Modern analytical ultracentrifugation in protein science: a tutorial review

Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein-protein, protein-nucleic acid, and protein-small molecule binding.     

Evaluating the stoichiometry of macromolecular complexes using multisignal sedimentation velocity

Gleaning information regarding the molecular physiology of macromolecular complexes requires knowledge of their component stoichiometries. In this work, a relatively new means of analyzing sedimentation velocity (SV) data from the analytical ultracentrifuge is examined in detail. The method depends on collecting concentration profile data simultaneously using multiple signals, like Rayleigh interferometry and UV spectrophotometry. If the cosedimenting components of a complex are spectrally distinguishable, continuous sedimentation-coefficient distributions specific for each component can be calculated to reveal the molar ratio of the complex's components. When combined with the hydrodynamic information available from the SV data, a stoichiometry can be derived. Herein, the spectral properties of sedimenting species are systematically explored to arrive at a predictive test for whether a set of macromolecules can be spectrally resolved in a multisignal SV (MSSV) experiment. Also, a graphical means of experimental design and criteria to judge the success of the spectral discrimination in MSSV are introduced. A detailed example of the analysis of MSSV experiments is offered, and the possibility of deriving equilibrium association constants from MSSV analyses is explored. Finally, successful implementations of MSSV are reviewed.     

Characterization of heterologous protein-protein interactions using analytical ultracentrifugation.

Methods for quantitative characterization of heterologous protein-protein interactions by means of analytical ultracentrifugation (AUC) include sedimentation equilibrium, tracer sedimentation equilibrium, sedimentation velocity, and analytical band sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.     

The temperature jump relaxation technique.

The temperature jump relaxation technique is a convenient and general means of studying rapid reversible reactions of biological macromolecules. Recent advances in automatic data acquisition and the introduction of different optical detection systems will soon allow us to exploit the full potential of kinetic measurements near equilibrium. On the other hand, the kinetic approach can be quite limited if not accompanied by detailed structural and thermodynamic studies. Finally, we must accept the fact that one can rarely demonstrate a reaction mechanism to the exclusion of all plausible alternative models.     

Differential modulation of the active site environment of human carbonic anhydrase XII by cationic quantum dots and polylysine

Due to prevalence of negative charges on the protein surface, opposite to the active site pocket of human carbonic anhydrase XII (hCA XII), both positively charged CdTe quantum dots (Qds⁺) and polylysine electrostatically interact with the enzyme, and such interaction does not influence the catalytic activity of the enzyme. However, both these cationic macromolecules differently modulate the active site environment of the enzyme. The steady-state kinetic data revealed that whereas polylysine exhibited no influence on dansylamide (DNSA) dependent inhibition of the enzyme, Qds⁺ overcame such an inhibitory effect, leading to almost 70% restoration of the catalytic activity of the enzyme. We provide evidence that DNSA remains bound to the enzyme upon interaction with both polylysine and Qds⁺. Arguments are presented that the above differential feature of polylysine and Qds⁺ on hCA XII is encoded in the “rigidity” versus “flexibility” of these cationic macromolecules.     

Determination of macromolecular folding and structure by synchrotron x-ray radiolysis techniques

Radiolysis of water by synchrotron X-rays generates oxygen-containing radicals that undergo reactions with solvent accessible sites of macromolecules inducing stable covalent modifications or cleavage on millisecond time scales. The extent and site of these reactions are determined by gel electrophoresis and mass spectrometry analysis. These data are used to construct a high-resolution map of solvent accessibility at individual reactive sites. The experiments can be performed in a time-resolved manner to provide kinetic rate constants for dynamic events occurring at individual sites within macromolecules or can provide equilibrium parameters of binding and thermodynamics of folding processes. The application of this synchrotron radiolysis technique to the study of lysozyme protein structure and the equilibrium urea induced unfolding of apomyoglobin are described. The Mg2+-induced folding of Tetrahymena thermophila group I ribozyme shows the capability of the method to study kinetics of folding.     

