见微知著:单分子力谱技术窥探蛋白质分子动态功能北大核心CSCD

蛋白质的动态功能调控并决定着细胞的生理和病理过程。其不仅受到蛋白质本身生物化学特性的影响,还受到生物体内复杂的生物力学微环境的动态调控。这些生物力因素主要通过耦联生物化学特性来改变蛋白质的动态相互作用、构象变化以及后续的信号传导。近些年来,单分子力谱检测技术突破了传统生物化学技术的限制,在单分子水平有效地研究生物力学——化学耦联调控下的蛋白质动态功能。本文详细介绍了4种代表性的单分子力谱检测技术(原子力显微镜、光镊、生物膜力学探针以及磁镊),着重介绍这些技术在蛋白质动态功能研究方面的典型应用,主要包括蛋白质动态相互作用,蛋白质动态构象变化以及信号传导等。同时,本文还介绍了几种常用的基于上述单分子检测技术的单分子力谱检测方法,主要用于定量检测蛋白质相互作用、构象变化等生物化学过程的分子动力学参数。最后,本文还简要讨论了单分子力谱检测技术的未来发展方向,特别是如何与其他研究手段的有机整合,更全面地研究蛋白质的动态功能。我们希望该综述能够给更多的生物化学家带来新的概念和工具,帮助更全面地研究蛋白质的动态特性。     

见微知著:单分子力谱技术窥探蛋白质分子动态功能

蛋白质的动态功能调控并决定着细胞的生理和病理过程。其不仅受到蛋白质本身生物化学特性的影响,还受到生物体内复杂的生物力学微环境的动态调控。这些生物力因素主要通过耦联生物化学特性来改变蛋白质的动态相互作用、构象变化以及后续的信号传导。近些年来,单分子力谱检测技术突破了传统生物化学技术的限制,在单分子水平有效地研究生物力学——化学耦联调控下的蛋白质动态功能。本文详细介绍了4种代表性的单分子力谱检测技术(原子力显微镜、光镊、生物膜力学探针以及磁镊),着重介绍这些技术在蛋白质动态功能研究方面的典型应用,主要包括蛋白质动态相互作用,蛋白质动态构象变化以及信号传导等。同时,本文还介绍了几种常用的基于上述单分子检测技术的单分子力谱检测方法,主要用于定量检测蛋白质相互作用、构象变化等生物化学过程的分子动力学参数。最后,本文还简要讨论了单分子力谱检测技术的未来发展方向,特别是如何与其他研究手段的有机整合,更全面地研究蛋白质的动态功能。我们希望该综述能够给更多的生物化学家带来新的概念和工具,帮助更全面地研究蛋白质的动态特性。     

AFM单分子力谱技术及其在活体细胞和菌体表面生物大分子研究中的最新进展

生命活动过程与生物分子内或生物分子间机械力的作用密不可分.原子力显微镜具有极高的力学分辨率,可以在近生理条件下对生物样品进行力学测量,是研究生物体系力学相互作用的理想工具.基于原子力显微镜的单分子力谱(AFM-SMFS)技术可以在单分子、单细胞水平测量生物分子内或生物分子间的相互作用.本文首先扼要介绍了AFM-SMFS技术,包括AFM-SMFS的基本原理、力谱测量及分析方法(蠕虫链模型、自由连接链模型和自由旋转链模型)以及探针的化学修饰方法(硅/氮化硅探针和镀金探针的修饰);重点介绍了利用AFM-SMFS技术对活体细胞表面蛋白(转化生长因子β1、CD20、热休克蛋白以及蛋白酪氨酸激酶)和糖类分子(葡萄糖和甘露糖)的近期研究进展;随后介绍了利用AFM-SMFS技术对活菌体表面蛋白(肝素结合血凝黏附素和Als5p黏附蛋白)和糖类分子(半乳糖、甘露糖、B族碳水化合物、荚膜多糖、α-甘露聚糖、β-甘露聚糖、β-葡聚糖以及几丁质)的近期研究进展;最后对AFM-SMFS技术的缺点和发展前景进行了总结和展望.     

