分子间相互作用对蛋白质晶体生长的影响

采用原子力显微镜对溶菌酶和刀豆蛋白A的分子间相互作用力的情况进行了研究, 并用动态光散射研究了此二种分子间相互作用力有较大差异的蛋白质在晶体生长条件和非生长条件下, 溶液中的聚集体的状态(大小和分散度)随浓度和温度的变化情况. 实验结果表明, 范德华力强的刀豆蛋白A在成核前, 溶液中的聚集体不能很快转变为生长基元, 导致晶体生长时间长; 而范德华力弱的溶菌酶, 溶液中的聚集体可以很快转变成生长基元, 晶体生长时间也较短.     

Equilibrium clusters in concentrated lysozyme protein solutions

We have studied the structure of salt-free lysozyme at 293 K and pH 7.8 using molecular simulations and experimental SAXS effective potentials between proteins at three volume fractions, ?=0.012, 0.033, and 0.12. We found that the structure of lysozyme near physiological conditions strongly depends on the volume fraction of proteins. The studied lysozyme solutions are dominated by monomers only for ?≤0.012; for the strong dilution 70% of proteins are in a form of monomers. For ?=0.033 only 20% of proteins do not belong to a cluster. The clusters are mainly elongated. For ?=0.12 almost no individual particles exits, and branched, irregular clusters of large extent appear. Our simulation study provides new insight into the formation of equilibrium clusters in charged protein solutions near physiological conditions.     

溶液热历史对蛋白质晶体生长影响的内在原因研究

从溶液中聚集体的角度研究了溶液的热历史改变生长出的蛋白质晶体的数目和尺寸的内在原因. 将在281 和309 K下保存1 d的两组溶菌酶溶液按不同比例混合, 加入沉淀剂生长晶体. 随着高温溶液的比例增加, 生长出的晶体数目减少, 同时溶液中生长基元的尺寸增大. 在5周内, 采用动态光散射对281, 293和309 K三种温度下保存的溶菌酶溶液中聚集体的变化情况进行监测, 发现溶液中均存在大小不同的两部分聚集体, 称之为小聚集体与多聚体. 前者的尺寸基本不随保存时间而变化, 而后者尺寸随保存时间增加而减小, 减小的速度与保存温度有关. 多聚体的尺寸经过5周后和小聚集体基本相同. 研究结果表明, 处于无序聚集阶段的溶液的均一化程度和成核阶段生长基元的尺寸受到了溶液热历史的影响, 并最终对晶体的数目产生影响.     

Mass transfer limitations at crystallizing interfaces in an atomic force microscopy fluid cell: a finite element analysis

Although atomic force microscopy (AFM) has emerged as the preeminent experimental tool for real-time in situ measurements of crystal growth processes in solution, relatively little is known about the mass transfer limitations that may impact these measurements. We present a continuum analysis of flow and mass transfer in an atomic force microscope fluid cell during crystal growth, using data acquired from calcium oxalate monohydrate (COM) crystal growth measurements as a comparison. Steady-state flows and solute concentration fields are computed using a three-dimensional, finite element method implemented on a parallel supercomputer. Steady-state flow results are compared with flow visualization experiments to validate the model. Computations of the flow field demonstrate how nonlinear momentum transport alters the spatial structure of the flow with increasing flow volume, altering mass transport conditions near the AFM cantilever and tip. The simulations demonstrate that the combination of solute depletion from crystal growth and mass transfer resistance lowers the solute concentration in the region between the tip and the crystal compared with the solute concentration at the inlet of the AFM cell. For example, using experimentally measured growth rates for COM, the solute concentration in this region is 3.1% lower than the inlet value because the solute consumed by crystal growth beneath the AFM tip cannot be replenished fully due to mass transport limitations. The simulations also reveal that increasing the flow rate through the cell does not affect this difference significantly because of the inherent shielding by the AFM tip in proximity with the crystal surface. Models such as the one presented here, used in conjunction with AFM measurements, promise more precise interpretations of measurement data.     

Observation of crystal growth of lysozyme protein crystals by atomic force microscopy

Surface structure and the propagation of elementary growth layers over the (010) face of orthorhombic lysozyme crystal is examined at a molecular‐scale resolution by the method of atomic force microscopy (AFM). The steps have a small number of kinks spaced by about 150 growth units. The step motion occurs via successive deposition of rows of growth units. The data obtained are discussed in terms of the model of one‐dimensional nucleation.     