Ribosomal elongation cycle: energetic, kinetic and stereochemical aspects

As a preface to an analysis of the ribosomal elongation cycle, we examine the energetics of macromolecular structural transformations. We show that the kinetic barriers and changes of the energetic levels during these transformations are essentially determined by disruption of hydrogen and cation-ligand bonds, and by uncompensated losses of these bonds (ULBs). The disruption of a hydrogen or cation-ligand bond increases the heights of kinetic barriers by the energy of these bonds. The association and dissociation of macromolecules, and conformational transitions within macromolecules, can change the numbers of ULBs but cannot completely eliminate them. Two important general conclusions are drawn from this analysis. First, occupation of enzyme active centers by substrates should be accompanied by a reduction in the number of ULBs. This reduction decreases the activation barriers in enzyme reactions, and is a major contributor to catalysis. Second, the enzymic reactions of the ribosomal cycle (structural changes caused by transpeptidation and by GTP hydrolyses in EF-Tu and EF-G) disrupt kinetic traps that prevent tRNAs from dissociating into solution during their motion within the ribosome and are necessary for progression of the cycle. These results are general purpose structural-functional blocks for building a molecular model of the ribosomal elongation cycle. Here, we demonstrate the utility of these blocks for analysis of acceptance of cognate tRNAs into the ribosomal elongation cycle.     

Measuring low levels of protein aggregation by sedimentation velocity

The required performance of an analytical method depends on the purpose for which it will be used. As a methodology matures, it may find new application, and the performance demands placed on the method can increase. Sedimentation velocity analytical ultracentrifugation (SV-AUC) has a long and distinguished history with important contributions to molecular biology. Now the technique is transitioning into industrial settings, and among them, SV-AUC is now used to quantify the amount of protein aggregation in biopharmaceutical protein products, often at levels less than 1% of the total protein mass. In this paper, we review recent advances to SV methodology which have been shown to improve quantitation of protein aggregation. Then we discuss the performance of the SV method in its current state, with emphasis on the precision and quantitation limit of the method, in the context of existing industrial guidance on analytical method performance targets for quantitative methods.     

Surface plasmon resonance: towards an understanding of the mechanisms of biological molecular recognition

With the introduction of new instruments and improved sensor chip chemistries, surface plasmon resonance (SPR) is finding new applications for molecular interaction studies. Easy access to high-quality kinetic and thermodynamic data for macromolecular binding events is providing insights into the fundamental mechanisms of molecular recognition. Progress is being made to allow larger-scale interaction studies. In addition, combining SPR with other analytical methods is enabling SPR-based analysis of interaction proteomics.     

Parameter estimation for ligand binding systems kinetics applied to 1,25-dihydroxycholecalciferol

A mathematical analysis of the kinetics of the hormone-receptor interaction was applied to the 1,25-dihydroxycholecalciferol-intestinal receptor system. The exact analytical solution and the numerical integration of the kinetic equation were installed in a Statistical Analysis System (SAS) computer program to estimate the rate constants of the reaction. Estimates of the parameters obtained by these two methods are similar, demonstrating that the numerical integration can be combined with the nonlinear regression procedure for least-squares parameter fitting using a simple SAS program. This enables estimation of kinetics rate constants when the kinetic equation cannot be solved analytically. The ratio of the rate constants (ka/kd) found by the nonlinear procedure is close to the independently determined equilibrium (Scatchard) constant in the nonlinear analysis.     

SV2B regulates synaptotagmin 1 by direct interaction

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor (SV2C) isoform. SV2A and SV2B have been shown to be involved in the regulation of synaptic vesicle exocytosis. Previous studies found that SV2A, but not SV2B, can interact with the cytoplasmic domain of synaptotagmin 1, a Ca2+ sensor for synaptic vesicle exocytosis. To determine whether SV2B can interact with full-length synaptotagmin 1, we performed immunoprecipitations from brain protein extracts and found that SV2B interacts strongly with synaptotagmin 1 in a detergent-resistant, Ca2+ -independent manner. In contrast, an interaction between native SV2A and synaptotagmin 1 was not detectable under these conditions. The SV2B-synaptotagmin 1 complex also contained the synaptic t-SNARE proteins, syntaxin 1 and SNAP-25, suggesting that SV2B may participate in exocytosis by modulating the interaction of synaptotagmin 1 with t-SNARE proteins. Analysis of retinae in SV2B knock-out mice revealed a strong reduction in the level of synaptotagmin 1 in rod photoreceptor synapses, which are unique in that they express only the SV2B isoform. In contrast, other synaptic vesicle proteins were not affected by SV2B knock out, indicating a specific role for SV2B in the regulation of synaptotagmin 1 levels at certain synapses. These experiments suggest that the SV2B-synaptotagmin 1 complex is involved in the regulation of synaptotagmin 1 stability and/or trafficking. This study has demonstrated a new role of SV2B as a regulator of synaptotagmin 1 that is likely mediated by direct interaction of these two synaptic proteins.     

