全新设计蛋白质的折叠机制

蛋白质全新设计和折叠研究是从两个不同的方向来理解蛋白质序列-结构-功能关系这一结构生物学重要问题. 蛋白质全新设计取得的成功实例一定程度上检验了人们对蛋白质结构和相互作用理解的准确性, 但它们中多数所表现的不同于天然蛋白质的折叠动力学特征也表明, 要达到最终的功能化实现目标还面临着不少的挑战. 本文综述了蛋白质全新设计的发展过程及现状, 蛋白质折叠研究在实验、理论及模拟方面的研究进展, 以及全新设计蛋白质的折叠机制的研究现状. 阐述了深入了解全新设计蛋白质与天然蛋白质折叠机制的不同, 可以为进一步有效地合理化设计蛋白质提供有益的参考.     

准确快速计算含多肽酰胺和核酸碱基的氢键复合物的氢键强度和氢键作用势能曲线

N-H···O=C、C-H···O=C、N-H···N和C-H···N等氢键作用是蛋白质a-螺旋结构、b-折叠结构和DNA双螺旋结构形成的主要因素,在生物分子识别、蛋白质复制以及遗传信息传递等过程中起重要作用。准确快速计算生物体系中存在的N-H···O=C、C-H···O=C、N-H···N和C-H···N等氢键作用强度以及氢键强度随分子几何结构(距离和角度)变化的势能曲线对正确模拟(从而正确认识和理解)蛋白质折叠机制和DNA双螺旋结构形成机制等生物过程意义重大,对设计合成具有特殊功能的生物分子材料有重要指导价值。本文主要介绍了近年来建立的偶极-偶极氢键作用模型及其在快速预测多肽-多肽分子间和核酸碱基-多肽分子间氢键作用强度和氢键作用势能曲线方面的应用。     

蛋白折叠中的暂态结构与表面水分子慢尺度动力学

蛋白表面水的慢尺度动力学行为往往被认为与蛋白的结构稳定性、功能以及折叠过程有关, 但在分子水平上, 还不清楚水分子的慢尺度动力学如何参与蛋白折叠过程. 以Trp-cage蛋白作为个案, 本文利用40条100 ns(总长4 μs)的全原子分子动力学轨迹,分析了蛋白折叠过程中蛋白表面水分子的停留行为,并探究影响蛋白表面水分子慢尺度行为的微观因素. 结果发现, 即使在蛋白折叠过程中蛋白拓扑结构变化很大, 残基之间也会形成稳定的局部暂态结构. 这些结构为水分子提供饱和、稳定的氢键, 通过与水分子之间的极性相互作用, 以及凹形的几何结构, 约束水分子长时停留, 我们称之为“停留中心”. 停留中心的形成是引起水分子慢尺度行为的重要因素. 另外, 停留中心的分布与蛋白折叠的进程有密切关系, 特别地, 在折叠轨迹中, 疏水核周围的残基组成了一个主要的停留中心. 研究结果不但有助于解释水分子慢尺度特征行为的来源, 还可以为实验中通过研究水分子在蛋白附近的慢尺度行为, 揭示蛋白折叠过程中的关键步骤提供一些启发.     

静电排斥表面诱导溶菌酶分子站立

抑制蛋白质聚集是应用基因重组技术生产药用蛋白质过程中的关键. 实验研究发现与蛋白质带同种电荷的离子交换介质能够通过静电排斥作用有效抑制蛋白质折叠中间体的聚集. 但其微观细节尚不明晰, 且利用现有实验技术很难直接阐释. 分子动力学模拟是研究微观过程的有力工具. 因此, 本文构建了静电排斥表面模型以模拟同电荷离子交换介质, 采用分子动力学模拟和全原子模型, 研究溶菌酶在静电排斥表面上的空间取向及其变化过程, 并考察表面所带电荷数的影响规律. 结果表明, 溶菌酶受到表面的静电排斥作用而远离. 在此过程中, 溶菌酶逐渐“站立”, 形成其偶极和表面相站立垂直的空间取向. 而当蛋白质远离表面时, 由于静电排斥作用衰减, 形成“站立”取向的趋势减弱. 同时, 研究发现静电排斥表面所带电荷数增加有利于蛋白质形成“站立”取向. 本文的模拟结果从微观揭示蛋白质在静电排斥表面上的空间取向及其影响因素, 将有助于推动蛋白质在荷电表面折叠和分子相互作用研究.     

