用全内反射瞬逝场照明磁镊研究Bloom解旋G-四联体

G-四联体(G-quadruplex,G4)是广泛存在于细胞基因组中的一种DNA结构,在DNA的代谢如复制、转录、同源重组等过程中起重要作用.G4解旋酶近年来受到广泛研究,其中Bloom(BLM)解旋酶的研究已经相当丰富,但仍有一些基本问题不清楚.我们应用全内反射瞬逝场照明磁镊对BLM解旋G4的动力学过程进行了深入研究,观察到了BLM解旋G4的分步过程.相对于单分子荧光共振能量转移技术而言,借助磁镊的长时间观测性能,我们在近饱和三磷酸腺苷(ATP)浓度的实验体系中观察到BLM长时间反复解开G4或者长时间维持G4于打开状态的两种作用方式.最后,使用相同的实验条件做了单分子荧光共振能量转移实验,确定了加载2-3 pN的外力对BLM解旋G4没有显著影响.     

钙离子调控微丝切割蛋白中A6亚基解折叠的单分子力谱研究

在生命活动中,金属离子扮演了非常重要的角色.微丝切割蛋白(adseverin)需要钙离子的活化才能行使其切割肌动蛋白微丝的功能.本文通过基于原子力显微镜的单分子力谱研究了微丝切割蛋白C端末的A6亚基在结合钙离子前后的力学解折叠机理.实验结果显示:在未结合钙离子时,A6的解折叠表现为两态过程;在结合钙离子后A6力学稳定性显著提高;同时,钙离子的结合使得A6解折叠过程中出现稳定的中间态.通过对中间态的链长的分析,我们推测了中间态对应着A6的N端部分解折叠.而这一部分的解折叠可以使得掩藏在该结构后的A5亚基中肌动蛋白微丝结合位点暴露,从而促使微丝切割蛋白执行功能.我们的实验结果为理解微丝切割蛋白的工作原理提供了新的实验证据.     

光谱法研究BLM解旋酶的稳定性

BLM解旋酶是RecQ家族DNA解旋酶中的一个重要成员,在维持染色体的稳定性中具有重要的作用,其突变会导致Bloom综合症.Bloom综合症是一种罕见隐性常染色体遗传疾病,患者易患各种类型癌症.本研究利用紫外吸收光谱法研究了pH、反应时间、NaCl浓度对BLM解旋酶的稳定性和ATP酶活性的影响.结果显示,pH会影响BL...     

pH Induces Thermal Unfolding of UTI: An Implication of Reversible and Irreversible Mechanism Based on the Analysis of Thermal Stability, Thermodynamic, Conformational Characterization

The thermal unfolding of Urinary Trypsin Inhibitor (UTI) was studied by several methods: Circular Dichroism (CD), Fluorescence and UV–Vis spectra. Thermal melting of UTI, dissolved in the neutral and basic buffers, was proved to be irreversible and two domains of UTI unfolded simultaneously, but the melting was reversible and the intermediate was observed when pH is lower than 4.2. The result suggested that heat and changes in pH, which had a more important impact on the stabilization of the domain I and the interaction between two domains, might cause different unfolding transitions. A reasonable explanation was deduced for the mechanism of reversible and irreversible thermal unfolding based on the effect of pH on the protein structure, the analysis of thermal transitions and the result of Electron Microscopy: In neutral and basic buffers, the Reactive Central Loop (RCL) in domain II can interact with or insert into the partial expanding domain I and UTI become self-polymerization, however, no aggregation can be observed in acid buffer since low pH and heat destabilized the structure of the domain I and the native conformation can restructure. The interaction between the special structural element RCL and domain I play an important role in the formation of polymer which was different from other two reasons given by other authors—the cleavage of disulfide and the formation of irregular polymer mainly based on hydrophobic interaction.     

Mössbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Mössbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Mössbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.     

