人亲环素18抑制肌酸激酶的去折叠

新生肽链折叠过程中容易出现错误折叠与聚沉,从而导致折叠病等病理现象. 分子伴侣具有辅助其他蛋白质正确折叠,保护蛋白质分子结构的功能.本文选用人肌肌酸激酶为靶蛋白,研究了肽基脯氨酰顺反异构酶人亲环素18（human cyclophilin 18,hCyp18）对人肌肌酸激酶去折叠的作用,发现hCyp18能够抑制人肌肌酸激酶在热变性与化学变性过程中的失活与构象变化,并抑制人肌肌酸激酶在化学变性过程中的聚沉,因此推断hCyp18具有针对人肌肌酸激酶的分子伴侣功能.本文同时研究了hCyp18与人肌肌酸激酶的结合作用,对hCyp18的作用机制进行了初步探讨.     

盐酸胍诱导的α淀粉酶去折叠与再折叠

利用蛋白质内源荧光和酶活性两种信号以及荧光偏振,HPLC和停流等方法研究了盐酸胍诱导的α淀粉酶去折叠与重折叠的平衡转变和动力学。实验结果表明α淀粉酶去折叠与重折叠是两个不同的过程;变性与复性过程中可能伴有聚集体生成;去折叠与重折叠均为双相过程,重折叠大约始于2秒之后。     

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。     

毛细管电泳及其在蛋白质折叠研究中的应用

简要介绍了毛细管电泳的常用分离模式及其原理,并对毛细管电泳在蛋白质化学领域中的新应用——研究蛋白质折叠和发展前景作了评述。     

人肌肌酸激酶在SDS溶液中失活与构象变化的比较研究

应用紫外差吸收光谱、荧光发射光谱、ＣＤ先谱等监测手段,研究了ＳＤＳ溶液滴定人肌肌酸激酶时的构象与活力变化的关系。结果表明酶的活力丧失先于以紫外差吸收先谱、荧先发射谱和巯基暴露数目所监测到的构象变化。ＳＤＳ滴定时引起的酶的荧光发射光谱的变化在低滴定度阶段随着ＳＤＳ滴定量的增加,荧光强度下降,发射峰位红移,当ＳＤＳ浓度达到２．１ｍｍｏｌ／Ｌ时,荧光强度增大,继续增加ＳＤＳ滴定量,荧光强度又降低,发射峰位红移直至终态。紫外差吸收光谱随着ＳＤＳ溶液的加入,２８１ｎｍ．２８７ｎｍ和２９２ｎｍ的负差吸收峰增大。ＣＤ光谱结果表明在本实验所用的ＳＤＳ浓度范围内,ＳＤＳ对人肌肌酸激酶的二级结构几乎没有影响。上述结果支持了酶的活性部位构象柔性的观点。     

结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件（100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h）将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件(100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h)将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

蛋白质折叠机理的理论研究

简要综述了近年来蛋白质折叠机理的理论研究。首先回顾了蛋白质折叠理论的发展历程,然后对折叠中间体的研究现状作了较详细的介绍。同时,对折叠机理理论研究中的几种理论模型和模拟算法作了细致评述,分析了其现状和存在的问题。最后,总结和讨论了折叠机理理论研究的现存问题及研究热点,并展望了该领域研究的发展趋势。     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ和脱铜锌ＳＯＤ在不同浓度胍溶液中的去折叠及活力变化。结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失。提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏     

