and pH-independent. The fluorescence decay parameters of Trp94 are pH-dependent, and an intramolecular collision frequency of 2 to 5 x 10(9) s(-1) between Trp94 and His18 is calculated. Fluorescence emission of Trp94 is red-shifted. Fluorescence anisotropy decay reveals the local mobility of the individual tryptophan residues and this result correlates well with their phosphorescence properties. Trp35 and Trp71 display a single phosphorescence lifetime, which reflects the rigidity of their environment. Surface Trp94 does not exhibit detectable phosphorescence emission. The existence of energy transfer between Trp71 and Trp94, as previously detected by fluorescence measurements, is also observed in the phosphorescence emission of barnase. Dynamic quenching causes the phosphorescence intensity to be protein-concentration dependent. In addition, fluorescence anisotropy shows concentration dependency, and this can be described by the formation of trimers in solution.     

Fluorescence properties of glutamine-binding protein from Escherichia coli and its complex with glutamine

In this work, the fluorescence of glutamine-binding protein (GlnBP) and its complex with glutamine (GlnBP/Gln) in native and unfolded forms was studied. The experimental data were interpreted on the basis of the results of the analysis of Trp and Tyr microenvironments taking into the account the data for GlnBP mutated forms Trp32Phe(Tyr) and Trp220Phe(Tyr), which have been obtained by Axelsen et al. (Biophys. J. 1991, 60, 650-659). This allowed us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of the fluorescence characteristics of GlnBP and GlnBP/Gln, and the uncommon effect of the excess of the fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm respect to the excitation at 280 nm. The last effect is explained by the spectral dependence of the Trp 32 and Trp 220 contributions to the protein absorption. The protein Trp fluorescence dependence on the excitation wavelength must be taken into account for the evaluation of the Tyr residues contribution to the bulk fluorescence of protein, and in principle, it also may be used for the development of an approach for the decomposition of a multicomponent protein fluorescence spectrum.     

A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.     

The structure and stability of the glutamine-binding protein from Escherichia coli and its complex with glutamine

A study was made of the conformational changes in the Escherichia coli glutamine-binding potein (GlnBP) induced by GdnHCl, and of the effect of glutamine (Gln) binding on these processes. Intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy were used. The obtained experimental data were interpreted, taking into the account results of the analysis of tryptophan and tyrosine residues microenvironments. This enabled us to explain the negligible contribution of Tyr residues to the bulk fluorescence of the native protein, the similarity of fluorescence characteristics of GlnBP and GlnBP/Gln, and an uncommon effect of the excess of fluorescence intensity at 365 nm (Trp emission) upon excitation at 297 nm compared to the excitation at 280 nm. The latter effect is explained by the spectral dependence of Trp 32 and Trp 220 contributions to protein absorption. The dependence of Trp fluorescence of protein on the excitation wavelength must be taken into account for the evaluation of Tyr residues contribution to the bulk fluorescence of protein, and in principle, it may also be used for the development of an approach to decomposition of multi-component protein fluorescence spectrum. The parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP/Gln unfolding are three-step processes (N-->I1-->I2-->U), though in the case of the GlnBP/Gln complex these stages essentially overlap. Despite its complex character, GlnBP unfolding is completely reversible. In comparison with GlnBP, in the case of GlnBP/Gln the dramatic shift of N-->I1 process to higher GdHCl concentrations is shown.     

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues

We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

Isolating and localizing ATP-sensitive tryptophan emission in skeletal myosin subfragment 1

The fluorescence intensity difference between rabbit skeletal myosin subfragment 1 (S1) and nucleotide-bound or trapped S1 isolates ATP-sensitive tryptophans (ASTs) emission from the total tryptophan signal. Neutral (acrylamide) quenching of the ASTs is sensitive to the binding or trapping of nucleotide to the active site of S1. Anion (I(-)) quenching of the ASTs, sensitive to charge separation in the tryptophan micro environment, is negligible. These findings suggest the ASTs sense conformational change during ATPase from negatively charged surroundings. Specific chemical modifications of S1 identified the location of the ASTs. Trp131 was quenched by chemical modification, and its emission was isolated by taking the intensity difference between unmodified and modified S1. Trp131 fluorescence intensity and quenching constant do not distinguish among the bound or trapped nucleotides, suggesting that the vicinity of Trp131 does not change conformation during the ATPase cycle and eliminating Trp131 as an AST. Trp510 fluorescence was quenched by 5'-iodoacetamidofluorescein (5'IAF) modification of the reactive thiol (SH1) of S1. The tryptophan emission enhancement increment due to active site trapping decreases linearly with SH1 modification and extrapolates to 0 for 100% modification. These data identify Trp510 as the primary AST in skeletal S1 in agreement with observations from Dictyostelium (Batra and Manstein (1999) Biol. Chem. 380, 1017-1023) and smooth muscle S1 (Yengo et al. (2000) Biophys. J. 78, 242A). With Trp510 identified as the sole AST, fluorescence difference spectroscopy provides a novel means to monitor the concentration of myosin transient intermediates in ATP hydrolysis.