生物单分子探测与成像的新进展

生物单分子研究是分子生物学向更深层次发展的自然趋势。从上世纪末开始,由于研究单分子技术的不断发展,这一领域已取得了许多重要成果。近年来活细胞内单分子过程的揭示成为关注的焦点。丈章仅从基因表达的研究、用受激发射损耗（STED）成像研究细胞膜、胞浆中单个分子的追踪以及单分子力谱在细胞生物学中的应用等几个方面说明本领域发展的现状。     

小分子有机化合物与DC-SIGN相互作用的荧光光谱分析

采用实验和理论化学研究相结合的方法研究分子间相互作用,这种在计算机上进行“模拟实验”与传统“有机化学实验”相辅相成的研究策略,正在成为有机化学研究的重要手段.本论文利用荧光光谱分析法来研究小分子有机化合物与蛋白底物之间的相互作用机制.利用先进的分子模拟软件,结合分子力学、量子力学和神经网络等方法与分子对接等理论化学手段,建立和优化相互作用的分子间形成复合物的空间构象,并且预测分子间相互作用的稳定性.用荧光光谱分析法对12种小分子有机化合物与DC-SIGN(DC-specific ICAM-3 grabbing nonintegrin)之间的相互作用进行了研究,结果与理论分子模型计算十分吻合.在这12种化合物中,1-脲基氨基甘露糖与DC-SIGN络合的效果最好.这一发现可能对研究新一代抗艾滋病药物有重要意义.     

原子力显微镜在纳米生物学研究中的应用

原子力显微镜(atomic force microscope,AFM)自从1986年发明以来,作为一种重要的单分子研究工具,在包括细胞粘附、蛋白折叠以及蛋白间的相互作用等生物学过程的研究中得到了广泛应用。本文主要介绍原子力显微镜的原理,着重于研究相互作用时能垒大小的确定,以及常用的单分子表面修饰方法。     

纳米狭缝中Aβ蛋白构象变化的间距作用

目的 金属表面对蛋白质分子具有吸附作用,然而在纳米尺度内,蛋白质分子构象受到狭缝的间距作用尚未明确。本文通过在分子动力学模拟中建立不同间距的金原子层,研究纳米级金属狭缝中蛋白质分子构象变化。方法 使用GOIPCHARMM力场在Au (111)金原子界面间对Aβ1-42蛋白单体进行分子动力学仿真,研究无狭缝的水溶液环境下和由5.0、5.5以及8.5 nm狭缝结构与Aβ蛋白相互作用及蛋白质构象变化。结果 当金狭缝结构间距从5.0 nm增加到8.5 nm,Aβ蛋白分子与两侧金层相互作用可由单表面吸附、双表面吸附过渡到无吸附。结论 Aβ蛋白分子在金狭缝结构中与表面发生相互作用,随狭缝间距和蛋白质分子距界面距离的变化,蛋白质分子的状态可能表现为单表面吸附、双表面吸附和无吸附3种状态。     

乳腺癌细胞中p27kip1分子相互作用蛋白质谱的改变

p27kipl是细胞周期重要的负性调控因子,在乳腺癌等多种肿瘤发生发展过程中发挥重要作用.乳腺癌细胞中p27kipl蛋白通常是低丰度表达和错位分布,导致这种分布和表达改变的确切机制并不明确.已有研究表明,p27kipl磷酸化是重要的调节途径之一,细胞内外信号分子通过多种途径调节p27kipl的分布和表达.为了进一步阐明肿瘤细胞内调节p27kipl功能的分子机制,必须首先明确p27kipl在肿瘤细胞与正常细胞中相互作用蛋白质谱的差异.包括细胞周期素、周期素依赖性激酶、CRM1、jab1、Skp2等在内的多种分子可以与p27kipl发生相互作用.在乳腺癌细胞中还有儿种特异的作用分子.在不同细胞周期和不同细胞内分布状态下,p27kipl蛋白有不同的相互作用蛋白质谱.因此,我们推断在乳腺癌细胞内p27kipl分子相互作用蛋白质谱的差异可能是导致其低表达和错位分布的主要机制.     