AUT凝胶电泳在牛胰核糖核酸酶折叠研究中的应用

目的:牛胰核糖核酸酶是一种用于蛋白折叠研究的经典模式蛋白,在折叠研究过程中主要使用高效液相色谱用于分离检测不同阶段的蛋白折叠中间体。高效液相色谱具有自动化、分离效果好、样品可回收等优点,同时也存在检测通量较低、仪器设备较为昂贵等不足。AUT凝胶电泳简便、快捷、检测通量较高,本文尝试将其应用于牛胰核糖核酸酶的折叠研究。方法:使用AUT凝胶电泳、酶活性检测、质谱对牛胰核糖核酸酶还原变性过程及产生的折叠中间体进行检测;通过高效液相色谱和质谱对折叠中间单体进行分离检测,并分别进行AUT凝胶电泳检测以解析各折叠中间单体在电泳中的条带位置;通过AUT凝胶电泳和酶切后质谱鉴定各折叠中间单体的二硫键配对方式。结果:AUT凝胶电泳可以有效区分不同条件下的牛胰核糖核酸酶还原变性过程,检测结果与酶活性、质谱结果相符,并可以很好地区分牛胰核糖核酸酶还原变性过程折叠中间体。高效液相色谱将牛胰核糖核酸酶还原变性过程折叠中间体分离为13个色谱峰,并与AUT凝胶电泳中的11个条带位置进行匹配。确认牛胰核糖核酸酶还原变性过程折叠中间单体的二硫键配对方式,并与AUT凝胶电泳条带进行匹配,Cys58-Cys110和Cys26-Cys84构象熵减作用强于Cys40-Cys95和Cys65-Cys72。结论:AUT凝胶电泳适用于检测牛胰核糖核酸酶折叠中间体,可以与高效液相色谱、质谱等检测技术相互补充,共同应用于牛胰核糖核酸酶的折叠研究。     

Equilibrium and kinetics of the unfolding and refolding of Escherichia coli Malate Synthase G monitored by circular dichroism and fluorescence spectroscopy

The equilibrium and kinetics studies of an 82 kDa large monomeric Escherichia coli protein Malate Synthase G (MSG) was investigated by far and near-UV CD, intrinsic tryptophan fluorescence and extrinsic fluorescence spectroscopy. We find that despite of its large size, folding is reversible, in vitro. Equilibrium unfolding process of MSG exhibited three-state transition thus, indicating the presence of at least a stable equilibrium intermediate. Thermodynamic parameters suggest this intermediate resembles the unfolded state. However, the equilibrium intermediate exhibits pronounced secondary structure as measured by far-UV CD, partial tertiary structure as delineated by near-UV CD, compactness (m value) and exposed hydrophobic surface area as assessed by ANS binding, typically depicting a molten globule state. The stopped-flow kinetic data provide clear evidence for the presence of a burst phase during the refolding pathway due to the formation of an early Intermediate, within the dead time of the instrument. Refolding from 4 M to various lower concentrations until 0.4 M of GdnHCl follow biphasic kinetics at lower concentrations of GdnHCl (<0.8 M), whereas monophasic kinetics at concentrations above 1.5 M. Also, rollover in the refolding and unfolding limbs of chevron plot verifies the presence of a fast kinetic intermediate at lower concentration of GdnHCl. Based upon the above observations we hereby propose the folding pathway of a large multi-domain protein Malate Synthase G.     

Effect of a specific inhibitor on the unfolding and refolding kinetics of dimeric triosephosphate isomerase: establishing the dimeric and similarly structured nature of the main transition states on the forward and backward reactions

2-Phosphoglycolate (PGA), a strong competitive inhibitor of the dimeric enzyme triosephosphate isomerase (TIM), brings about a large decrease in the unfolding rate constant of the protein. The data set of rate constants versus ligand concentration may be equally well explained by regarding either a monomeric or a dimeric transition state (TS). However, if the thermodynamics for binding of PGA to native TIM is taken into account, it becomes clear that a dimeric TS is the right assumption. Furthermore, by studying the effect of the ligand on the second-order refolding reaction, we found results indicating similar PGA-binding affinities to be present in the transition states for the rate-limiting steps of the forward and backward reactions. Most likely, therefore, both TS resemble each other in respect to the active site architecture. It should be mentioned, however, that our data do not rule out the possible occurrence of an unstable, (partially) folded monomeric intermediate, which would rapidly interconvert with the unfolded monomer.     

Effect of Zn2+ during reactivation and refolding of urea-denatured creatine kinase

The courses of refolding and reactivation of urea-denatured creatine kinase (CK) (ATP:creatine N-phosphotrans-ferase, EC 2.7.3.2) have been studied in the absence and presence of zinc ions. The presence of Zn²⁺ at low concentrations blocks the reactivation and refolding of urea-denatured CK and keeps it in a partially folded state. The partially folded state proved to be a monomeric state which resembles the molten globule state in the CK folding pathway. During refolding in the presence of Zn²⁺ , creatine kinase forms aggregates with the aggregation dependent on zinc concentration and temperature. In the presence of EDTA, the partially folded creatine kinase can be reactivated and refolded following a biphasic course, suggesting the existence of a monomeric intermediate during the refolding of CK. The results also suggest that low concentrations of zinc ions might be toxic to some proteins such as creatine kinase by disrupting their proper folding.     

