Isothermal titration calorimetry of RNA

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA.     

Membrane catalysis: synchronous multielectron reactions at the interface between two liquid phases. Bioenergetic mechanisms

Kinetics of multi-electron reactions at the interface between two immiscible liquids are considered. Calculations of the energy of solvent reorganization, of the work required to bring reactants and reaction products together, and of the electrostatic contributions to the Gibbs free energy of the reaction during electron transfer between reactants which are in different dielectric media are reported. Conditions under which the free energy of activation of the interfacial reaction of electron transfer decreases are established. The influence of the distance between reactants and of the dielectric permittivity of the non-aqueous phase on the solvent reorganization energy value is studied. Conditions under which multielectron reactions at the interface proceed are discussed. The biophysics and biochemistry of photosynthesis and respiration are considered as examples of multielectron processes.     

Investigating a macromolecular complex: the toolkit of methods

Structural biologists studying macromolecular complexes spend considerable effort doing strictly "non-structural" work: investigating the physiological relevance and biochemical properties of a complex, preparing homogeneous samples for structural analysis, and experimentally validating structure-based hypotheses regarding function or mechanism. Familiarity with the diverse perspectives and techniques available for studying complexes helps in the critical assessment of non-structural data, expedites the pre-structural characterization of a complex and facilitates the investigation of function. Here we survey the approaches and techniques used to study macromolecular complexes from various viewpoints, including genetics, cell and molecular biology, biochemistry/biophysics, structural biology, and systems biology/bioinformatics. The aim of this overview is to heighten awareness of the diversity of perspectives and experimental tools available for investigating complexes and of their usefulness for the structural biologist.     

High-throughput protein crystallization

High-throughput structural biology is a focus of a number of academic and pharmaceutical laboratories around the world. The use of X-ray crystallography in these efforts is critically dependent on high-throughput protein crystallization. The application of current protocols yields crystal leads for approximately 30% of the input proteins and well-diffracting crystals for a smaller fraction. Increasing the success rate will require a multidisciplinary approach that must invoke techniques from molecular biology, protein biochemistry, biophysics, artificial intelligence, and automation.     

Thermodynamic calculations in biological systems

The ability to compute intra- and inter-molecular interactions provides the opportunity to gain a deeper understanding of previously intractable problems in biochemistry and biophysics. This review presents three examples in which molecular dynamics calculations were used to gain insight into the atomic detail underlying important experimental observations. The three examples are the following: (1) Entropic contribution to rate acceleration that results from conformational constraints imposed on the reactants; (2) Mechanism of force unfolding of a small protein molecule by the application of a force that separates its N- and C-terminals; and (3) Loss of translational entropy experienced by small molecules when they bind to proteins.     

The measurement of volume change by capillary dilatometry

Capillary dilatometry enables direct measurement of changes in volume, an extensive thermodynamic property. The results provide insight into the changes in hydration that occur upon protein folding, ligand binding, and the interactions of proteins with nucleic acids and other cellular components. Often the entropy change arising from release of hydrating solvent provides the main driving force of a binding reaction. For technical reasons, though, capillary dilatometry has not been as widely used in protein biochemistry and biophysics as other methods such as calorimetry. Described here are simple apparatus and simple methods, which bring the technique within the capacity of any laboratory. Even very simple results are shown to have implications for macromolecular‐based phenomena. Protein examples are described.     

Simultaneous determination of equilibrium constants and enthalpy changes by titration calorimetry: Methods, instruments, and uncertainties

Calorimetric methods have been used to determine equilibrium constants since 1937, but no comprehensive review of the various calorimeters and methods has been done previously. This article reports methods for quantitative comparison of the capabilities of calorimeters for simultaneous determination of equilibrium constants and enthalpy changes, for determining optimal experimental conditions, and for assessing the effects of systematic and random errors on the accuracy and precision of equilibrium constants and enthalpy changes determined by this method.     

蛋白质双向电泳图谱的计算机处理和数量化分析

本文叙述了通过计算机硬件和软件的方法,对蛋白质的二维电泳图谱进行图像处理和数量化分析的工作.采用计算机图像处理的方法从电泳图谱的斑点图中抽取有用的信息,采用图像分析技术确定电泳图谱蛋白斑点的强度,比较几个凝胶的斑点图案.图谱的计算机处理和数量化分析技术,在许多生物物理和生物化学的研究中都可以应用,以给研究工作带来帮助.     

Superoxide anion radical induces lipid phase transitions that mimic temperature-induced phase-transition phenomenon of membrane lipids

A biological membrane is shown to undergo lipid-phase transition leading to increased membrane fluidity when exposed to extraneously generated superoxide anion radical. This phase-transition is several folds higher in magnitude when compared to the temperature-induced fluidity change at the transition temperatures. This finding could have significant importance since an altered membrane configuration may involve aspects of biochemistry, biophysics or physiology.     

Implicit solvent simulation models for biomembranes

Fully atomic simulation strategies are infeasible for the study of many processes of interest to membrane biology, biophysics and biochemistry. We review various coarse-grained simulation methodologies with special emphasis on methods and models that do not require the explicit simulation of water. Examples from our own research demonstrate that such models have potential for simulating a variety of biologically relevant phenomena at the membrane surface.     