VirB7去折叠的全原子分析

采用分子动力学模拟的方法,对VirB7蛋白的球状结构域在550 K高温下的去折叠行为进行了统计分析.结果表明,VirB7蛋白球状结构域的去折叠过程以一条路径为主,多条路径并存,其去折叠过程的发生顺序依赖于其内部疏水残基的分布及其独特的三明治结构.该研究揭示了VirB7蛋白球状结构域在去折叠过程中动力学特征,对于揭示其生物学分子机制具有一定价值.     

人类朊蛋白的动力学稳定性研究（英文）

朊蛋白病是一种能在人类或者动物之间传播的致命的神经退行性疾病.尤其是人类朊蛋白疾病在近几年蔓延迅速,已经威胁到人类的健康.在本文中,我们使用分子动力学(MD)和流体分子动力学(FMD)模拟相结合的方法研究了人类朊蛋白(hPrPc)的动力学稳定性.我们通过FMD模拟产生了两个典型的hPrPc的变性结构,并进一步研究了在自然状态下这两个变性结构重折叠的过程,从关键残基、二级结构、残基-残基相互作用图等方面详细讨论了hPrPc的解折叠和重折叠路径.研究发现hPrPc的三个α-螺旋结构组成了一个疏水核心,在蛋白质的解折叠和重折叠过程中发挥了重要的作用.刚性的疏水核心就像是脚手架一般为hPrPc的重折叠提供便利.在重折叠过程中,π-螺旋和310螺旋出现几率较高,并且β-折叠的延长也更多地出现在完全解折叠的hPrPc体系中.     

无序随机多肽组分相关的结构转变的蒙特卡洛模拟:以赖氨酸、谷氨酸和异亮氨酸组成的随机多肽为例

无序蛋白和折叠蛋白二者在结构和序列组成上存在着明显的差异.是疏水相互作用还是静电相互作用诱导了多肽结构的转变?在多肽结构转变过程中,疏水相互作用和静电相互作用各自发挥着什么样的作用?本工作以正（赖氨酸）、负（谷氨酸）和疏水性（异亮氨酸）的三种氨基酸为组分,产生了一系列电中性的无序随机多肽系统.利用全原子模型并采用蒙特卡洛方法进行了大规模计算模拟.结果表明,随着温度升高,多肽将从紧密构象转变到扩展构象.不同的多肽其转变温度依赖于疏水性氨基酸和带电氨基酸的比例.当平均疏水性低于临界疏水性时,转变温度低于室温;当平均疏水性大于临界疏水性时,转变温度高于室温.定量分析发现,临界疏水性数值与生物信息研究的结论是吻合的.此外,统计氨基酸残基之间的接触对数目表明,在多肽结构的转变过程中疏水作用发挥着主要作用.研究结果对蛋白质序列与结构关系的研究具有一定的理论指导意义,期望对基于序列的蛋白质全新设计提供参考.     

EK多肽折叠以及离子浓度和pH值依赖性的计算研究

我们分别运用了传统AMBER03力场以及极化力场研究了EK多肽在不同离子浓度和pH值条件下的折叠.其中极化力场源于我们近期发展的可调的氢键专一性电荷方案.这两种力场的差别仅仅在于原子电荷的不同.溶剂化效应用推广的波恩模型描述.结果表明,当使用AMBER03电荷时,尽管多肽倾向于形成螺旋结构,但是对离子浓度和pH值的依赖关系定性上是错误的.而当使用可调的氢键专一性电荷方案时,EK多肽在10ns内就达到了折叠态.在高离子浓度或者极端pH条件下,计算得到的螺旋结构的概率降低.对于原子电荷以及主链氢键相互作用的分析表明极化效应增加了螺旋结构的稳定性.该研究结果再一次证明了静电极化效应对提高传统力场的精度以及对蛋白质折叠的研究都是非常必要的.进一步的分析说明,间隔4个残基的盐桥作用虽然对稳定蛋白质结构起到了一定的效果,但并不是关键作用,这和实验是吻合的.     