BK通道在细胞膜上的单分子定位及空间分布*

摘要目的：研究大电导、钙离子和电压激活的钾离子通道（BK通道）在HEK293 细胞膜上的单分子定位及其总体空间分布情况。 方法：分别用mEos2、Dronpa 等荧光蛋白标记BK通道的α亚基和辅助性β2 亚基,将这些质粒在HEK293 细胞内瞬时转染以表 达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及 单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果：激光共聚焦荧光显微成像和全内反射荧光显微成像技 术只能在亚细胞水平定位通道蛋白,BK 通道在细胞膜上聚集并形成不规则的蛋白簇,它的α亚基和β2 亚基在细胞膜上完全共 定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2 亚基紧紧靠在一起,它们之间依然存在空间 距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论：BK通道蛋白簇里面包含大量的α和β2 亚基的蛋白单分子, 它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和β亚基功能耦合的结构基础,为以后研究大 分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表 达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。     

SpA蛋白与磷脂单分子膜相互作用的研究

利用石英表面沉积ＬＢ膜的紫外吸收特性与ＣＤ谱特性研究了磷脂单分子膜与ＳｐＡ的相互作用,实验结果表明,单分子膜的疏密状态,蛋白质与膜表面的电荷特性及亚相Ｃａ＾２＋离子浓度均对该蛋白质与磷脂的相互作用有显著的影响,蛋白质在磷膜中的镶嵌与吸附作用为生物膜的重组提供了新的途径。     

Atomic force microscope-based single-molecule force spectroscopy of RNA unfolding

Single-molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) has emerged as an important tool for probing biomolecular interaction and exploring the forces, dynamics, and energy landscapes that underlie function and specificity of molecular interaction. These studies require attaching biomolecules on solid supports and AFM tips to measure unbinding forces between individual binding partners. Herein we describe efficient and robust protocols for probing RNA interaction by AFM and show their value on two well-known RNA regulators, the Rev-responsive element (RRE) from the HIV-1 genome and an adenine-sensing riboswitch. The results show the great potential of AFM–SMFS in the investigation of RNA molecular interactions, which will contribute to the development of bionanodevices sensing single RNA molecules.     

Nonspecific interactions in AFM force spectroscopy measurements

Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.     

Piezoelectric tuning fork probe for atomic force microscopy imaging and specific recognition force spectroscopy of an enzyme and its ligand

Piezoelectric quartz tuning fork has drawn the attention of many researchers for the development of new atomic force microscopy (AFM) self‐sensing probes. However, only few works have been done for soft biological materials imaging in air or aqueous conditions. The aim of this work was to demonstrate the efficiency of the AFM tuning fork probe to perform high‐resolution imaging of proteins and to study the specific interaction between a ligand and its receptor in aqueous media. Thus, a new kind of self‐sensing AFM sensor was introduced to realize imaging and biochemical specific recognition spectroscopy of glucose oxidase enzyme using a new chemical functionalization procedure of the metallic tips based on the electrochemical reduction of diazonium salt. This scanning probe as well as the functionalization strategy proved to be efficient respectively for the topography and force spectroscopy of soft biological materials in buffer conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.     

Direct force measurements between siRNA and chitosan molecules using force spectroscopy

Information on the interaction strength between small interfering RNA (siRNA) and chitosan can contribute to the understanding of the formation and stability of chitosan/siRNA nanoparticles used as siRNA delivery systems for gene silencing. In this study, we utilize atomic force microscopy to obtain force spectroscopy results of the interaction strengths between siRNA and chitosan measured in physiological phosphate buffered saline buffer at different pH. The force measurements revealed that the adhesive interactions decreased in force strength and force frequency as the pH was increased from 4.1 to 6.1, 7.4, and 9.5, exhibiting distinct multimodal distributions of the interaction forces between siRNA and chitosan molecules at acidic pH and only negligible adhesive forces were observed at neutral or high pH. The strong pH dependence of siRNA-chitosan interactions can provide a convincing rationale for siRNA/chitosan complex formation and nanoparticle stability under low acidic conditions. These findings demonstrate that the use of force spectroscopy for the adhesive force measurements allows an evaluation of the complexing ability between siRNA and chitosan that can be utilized to predict nanoparticle stability.     