Single-cell dissection and microdroplet chemistry

The unique roles of individual cells may be critical to the physiology of an organism. In such cases, micromethods are essential to elucidating the molecular biology, biochemistry and biophysics of the specialized cells or even subcellular compartments of the important cells. The great proliferation of micromethods testifies to their value and no single review can be comprehensive. This review therefore provides only a generalized overview of one approach, namely dissection that provides a pure sample for subsequent extraction and analysis by microdroplet chemistry. As a means of illustrating the utility of this approach, an application-study of the interaction of cytosolic malate concentration and guard-cell phosphoenolpyruvate carboxylase-is provided.     

Irrigated radiofrequency-biophysics and application to surgical ablation

Surgical ablation has become a routine procedure in many institutions and is a nearing standard of care for certain conditions. A variety of energy sources are available to the surgeon to induce cell death and create a line of conduction block. This article provides a brief review of the biophysics of irrigated radiofrequency and its application to monopolar and bipolar ablation.     

一种新的DNA复制和转录模型

DNA复制和转录有两种模型,一种是传统的滑动模型,复制和转录发生时参与反应的蛋白质沿DNA模板滑动.在另一种新提出的工厂模型中,固定在核结构上的蛋白质拉动模板来完成DNA的复制和转录.来自生物化学、生物物理学和细胞生物学等的实验证据表明,新的工厂模型是生物活体细胞内真实的复制和转录模式.     

Giant unilamellar vesicle formation under physiologically relevant conditions

We present an upgrade to the giant unilamellar vesicle (GUV) electroformation method allowing easy GUV production in different buffers and with various membrane compositions. Our experimental results reveal that lipid deposits obtained from aqueous liposome or proteoliposome dispersions are highly efficient for GUV electroformation. This is related to the ability of such dispersions to produce readily well-oriented membrane stacks. Furthermore, we present a protocol for GUV electroformation in various aqueous media, including electrolyte-containing buffers at characteristic concentrations of biological fluids. This work unlocks historical barriers to GUV applications in scientific fields like biology, biochemistry, or biophysics where membrane composition, as well as its aqueous environment, should be adapted to biological significance.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Calibration of nanowatt isothermal titration calorimeters with overflow reaction vessels

Obtaining accurate results with nanowatt titration calorimeters with overflow cells requires mass calibration of the buret injection volume, chemical calibration of the reaction vessel effective volume, and chemical calibration of the calorimetric factor used to convert the measured electrical signal to heat rate. Potential errors in electrical calibration of power compensation calorimeters require validation of the calorimetric factor with chemical reactions with accurately known stoichiometries and enthalpy changes. The effective volume of the reaction vessel can be determined from the endpoint of a quantitative reaction with known stoichiometries. Methods for calibration and potential calibration errors to be avoided are described. Publication of results obtained must include data on calibrations and sufficient raw data to assess precision and accuracy of the results.     

Analysis of the microbial proteome

Proteomics has begun to provide insight into the biology of microorganisms. The combination of proteomics with genetics, molecular biology, protein biochemistry and biophysics is particularly powerful, resulting in novel methods to analyse complex protein mixtures. Emerging proteomic technologies promise to increase the throughput of protein identifications from complex mixtures and allow for the quantification of protein expression levels.     

Problems associated with determining protein concentration: a comparison of techniques for protein estimations

Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric analysis and densitometry of stained protein gels, are applied, but perhaps to a lesser extent. However, the reliability of all of the above techniques is questionable and dependent to some extent on the protein to be assayed. In this paper we describe problems we encountered when using some of the foregoing techniques to quantify the concentration of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), a nuclear enzyme found in most eukaryotes. We also describe how, by using a fluorescence-based assay and amino acid analysis, we overcame the problems we encountered.     

Statcking and separatio of photosystem I and photosystem II in plant thylak oid membranes: A physico-chemical view

The main objective of this article is to highlight important experimental findings and discuss current theories on photosystem segregation and stacking in thylakoid membranes. The facts are put in a framework of recent theoretical developments in the field of membrane biochemistry and biophysics. Some important experiments not considered by the currently accepted theories are discussed. Modifications of these theories in order to incorporate new results are proposed. The currently accepted theories on formation of membrane domains (probably responsible for segregation of photosystem 1 and II) and on interlamellar interactions (stacking) are discussed. Finally, a scheme is put forward summarising the forces which are responsible for organisation of functional structure of thylakoid membranes.     

现代生物技术在乳品工业中的应用研究

随着细胞生物学和分子生物学的发展及对生物物理、生物化学、遗传学和免疫学研究的深入,培育了基因工程、细胞工程、酶工程、发酵工程等改变生物特性进行物质转化的现代生物技术,形成了DNA探针、PCR技术、分子标记、生物荧光技术、基因芯片技术等前沿性的生物检测技术,其在乳品工业中的广泛应用,推动了乳业的技术变革,对乳品生产、研究和乳品安全意义重大。