温控分子动力学研究微管蛋白活性肽链的折叠机制

运用温控和常温分子动力学方法, 研究了微管蛋白活性部位Pep1-28肽链的折叠机制, 总模拟时间为380.0 ns. 对于温控分子动力学, 逐渐降温可以清晰显示Pep1-28肽链的折叠途径, 发生明显折叠的温度约为550 K, 其折叠和去折叠可逆机制为U(>1200 K)←→I1(1200-1000 K)←→I2(800 K)←→I3(600 K)←→I4(450 K)←→F1(400 K)←→F2(300 K), 其中U为去折叠态构象, I1、I2、I3和I4是折叠过程中的四个重要的中间态构象, F1和F2是两个结构相近的折叠态构象. 对于常温(300 K)分子动力学, 其构象转变和折叠过程相当迅速, 很难观察到有效、稳定的中间态构象. 尤其引人注意的是, 其折叠态结构陷入了能量局域极小点, 与温控(300 K)的有较大差异, 两者能量差高达297.53 kJ·mol-1. 可见, 温控分子动力学方法不仅清晰地显示多肽和蛋白质折叠过程的重要中间态构象, 为折叠和去折叠机制提供直接、可靠的依据, 而且还有助于跨越较高的构象能垒, 促使多肽和蛋白质折叠以形成全局能量最低的稳定结构.     

甘氨酸从水到有机物水溶液的迁移焓研究

蛋白质是各种生物形态结构和生命活动所依赖的物质基础,在水溶液中蛋白质天然结构的稳定性归结于氨基酸残基之间以及与溶液中其它组分的相互作用．天然环境中存在的众多物质对蛋白质的溶解度、变性行为和解缔等都有很大的影响．为深入了解蛋白质折叠与解折叠过程中的物理化学现象,以氨基酸、肽、酰胺及其衍生物作为蛋白质模型分子的热力学研究引起了广泛重视．     

Folding dynamics of Trp-cage in the presence of chemical interference and macromolecular crowding. I

Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.     

Simulation of the thermodynamics of folding and unfolding of the Trp-cage mini-protein TC5b using different combinations of force fields and solvation models

Molecular dynamics simulations based on AMBER force fields(ff96 and ff03) and generalized Born models(igb1 and igb5) have been carried out in order to study folding/unfolding of the Trp-cage mini-protein TC5b.The thermodynamic properties of TC5b were found to be sensitive to the specific version of the solvation model and force field employed.When the ff96/igb5 combination was used,the predicted melting temperature from unfolding simulations was in good agreement with the experimental value of 315 K,but the...     

The equilibrium properties and folding kinetics of an all-atom Go model of the Trp-cage

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial alpha-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.     

Shedding light on biomolecule conformational dynamics using fluorescence measurements of trapped ions

Biomolecule conformational change has been widely investigated in solution using several methods; however, much less experimental data about structural changes are available for completely isolated, gas-phase biomolecules. Studies of conformational change in unsolvated biomolecules are required to complement the interpretation of mass spectrometry measurements and in addition, can provide a means to directly test theoretical simulations of biomolecule structure and dynamics independent of a simulated solvent. In this Feature Article, we review our recent introduction of a fluorescence-based method for probing local conformational dynamics in unsolvated biomolecules through interactions of an attached dye with tryptophan (Trp) residues and fields originating on charge sites. Dye-derivatized biomolecule ions are formed by electrospray ionization and are trapped in a variable-temperature quadrupole ion trap in which they are irradiated with either continuous or short pulse lasers to excite fluorescence. Fluorescence is measured as a function of temperature for different charge states. Optical measurements of the dye fluorescence include average intensity changes, changes in the emission spectrum, and time-resolved measurements of the fluorescence decay. These measurements have been applied to the miniprotein, Trp-cage, polyproline peptides and to a beta-hairpin-forming peptide, and the results are presented as examples of the broad applicability and utility of these methods. Model fits to Trp-cage fluorescence data measured as a function of temperature provide quantitative information on the thermodynamics of conformational changes, which are reproduced well by molecular dynamics. Time-resolved measurements of the fluorescence decays of Trp-cage and small polyproline peptides definitively demonstrate the occurrence of fluorescence quenching by the amino acid Trp in unsolvated biomolecules.     