全内反射瞬逝场照明高精度磁镊及其在DNA解旋酶研究中的应用

传统磁镊的测量精度受限于磁球的布朗涨落, 当磁力小于约10 pN时, 磁球的布朗涨落明显增大, 对应磁镊的空间分辨率显著下降. 为了提高传统磁镊在小力条件下的测量精度, 本文将全内反射荧光技术引入到磁镊技术中, 并建立相适应的“磁球-手柄-荧光微球-待测生物分子”单分子连接系统, 在小力条件下(小于10 pN)获得纳米量级的测量精度. 应用改进的磁镊对DNA发卡的折叠-去折叠态的转变过程进行了研究, 依据DNA发卡的折叠-去折叠态转变的性质对全内反射场的穿透深度进行了校正, 并结合实验结果对改进后的磁镊的测量精度进行分析. 观察了Bloom解旋酶的解旋动力学过程, 获得初步实验结果, 证实了改进的磁镊在单分子研究中的实用性. 关键词： 磁镊 全内反射荧光 DNA发卡 解旋酶     

Pressure-assisted cold unfolding of proteins and its effects on the conformational stability compared to pressure and heat unfolding

Abstract

The pressure-assisted cold unfolding of metmyoglobin was investigated and the cold unfolded state was compared to the heat and pressure unfolded states. Conformationally and mechanistically the pressure and cold unfolding processes are found to be very alike, while the heat unfolding shows some pronounced differences. We also propose a hypothesis on protein aggregation.     

单分子技术研究T7解旋酶的解旋与换链

单分子荧光共振能量转移(smFRET)和磁镊(MT)技术目前广泛应用于研究分子马达.相较于常规技术,其具有高精度及动态观测的优点.本文研究对象为T7解旋酶,是六聚体解旋酶的典型代表.研究表明,这种解旋酶主要消耗脱氧胸苷三磷酸(dTTP)提供能量,且仅能沿着5′-3′单向进行行走和解旋工作.目前对于六聚体解旋酶的解旋和换链机制的认知仍然存在着诸多问题,因此本文主要以此作为切入点开展研究.首先通过运用smFRET技术研究T7解旋酶在不同DNA底物上的解旋现象,发现其需要3′-尾链参与到解旋工作中,但其为单链或双链结构并无明显区别;通过改变脱氧核糖核酸(DNA)序列中的GC含量,发现T7解旋酶随着序列中GC含量的升高会更容易在解旋过程中发生回退现象,导致解旋长度明显缩短;通过进一步分析发生回退先的实验数据,发现T7解旋酶除了可以瞬时回退到叉形DNA岔口或脱落外,还可以缓慢回退到叉形DNA岔口;运用MT技术研究该解旋酶,同样发现这种缓慢回退现象的存在.根据T7解旋酶解旋DNA遵循的单向性和极性,其只能沿着5′到3′方向进行行走和解旋.因此,本文推测这种缓慢回退的现象可能是解旋酶从5′-链转移至3′-链上,即发生换链过程;最后,本文提出了T7解旋酶在解旋过程中进行换链的模型,将有助于进一步理解环状六聚体解旋酶行使其功能的分子机制.     

Accurate reconstruction of soft X-ray energy spectrum in detector response matrix

In this paper, we report the design of a Monte Carlo simulation for the energy spectrum measurement system based on the ladderabsorption method. Herein, the detector response matrix can be calculated using the detector responses to several monochromatic X-ray beams. A novel soft X-ray spectrum unfolding method based on the two-step reverse iteration(TSRI) algorithm is applied to acquire the primary spectrum. This paper provides examples of the use of TSRI, and the unfolded energy spectra for the soft Xray beams show excellent agreement with the references. The unfolded energy spectra obtained using the TSRI exhibit better accuracy than those obtained from the commonly used unfolding code GRAVEL.     

Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.     