配液结晶法制备溶菌酶蛋白质晶体的生长机理研究

采用配液结晶法制取了溶菌酶蛋白质晶体, 使用动态光散射测量了溶液中聚集体的颗粒度几率分布; 使用Zeiss显微镜测定了溶菌酶(110)晶面的生长速度. 实验表明: 随着蛋白质和NaCl浓度的增加, 溶液中聚集体的颗粒尺寸也相应增加. 随着反应时间的增加, 溶菌酶分子在溶液中的聚集反应, 逐渐达到平衡; 在蛋白质和NaCl浓度较高时, 溶菌酶晶体的(110)面生长较快, 而在蛋白质和NaCl浓度较低时, 该晶面生长较慢. 基于二维成核生长机理, 从晶体生长动力学理论方程出发, 计算了二维成核的形成能α＝4.01×10^－8 J•cm^－2.     

Crystal Growth Mechanisms and Morphological Control of the Prototypical Metal–Organic Framework MOF‐5 Revealed by Atomic Force Microscopy

Crystal growth of the metal–organic framework MOF‐5 was studied by atomic force microscopy (AFM) for the first time. Growth under low supersaturation conditions was found to occur by a two‐dimensional or spiral crystal growth mechanism. Observation of developing nuclei during the former reveals growth occurs through a process of nucleation and spreading of metastable and stable sub‐layers revealing that MOFs may be considered as dense phase structures in terms of crystal growth, even though they contain sub‐layers consisting of ordered framework and disordered non‐framework components. These results also support the notion this may be a general mechanism of surface crystal growth at low supersaturation applicable to crystalline nanoporous materials. The crystal growth mechanism at the atomistic level was also seen to vary as a function of the growth solution Zn/H₂bdc ratio producing square terraces with steps parallel to the <100> direction or rhombus‐shaped terraces with steps parallel to the <110> direction when the Zn/H₂bdc ratio was >1 or about 1, respectively. The change in relative growth rates can be explained in terms of changes in the solution species concentrations and their influence on growth at different terrace growth sites. These results were successfully applied to the growth of as‐synthesized cube‐shaped crystals to increase expression of the {111} faces and to grow octahedral crystals of suitable quality to image using AFM. This modulator‐free route to control the crystal morphology of MOF‐5 crystals should be applicable to a wide variety of MOFs to achieve the desired morphological control for performance enhancement in applications.     

UV laser induced proton-transfer of protein molecule in the gas phase produced by droplet-beam laser ablation

Droplet-beam laser-ablation mass-spectrometry was applied for a study of the UV-laser induced proton-transfer reaction of protonated lysozyme hydrated clusters in the gas phase. Protonated lysozyme hydrated clusters were produced by irradiation of an IR laser onto a droplet-beam of an aqueous solution of lysozyme and were subsequently irradiated by a UV laser. It is found that H⁺ and H₃O⁺ are produced through photodissociation of protonated lysozyme hydrated clusters. The mechanism of the proton-transfer reaction is discussed.     

Scanning-induced growth on single crystal calcite with an atomic force microscope

We report observations of localized growth on the (1014) surface of single-crystal CaCO3 in supersaturated solutions while scanning with the tip of an atomic force microscope (AFM). At low contact forces, AFM scanning strongly enhances deposition along preexisting steps. This enhancement increases rapidly with increasing solution supersaturation, and is capable of filling in multilayer etch pits to produce defect-free surfaces at the resolution of the AFM. Attempts to achieve similar deposition rates in the absence of scanning require high supersaturations that produce three-dimensional crystal nuclei, which are important defects. Localized deposition produced by drawing the AFM tip back and forth across step edges can produce monolayer deposits extending well over a micron from the scanned area. These tip-induced deposits provide convincing evidence for the importance of ledge diffusion in calcite crystal growth.     

气相扩散法生长溶菌酶晶体的动态光散射研究

首次采用动态光散射研究了气相扩散法生长溶菌酶晶体.实验中采用了两种溶解溶菌酶的方法,所得实验结果是有区别的.这种区别表明了NaCl对溶菌酶分子间相互作用产生十分重要的影响.实验结果表明,晶体生长过程中,溶液中溶菌酶始终保持单分子与两分子聚集体的状态,这种状态是生长晶体的基础.     

谷光甘肽-Cd(Ⅱ)、Cu(Ⅱ)配合物在硅片表面形貌的AFM分析

用原子力显微镜观察了谷胱甘肽-镉、铜配合物在硅片表面的形貌. 低浓度时,谷胱甘肽-镉配合物主要以类球体颗粒无序地分散在基底表面,其表观高度和长度分别为(3.6±0.1) nm和(60±10) nm. 随着浓度的增加,出现由小颗粒聚集而成的簇体. 不同的缓冲溶液体系对谷胱甘肽-镉配合物的形貌无明显影响. 谷胱甘肽-铜配合物首先由均匀的球体颗粒聚集成长链,然后随链延伸方向的不同,形成类似网状和线团状2种表面形貌,显示良好的方向性和均匀性.     