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF₁₆₅ was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF₁₆₅ was dependent on the concentration of VEGF₁₆₅. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF₁₆₅ complexes was revealed, which was similar to that of the intact aptamer/VEGF₁₆₅. Besides, the dissociation rate constant (k_off) of split aptamer/VEGF₁₆₅ was close to that of intact aptamer/VEGF₁₆₅, and the interaction force of split aptamer/VEGF₁₆₅ was higher than the force of intact aptamer/VEGF₁₆₅. It indicated that split aptamer also possessed high affinity with VEGF₁₆₅. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.     

Two-dimensional kinetics of inter-connexin interactions from single-molecule force spectroscopy

Gap junction channels are intercellular channels that form by docking the extracellular loops of connexin protein subunits. While the structure and function of gap junctions as intercellular channels have been characterized using different techniques, the physics of the inter-connexin interaction remain unknown. Moreover, as far as we know, the capacity of gap junction channels to work as adhesion complexes supporting pulling forces has not yet been quantitatively addressed. We report the first quantitative characterization of the kinetics and binding strength of the interaction of a short peptide mimicking extracellular loop 2 of Cx26 with membrane-reconstituted Cx26, combining the imaging and force spectroscopy capabilities of atomic force microscopy. The fast dissociation rate inferred a dynamic bond, while the slow association rate reflected the reduced flexibility and small size of extracellular loops. Our results propose the gap junction channel as an adhesion complex that associates slowly and dissociates fast at low force but is able to support important pulling forces in its native, hexameric form.     

Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy

SfiI belongs to a family of restriction enzymes that function as tetramers, binding two recognition regions for the DNA cleavage reaction. The SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site-specific binding. The enzymatic action of SfiI has been very well characterized. However, the properties of the complex before the cleavage reaction are not clear. We used single-molecule force spectroscopy to analyze the strength of interactions within the SfiI-DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and the other was tethered to the probe. The complex was formed by the protein present in the solution. An alternative setup, in which the protein was anchored to the surface, allowed us to probe the stability of the complex formed with only one duplex in the approach-retraction experiments, with the duplex immobilized at the probe tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy) and the results suggest that the dissociation reaction of the SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was instrumental in revealing the role of the 5 bp spacer region within the palindromic recognition site on DNA-SfiI in the stability of the complex. The data show that, although the change of non-specific sequence does not alter the position of the activation barrier, it changes values of the off rates significantly.     

原子力显微镜力谱研究大肠杆菌启动子与RNA聚合酶的相互作用

【目的】本研究通过原子力显微镜(AFM)力谱技术研究了大肠杆菌启动子与RNA聚合酶(RNAp)间的相互作用,目的是建立一种高效的体外表征启动子的新方法。【方法】优化了用于单分子AFM力谱分析的蛋白固定化策略,建立AFM力谱分析启动子的策略,以缺失识别启动子序列的σ亚基核心RNA聚合酶(RNAp-C)为对照,研究启动子/RNAp间相互作用的特异性。最后比较了序列较典型的Ls1启动子和缺失–10区的Ls2启动子的力谱。【结果】基于建立的方法,验证了Ls1与大肠杆菌RNAp结合的特异性,其相互作用力为(331.10±5.10)p N。与Ls1相比,Ls2启动子与RNAp结合显著减少。利用启动子探针质粒,以报告基因cat的表达产物氯霉素乙酰转移酶(CAT)的酶活验证Ls1、Ls2启动子强度,分别为(181.70±4.10)、(0.30±0.20)U/mg。【结论】本研究建立的基于AFM力谱技术的启动子分析技术,是一种高效的、直接定量表征启动子活性的新方法。