Unfolding the singularities in superspace

A method is described for unfolding the singularities in superspace, \(\mathcal{G} = \mathfrak{M}/\mathfrak{D}\) , the space of Riemannian geometries of a manifoldM. This unfolded superspace is described by the projection $$\mathcal{G}_{F\left( M \right)} = \frac{{\mathfrak{M} \times F\left( M \right)}}{\mathfrak{D}} \to \frac{\mathfrak{M}}{\mathfrak{D}} = \mathcal{G}$$ whereF(M) is the frame bundle ofM. The unfolded space \(\mathcal{G}_{F\left( M \right)}\) is infinite-dimensional manifold without singularities. Moreover, as expected, the unfolding of \(\mathcal{G}_{F\left( M \right)}\) at each geometry [g _o] ∈ \(\mathcal{G}\) is parameterized by the isometry groupIg _o (M) of g₀. Our construction is natural, is generally covariant with respect to all coordinate transformations, and gives the necessary information at each geometry to make \(\mathcal{G}\) a manifold. This construction is a canonical and geometric model of a nonrelativistic construction that unfolds superspace by restricting to those coordinate transformations that fix a frame at a point. These particular unfoldings are tied together by an infinite-dimensional fiber bundleE overM, associated with the frame bundleF(M), with standard fiber \(\mathcal{G}_{F\left( M \right)}\) , and with fiber at a point inM being the particular noncanonical unfolding of \(\mathcal{G}\) based at that point. ThusE is the totality of all the particular unfoldings, and so is a grand unfolding of \(\mathcal{G}\) .     

Compaction and tensile forces determine the accuracy of folding landscape parameters from single molecule pulling experiments

We establish a framework for assessing whether the transition state location of a biopolymer, which can be inferred from single molecule pulling experiments, corresponds to the ensemble of structures that have equal probability of reaching either the folded or unfolded states (P(fold)=0.5). Using results for the forced unfolding of a RNA hairpin, an exactly soluble model, and an analytic theory, we show that P(fold) is solely determined by s, an experimentally measurable molecular tensegrity parameter, which is a ratio of the tensile force and a compaction force that stabilizes the folded state. Applications to folding landscapes of DNA hairpins and a leucine zipper with two barriers provide a structural interpretation of single molecule experimental data. Our theory can be used to assess whether molecular extension is a good reaction coordinate using measured free energy profiles.     

透射光栅测量的解谱方法

对透射光栅谱仪配Ｘ射线ＣＣＤ的软Ｘ射线谱测量系统ＴＧ－ＸＣＣＤ进行了简要描述,提出了正交函数展开法用于透射光栅谱仪配Ｘ射线ＣＣＤ测量的谱回推。将正交展开法用于激光打击金盘靶的软Ｘ射线谱回推,并与迭代法的解谱结果进行了比较。结果基本一致,不对此测量系统进行了误差分析。     

Effect of unfolding on the thickness of the hydration layer of a protein

Properties of water in the hydration layer around a protein is intimately correlated with its function. A knowledge of the thickness of the hydration layer is important to understand its role in guiding the folding-unfolding of the protein. We have performed atomistic molecular dynamics simulations of the folded native and a partially unfolded molten globule structure of the villin headpiece subdomain or HP-36 in aqueous solution to estimate the effect of unfolding on the thickness of hydration layer around different segments of the protein. In particular, several dynamic properties of water around different segments of the folded native and the unfolded structure have been calculated by varying the thickness of the hydration layers. It is found that unfolding of a segment of the protein is correlated with the dynamics of water around it, i.e., within the first hydration layer. The effect of unfolding on water properties has been found to diminish when water molecules present beyond the first hydration layer were included in the calculations.      

中子深度分析反演算法研究

针对中子深度分析技术,研究几类反演算法：概率迭代法、奇异值分解求解最小二乘法、线性正则化方法及约束线性正则化法.开展相关数值实验,分别在欠定方程、超定方程情况下,对反演算法的结果进行比较.一般情况下几种算法都能得到较为理想的结果,概率迭代法和约束线性正则化法由于有迭代过程,因此在离子源强度发生阶跃处得到的结果并不理想.在超定方程情况下,当选取的反演范围较为随意时,线性正则化方法可能使得解的稳定性较差,而约束线性正则化法能够很好地抑制求解过程中的不稳定性.对实际测量能谱进行反演时,由于能谱有随机误差,线性正则化方法不能很好地抑制误差的影响,反演结果振荡较强,其余方法的结果与参考值符合很好.     