A small-angle scattering study on equilibrium clusters in lysozyme solutions

We use small-angle scattering experiments to investigate the structural properties of aqueous lysozyme solutions under conditions where the existence of equilibrium clusters has recently been demonstrated (Nature 2004, 432, 492). We also discuss the possible emergence of a low angle scattering contribution, which recently attracted interest due to its appearance in solutions of various proteins. We demonstrate that in lysozyme solutions under our experimental conditions such rising low q intensities can only be observed under special circumstances and can thus not be attributed to the existence of a universal long-range attraction. We then focus on the structural properties of the equilibrium clusters as a function of protein concentration, temperature, and ionic strength. We show that the experimental structure factors obtained from the scattering measurements exhibit the typical cluster-cluster peak q(c) reflecting the mean distance between charged clusters as well as a monomer-monomer peak q(m), which represents the nearest neighbor shell of monomers within a single cluster. The underlying principle for the formation of these structures is the coexistence of two opposing forces, a short-range attraction and a long-range repulsion due to residual charges. We can quantitatively analyze our scattering data by applying a simple equilibrium cluster model and calculate an average cluster aggregation number, N(c). The thus obtained cluster aggregation number increases linearly with volume fraction. We also observe an increasing N(c) as temperature decreases and as the screening of residual charges increases. We point out the importance of the existence of equilibrium clusters and the universality of this phenomenon for self-assembling processes observed in nature. Finally, we discuss the limitations of our simple globular cluster model in view of recent findings from computer simulations.     

Dependence of the Nanoscale Composite Morphology of Fe3O4 Nanoparticle-Infused Lysozyme Amyloid Fibrils on Timing of Infusion: A Combined SAXS and AFM Study

Understanding the formation process and the spatial distribution of nanoparticle (NP) clusters on amyloid fibrils is an essential step for the development of NP-based methods to inhibit aggregation of amyloidal proteins or reverse the assembling trend of the proto-fibrillary complexes that prompts pathogenesis of neuro degeneration. For this, a detailed structural determination of the diverse hybrid assemblies that are forming is needed, which can be achieved by advanced X-ray scattering techniques. Using a combined solution small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) approach, this study investigates the intrinsic trends of the interaction between lysozyme amyloid fibrils (LAFs) and Fe₃O₄ NPs before and after fibrillization at nanometer resolution. AFM images reveal that the number of NP clusters interacting with the lysozyme fibers does not increase significantly with NP volume concentration, suggesting a saturation in NP aggregation on the fibrillary surface. The data indicate that the number of non-adsorbed Fe₃O₄ NPs is highly dependent on the timing of NP infusion within the synthesis process. SAXS data yield access to the spatial distribution, aggregation manner and density of NP clusters on the fibrillary surfaces. Employing modern data analysis approaches, the shape and internal structural morphology of the so formed nanocomposites are revealed. The combined experimental approach suggests that while Fe₃O₄ NPs infusion does not prevent the fibril-formation, the variation of NP concentration and size at different stages of the fibrillization process can impose a pronounced impact on the superficial and internal structural morphologies of these nanocomposites. These findings may be applicable in devising advanced therapeutic treatments for neurodegenerative diseases and designing novel bio-inorganic magnetic devices. Our results further demonstrate that modern X-ray methods give access to the structure of—and insight into the formation process of—biological–inorganic hybrid structures in solution.     

ＧＳＨ-Ｃｄ（Ⅱ）、Ｃｕ（Ⅱ）配合物在硅片表面形貌的ＡＦＭ 分析

用原子力显微镜观察了谷胱甘肽-镉、铜配合物在硅片表面的形貌。低浓度时,谷胱甘肽-镉配合物主要以类球体颗粒无序地分散在基底表面,其表观高度和长度分别为(3.6±0.1) nm 和(60±10) nm 。随着浓度的增加,出现由小颗粒聚集而成的簇体。不同的缓冲溶液体系对谷胱甘肽-镉配合物的形貌无明显影响。谷胱甘肽-铜配合物首先由均匀的球体颗粒聚集成长链,然后岁链延伸方向的不同,形成类似于网状和线团状 2 种表面形貌,显示良好的方向性和均匀性。     

In Situ AFM Investigation of Electrochemically Induced Surface‐Initiated Atom‐Transfer Radical Polymerization

Electrochemically induced surface‐initiated atom‐transfer radical polymerization is traced by in situ AFM technology for the first time, which allows visualization of the polymer growth process. It affords a fundamental insight into the surface morphology and growth mechanism simultaneously. Using this technique, the polymerization kinetics of two model monomers were studied, namely the anionic 3‐sulfopropyl methacrylate potassium salt (SPMA) and the cationic 2‐(metharyloyloxy)ethyltrimethylammonium chloride (METAC). The growth of METAC is significantly improved by screening the ammonium cations by the addition of ionic liquid electrolyte in aqueous solution.     