Folding and thermodynamic studies of Trp-cage based on polarized force field

Two replica exchange molecular dynamics (REMD) simulations were carried out to study the thermodynamics of a 20-residue Trp-cage folding based on a newly developed polarized protein-specific charge (PPC). Starting from a fully extended conformation, Trp-cage native conformation was successfully sampled using REMD based on a 3-step PPC update. Next, the obtained Trp-cage folded conformation was then used to calculate the PPC in which another REMD was performed to explore the thermodynamic stability of Trp-cage. The theoretical melting temperature T _m of ≈325 K was found to be in close agreement with experimental melting temperature, T _m of 315 K. This indicates that the PPC was correctly predicting the temperature dependence. The current study provides a direct proof of how electrostatic polarization affects protein folding.     

UV-resonance raman thermal unfolding study of Trp-cage shows that it is not a simple two-state miniprotein

Trp-cage, a synthetic 20 residue polypeptide, is proposed to be an ultrafast folding synthetic miniprotein which utilizes tertiary contacts to define its native conformation. We utilized UV resonance Raman spectroscopy (UVRS) with 204 and 229 nm excitation to follow its thermal melting. Our results indicate that Trp-cage melting is complex, and it is not a simple two-state process. Using 204 nm excitation we probe the peptide secondary structure and find the Trp-cage's alpha-helix shows a broad melting curve where on average four alpha-helical amide bonds melt upon a temperature increase from 4 to 70 degrees C. Using 229 nm excitation we probe the environment of the Trp side chain and find that its immediate environment becomes more compact as the temperature is increased from 4 to 20 degrees C; however, further temperature increases lead to exposure of the Trp to water. The chi(2) angle of the Trp side chain remains invariant throughout the entire temperature range. Previous kinetic results indicated a single-exponential decay in the 4-70 degrees C temperature range, suggesting that Trp-cage behaves as a two-state folder. However, this miniprotein does not show clear two-state behavior in our steady-state studies. Rather it shows a continuous distribution of steady-state spectral parameters. Only the alpha-helix melting curve even hints of a cooperative transition. Possibly, the previous kinetic results monitor only a small region of the Trp-cage which locally appears two-state. This would then argue for spatially decoupled folding even for this small peptide.     

Effects of amino acid phi,psi propensities and secondary structure interactions in modulating H alpha chemical shifts in peptide and protein beta-sheet.

H alpha chemical shifts are often used as indicators of secondary structure formation in protein structural analysis and peptide folding studies. On the basis of NMR analysis of model beta-sheet and alpha-helical peptides, together with a statistical analysis of protein structures for which NMR data are available, we show that although the gross pattern of H alpha chemical shifts reflects backbone torsion angles, longer range effects from distant amino acids are the dominant factor determining experimental chemical shifts in beta-sheets of peptides and proteins. These show context-dependent variations that aid structural assignment and highlight anomalous shifts that may be of structural significance and provide insights into beta-sheet stability.     

The interplay of turn formation and hydrophobic interactions on the early kinetic events in protein folding

While both turn formation and hydrophobic interactions play dominant roles in the initiation of protein folding, their individual contributions to the folding kinetics and to the structural stability of the protein still remain poorly understood. Here, we applied a photolabile linker to "cage" some important structural motifs, including both α-helices and β-sheets, into their non-native states. These "caged" structural motifs are then relaxed by laser-flash photolysis and their refolding events followed by photoacoustic calorimetry (PAC) and photothermal beam deflection (PBD). These experiments, combined with our previous results, revealed that spontaneous α-helix formation can occur extremely rapidly (10(8)-10(9) s(-1)) if the process is driven solely by turn formation followed by helix propagation. However, if sequestering of the side chains of hydrophobic amino acid residues participates in the refolding process, which may provide additional driving force beyond that afforded by turn formation alone, the refolding rate will be retarded, often by many orders of magnitude. This is usually the case in the formation of three-stranded β-sheets (10(7)-10(8) s(-1)) and β-hairpins (10(5)-10(6) s(-1)). Thus, we propose that proteins take advantage of the hierarchy of timescales associated with either turn formation, hydrophobic interactions, or global collapse of tertiary structure to accomplish the folding process in an orderly fashion, as these events are sufficiently separated in time and do not interfere with one another.