Abstracts: Albany 2007, The 15th Conversation

Abstract

Forty nine molecular dynamics simulations of unfolding trajectories of the segment B1 of streptococcal protein G (GB1) provide a direct demonstration of the diversity of unfolding pathway and give a statistically utmost unfolding pathway under the physical property space. Twelve physical properties of the protein were chosen to construct a 12-dimensional property space. Then the 12-dimentional property space was reduced to a 3-dimentional principle component property space. Under the property space, the multiple unfolding trajectories look like “trees”, which have some common characters. The “root of the tree” corresponds to the native state, the “bole” homologizes the partially unfolded conformations, and the “crown” is in correspondence to the unfolded state. These unfolding trajectories can be divided into three types. The first one has the characters of straight “bole” and “crown” corresponding to a fast two-state unfolding pathway of GB1. The second one has the character of “the standstill in the middle tree bole”, which may correspond to a three-state unfolding pathway. The third one has the character of “the circuitous bole” corresponding to a slow two-state unfolding pathway. The fast two-state unfolding pathway is a statistically utmost unfolding pathway or preferred pathway of GB1, which occupies 53% of 49 unfolding trajectories. In the property space all the unfolding trajectories construct a thermal unfolding pathway ensemble of GB1. The unfolding pathway ensemble resembles a funnel that is gradually emanative from the native state ensemble to the unfolded state ensemble. In the property space, the thermal unfolded state distribution looks like electronic cloud in quantum mechanics. The unfolded states of the independent unfolding simulation trajectories have substantial overlaps, indicating that the thermal unfolded states are confined by the physical property values, and the number of protein unfolded state are much less than that was believed before.     

Acid induced unfolding of anthrax protective antigen

Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.     

Despite its high similarity with monomeric arginine kinase, muscle creatine kinase is only enzymatically active as a dimer

Although having highly similar primary to tertiary structures, the different guanidino kinases exhibit distinct quaternary structures: monomer, dimer or octamer. However, no evidence for communication between subunits has yet been provided, and reasons for these different levels of quaternary complexity that can be observed from invertebrate to mammalian guanidino kinases remain elusive. Muscle creatine kinase is a dimer and disruption of the interface between subunits has been shown to give rise to destabilized monomers with slight residual activity; this low activity could, however, be due to a fraction of protein molecules present as dimer. CK monomer/monomer interface involves electrostatic interactions and increasing salt concentrations unfold and inactivate this enzyme. NaCl and guanidine hydrochloride show a synergistic unfolding effect and, whatever the respective concentrations of these compounds, inactivation is associated with a dissociation of the dimer. Using an interface mutant (W210Y), protein concentration dependence of the NaCl-induced unfolding profile indicates that the active dimer is in equilibrium with an inactive monomeric state. Although highly similar to muscle CK, horse shoe crab (Limulus polyphemus) arginine kinase (AK) is enzymatically active as a monomer. Indeed, high ionic strengths that can monomerize and inactivate CK, have no effect on AK enzymatic activity or on its structure as judged from intrinsic fluorescence data. Our results indicate that expression of muscle creatine kinase catalytic activity is dependent on its dimeric state which is required for a proper stabilization of the monomers.     

Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg²⁺ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg²⁺ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg²⁺; however, the rates of reactivation are independent of the Mg²⁺ concentration. In addition, Mg²⁺ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg²⁺ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg²⁺ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.     

The protective effects of osmolytes on arginine kinase unfolding and aggregation

Osmolytes are a series of different kinds of small molecules that can maintain the correct conformation of protein by acting as molecular chaperons. In this study, the protective effects of four compatible osmolytes, i.e., proline, sucrose, DMSO and glycerol, were studied during arginine kinase (EC 2.7.3.3) unfolding and aggregation. The results showed that all the osmolytes applied in this study obviously prevented AK unfolding and inactivation that was due to a GdnHCl denaturant by reducing the inactivation rate constants (k_i), increasing the transition free energy changes (ΔΔG_i) and increasing the value for the midpoint of denaturation (C_m). Furthermore, the osmolytes remarkably prevented AK aggregation in a concentration-dependent manner during AK refolding. Our results strongly indicated that osmolytes were not only metabolism substrates, but they were also important compounds with significant physiological protective functions for proteins, especially in some extremely harsh environments.