A motor that makes its own track: helicase unwinding of DNA

We study the unwinding of DNA by helicase proteins as a representative system in which a motor protein interacts with a mobile obstacle. In our discrete model, the interaction between the helicase and the DNA fork is characterized by an interaction potential. For the case of a hard-wall potential, the helicase opens the DNA by rectifying thermal fluctuations which spontaneously open base pairs. A potential with nonzero range describes the destabilization of the double strand by the enzymatic action of the helicase. We derive solutions for the opening speed as a function of the potential shape and relate our results to experiments on helicase motion.     

凝血酶适体DNA四螺旋构象去折叠机制的NMR研究

凝血酶适体DNA\[thrombin binding DNA aptamer d (G₁G₂T₃T₄G₅G₆T₇G₈T₉G₁₀G₁₁T₁₂T₁₃G₁₄G₁₅),TBA]是对凝血酶有极高亲和性、并能有效抑制凝血酶凝血功能的单链DNA适体. 凝血酶适体DNA在K⁺等离子存在时呈现出椅式构象,其中2个堆积的四碱基体（G-quartet）构成椅子的主体部分,而1个TGT loop环和2个TT loop环分别构成椅子的靠背和椅脚. 我们测定了在K⁺存在时凝血酶适体DNA中亚氨基质子的交换速率, 发现位于TGT loop环和TT loop环内的亚氨基质子G6、G5和G14由于受TGT loop环和TT loop环的保护有比较小的交换速率,而位于环之外的亚氨基质子G2、G11和G15有较大的交换速率;TGT loop环稳定性同TT loop环相似;碱基T4、T13和T9在稳定凝血酶适体DNA结构中起着很大的作用. 这些证据进一步支持了凝血酶适体DNA的去折叠机制：位于TGT loop环和TT loop环之外的碱基对G1G15、G2G14和G5G11首先断裂其Hoogsteen氢键,而TGT loop环、TT loop环和其内的Hoogsteen氢键保持完好;当温度进一步升高时,TGT loop环和两个TT loop环打开环状结构,凝血酶适体DNA的椅式构象彻底解体,转变为自由单链.      

The effects of shielding around the source on neutron energy spectra

The knowledge of neutron energy spectra contributes to unambiguous identification of neutron sources in the fields of nuclear safeguards and nuclear non-proliferation. Since a real scenario situation includes the presence of shielding around the source, we have investigated the influence of the potential shielding surrounding the source on the shape of energy spectra for a few neutron sources. We have applied the maximum-likelihood, expectation–maximisation (MLEM) method with one-step-late (OSL) algorithm for neutron spectra unfolding. The pulse height distributions used in the unfolding procedures were simulated with the high accuracy by using the MCNP-PoliMi code based on the Monte Carlo method. A possibility to identify the shielded neutron sources by using the unfolding method was examined with two continuous-in-energy sources, such as ²⁵²Cf and ²⁴¹Am–Be in source-shielding configurations with lead (Pb) and polyethylene (PE) blocks. The results of calculations have shown that the identification of ²⁵²Cf and ²⁴¹Am–Be sources with 2.5 cm of Pb and PE shield can be achieved successfully by using the MLEM method with the OSL algorithm. However, the unfolded results for ²⁵²Cf and ²⁴¹Am–Be sources with 10 cm of PE shield significantly deviate from the reference spectra and the sources cannot be correctly identified on the basis of their unfolded energy spectra.     

There and back again: Two views on the protein folding puzzle

The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10–11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: “U-to-N” and “N-to-U”. In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the “N-to-U” transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical “detailed balance” principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the “N-to-U” transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the “U-to-N” transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the side of the native state; and both theories agree with experiment. In addition, they predict the maximal size of protein domains that fold under solely thermodynamic (rather than kinetic) control and explain the observed maximal size of the “foldable” protein domains.