Direct observation of nucleus structure and nucleation pathways in apoferritin crystallization.

Using atomic force microscopy (AFM) in situ during the crystallization of the protein apoferritin from its solution, we imaged the arrangement of the molecules in near-critical clusters, larger or smaller than the crystal nucleus, that are representative of the nucleus structure. At supersaturations Delta mu/k(B)T of 1.1 -- 1.6 -- 2.3, the nuclei contain about 50 -- 20 -- 10 molecules. The molecular arrangement within the nuclei is similar to that in the crystal bulk. Contrary to the general belief, the observed nuclei are not compact molecular clusters, but are planar arrays of several rods of 4--7 molecules set in one or two monomolecular layers. Similarly unexpected nuclei structures might be common, especially for anisotropic molecules. Hence, the nucleus structure should be considered as a variable by advanced theoretical treatments.     

Kinetics of Lysozyme Crystallization From Solution

Abstract

We suggest that the growth of molecular aggregates is the rate-controlling step in the crystallization of lysozyme from pH buffered aqueous solutions of strong electrolytes. We propose that the aggregation reaction passes through a charged transition state whose rate of formation is accelerated by Debye-Huckel screening and whose charge is stabilized by ion exchange with the solution. Applying the theory of the “primary kinetic salt effect”, we predict that the half-life for decay of the lysozyme concentration in solution in contact with a growing crystal should decrease linearly with the square root of the ionic strength. This prediction is confirmed experimentally in the case of lysozyme crystals precipitating at 4°C from pH buffered aqueous solutions of sodium chloride.     

异硫氰酸荧光素-溶菌酶的制备及晶体生长预研究

迄今尚未有适用于光源为488 nm激光扫描共聚焦显微镜研究用的溶菌酶. 为此, 用异硫氰酸荧光素作为探针标记了溶菌酶, 测定了溶菌酶标记物的紫外-可见吸收光谱和荧光光谱, 摸索了其晶体的生长条件. 实验结果表明, 在标记过程中异硫氰酸荧光素没有影响溶菌酶的生化性质, 标记后的溶菌酶可用于激光扫描共聚焦显微镜进行后续研究.     

Molecular-scale observation of formation of nuclei in soap-free polymerization of styrene

To clarify the mechanism of the nucleation process in the soap-free polymerization of styrene in water, the polymerization process of styrene mainly at a temperature 46 degrees C was investigated on a molecular scale using an atomic force microscope (AFM) as well as a scanning electron microscope (SEM) and a dynamic light scattering apparatus (DLS). A cationic initiator was employed to make polymerized styrene adsorb electrostatically on the negatively charged mica plate with molecular-scale smoothness, and the mechanism was estimated from their AFM measurements in situ in water. The following was found. There exist two streams to produce PSL particles in the polymerization: one is the polymerization in monomer droplets fragmented by mixing from the main monomer reservoir floating above the solution, and the other is the polymerization of monomers dissolved in the solution by initiators. The former is normally neglected or not recognized because of the small contribution. The latter is the main production process of PSL particle, which is investigated in detail on a molecular scale, and a possible mechanism was proposed.     

Growth rates of protein crystals

Protein crystallization is important for structural biology. The rate at which a protein crystallizes is often the bottleneck in determining the protein's structure. Here, we give a physical model for the growth rates of protein crystals. Most materials crystallize faster under stronger growth conditions; however, protein crystallization slows down under the strongest conditions. Proteins require a crystallization slot of 'just right' conditions. Our model provides an explanation. Unlike simpler materials, proteins are orientationally asymmetrical. Under strong conditions, protein molecules attempt to crystallize too quickly, in wrong orientations, blocking surface sites for more productive crystal growth. The model explains the observation that increasing the net charge on a protein increases the crystal growth rate. The model predictions are in good agreement with experiments on the growth rates of tetragonal lysozyme crystals as a function of pH, salt concentration, temperature, and protein concentration.