A new approach based on monitoring of phase formation kinetics for examination of biological particles and cells,using aqueous two phase polymer systems

An original technique of use of two-phase polymer systems as an analytical research method is described. The technique is based on the absorbance change of two-phase systems in visible spectrum during formation of the phases. Dynamics of this process was demonstrated as the kinetic curves. Addition of studied objects (macromolecules or cells) to the two-phase system modified the shape of the kinetic curve, depending on their individual surface properties. The technique has the following advantages as compared with traditional procedures of the particle surface analysis with the help of two-phase polymer systems: examination of particles with partition coefficients approaching zero; multiple analyses of the same samples; use of interphase as a matrix for study of spontaneous formation of studied particle complexes. The opportunities of the technique were demonstrated in a series of previous authors' works.     

A new approach based on monitoring of phase formation kinetics for examination of biological particles and cells,using aqueous two phase polymer systems

An original technique of use of two-phase polymer systems as an analytical research method is described. The technique is based on the absorbance change of two-phase systems in visible spectrum during formation of the phases. Dynamics of this process was demonstrated as the kinetic curves. Addition of studied objects (macromolecules or cells) to the two-phase system modified the shape of the kinetic curve, depending on their individual surface properties. The technique has the following advantages as compared with traditional procedures of the particle surface analysis with the help of two-phase polymer systems: examination of particles with partition coefficients approaching zero; multiple analyses of the same samples; use of interphase as a matrix for study of spontaneous formation of studied particle complexes. The opportunities of the technique were demonstrated in a series of previous authors' works.     

Computational exploration of structural information from cryo-electron tomograms

Cryo-electron tomography aims to act as an interface between in vivo cell imaging and techniques achieving atomic resolution. This attempt to bridge the resolution gap is facilitated by recent software and hardware advances. Information provided by atomically resolved macromolecules and molecular interaction data need to be put into a common framework in order to create a hybrid multidimensional cellular image. A major partner in this enterprise is the development of regularization and pattern recognition techniques, which try to identify macromolecular complexes as a function of their structural signature in cryo-electron tomograms of living cells.     

A microdetermination method for assaying glycosaminoglycans and proteoglycans

A simple and reliable method is described for the determination of glycosaminoglycans and proteoglycans in tissue extracts as well as during preparative and analytical procedures for these molecules. It is particularly useful because it requires much less starting material for the identification of glycosaminoglycans or proteoglycans and, in addition, is several fold more sensitive than the currently used uronic acid assays. The procedure allows separation of macromolecules by ion-exchange chromatography, density gradient centrifugation, or molecular sieve chromatography and involves spotting onto cellulose acetate membrane, reaction with Alcian blue, and quantitation of color in a spectrophotometer. This method is particularly appropriate to use for the analysis of proteoglycans and glycosaminoglycans in tissues which are available in limited amounts or have low levels of these macromolecules.     

Determination of the association constants and maximum binding capacities between estradiol and cytosol of the uterus by the least-squares analysis of the Scatchard plot.

The association constants and the binding capacities of association of small molecules with macromolecules have been determined by the tangent analysis, the graphical analysis, and the computer data analysis, by trial and convergence of the Scatchard plot. The analytical method for the calculation of the binding parameters based on the Scatchard plot was derived and the optimum values of the binding parameters were obtained by the least squares calculation based on the analytical method. The errors by the analytical method were smaller than those by the graphical method in the equilibrium system between 3H-estradiol and some cytosols of uterus.     

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X‐ray crystallography and single‐particle cryo‐electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative‐staining, rotary‐shadowing and freeze‐etching EM, which are categorised here as ‘direct imaging EM methods’. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome–translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three‐dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.     

Enzymology in vivo using NMR and molecular genetics

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substates and effectors concentrations and possible interaction with other cellular macromolecules, which may modify their kinetic properties. These is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measured the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo