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Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by approximately 100-fold through the use of a quasi-continuous increase of rotor speed during the experiment. Such ‘gravitational sweep’ sedimentation approaches has previously been shown to be very effective in the study of nanoparticles with large size ranges. However, previously diffusion processes were not accounted for, therefore posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In the present work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extends the macromolecular size-range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitudes of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, for example, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar sized macromolecular species, in the study of broadly distributed nanoparticles.

Analytical ultracentrifugation (AUC) is a classical biophysical technique to characterize proteins and their interactions in solution. Traditional absorbance and interference detection systems lack specificity and high sensitivity, which limit AUC applications in complex and concentrated solutions. A recently introduced fluorescence detection system (FDS) strongly increases the sensitivity of the detection in AUC, and offers the potential to study proteins and protein interactions in biological fluids. By using enhanced green fluorescent protein (EGFP) as a model system, we explore the potential and challenges posed by the application of FDS-AUC to the study of protein interactions in E.coli and Cos7 whole cell extracts. Complex autofluorescence and quenching processes limit the concentration of E.coli cell extract to a few mg/mL. At these concentrations of E.coli cell extract, no impact of this environment on the sedimentation of EGFP is discernable. Autofluorescence also sets the detection limit of EGFP in the low nanomolar range. In comparison with E.coli, the mammalian Cos7 cell extract has less background autofluorescence, which lowers the detection limit for EGFP into the subnanomolar range. To investigate protein interactions, monoclonal anti-EGFP antibody was added to the E.coli cell extract with exogenous EGFP and the Cos7 cell extract with exogenous or endogenous EGFP. All three arrangements show shifts in the sedimentation coefficient distribution that reflect complex formation. Quantitatively, a small decrease of the complex sedimentation coefficient can be observed in cell extracts due to hydrodynamic nonideality, still allowing the determination of complex size and stoichiometry. In summary, this study demonstrates the potential for studying interactions of unpurified GFP-tagged proteins in bacterial and mammalian cell extracts using fluorescence detected AUC.

Sedimentation velocity (SV) analytical ultracentrifugation is a classical biophysical technique for the determination of the size-distribution of macromolecules, macromolecular complexes, and nanoparticles. SV has traditionally been carried out at a constant rotor speed, which limits the range of sedimentation coefficients that can be detected in a single experiment. Recently we have introduced methods to implement experiments with variable rotor speeds, in combination with variable field solutions to the Lamm equation, with the application to expedite the approach to sedimentation equilibrium. Here, we describe the use of variable-field sedimentation analysis to increase the size-range covered in SV experiments by ∼100-fold with a quasi-continuous increase of rotor speed during the experiment. Such a gravitational-sweep sedimentation approach has previously been shown to be very effective in the study of nanoparticles with large size ranges. In the past, diffusion processes were not accounted for, thereby posing a lower limit of particle sizes and limiting the accuracy of the size distribution. In this work, we combine variable field solutions to the Lamm equation with diffusion-deconvoluted sedimentation coefficient distributions c(s), which further extend the macromolecular size range that can be observed in a single SV experiment while maintaining accuracy and resolution. In this way, approximately five orders of magnitude of sedimentation coefficients, or eight orders of magnitude of particle mass, can be probed in a single experiment. This can be useful, for example, in the study of proteins forming large assemblies, as in fibrillation process or capsid self-assembly, in studies of the interaction between very dissimilar-sized macromolecular species, or in the study of broadly distributed nanoparticles.

Isothermal titration calorimetry (ITC) is a traditional and powerful method for studying the linkage of ligand binding to proton uptake or release. The theoretical framework has been developed for more than two decades and numerous applications have appeared. In the current work, we explored strategic aspects of experimental design. To this end, we simulated families of ITC data sets that embed different strategies with regard to the number of experiments, range of experimental pH, buffer ionization enthalpy, and temperature. We then re-analyzed the families of data sets in the context of global analysis, employing a proton linkage binding model implemented in the global data analysis platform SEDPHAT, and examined the information content of all data sets by a detailed statistical error analysis of the parameter estimates. In particular, we studied the impact of different assumptions about the knowledge of the exact concentrations of the components, which in practice presents an experimental limitation for many systems. For example, the uncertainty in concentration may reflect imperfectly known extinction coefficients and stock concentrations or may account for different extents of partial inactivation when working with proteins at different pH values. Our results show that the global analysis can yield reliable estimates of the thermodynamic parameters for intrinsic binding and protonation, and that in the context of the global analysis the exact molecular component concentrations may not be required. Additionally, a comparison of data from different experimental strategies illustrates the benefit of conducting experiments at a range of temperatures.

Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and post-injection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.

Accurate measurements of rotor temperature are critical for the interpretation of hydro dynamic parameters in analytical ultracentrifugation. We have recently developed methods for a more accurate determination of the temperature of a spinning rotor utilizing iButton® temperature loggers. Here we report that the temperature measured with the iButton on the counterbalance of a resting rotor, following thermal equilibration under high vacuum, closely corresponded to the temperature of the spinning rotor with a precision better than 0.2 °C. This strategy offers an inexpensive and straightforward approach to monitor the accuracy of the temperature calibration and determine corrective temperature offsets.

Sedimentation velocity (SV) analytical ultracentrifugation has re-emerged as an important tool in the characterization of biological macromolecules and nanoparticles. The computational analysis of the evolution of the macromolecular concentration profile allows the characterization of many hydrodynamic and thermodynamic properties of the macromolecules and their interactions. The Rayleigh interference optical system is often the detection method of choice, for its usually superior data quality and the wide applicability of refractive index sensitive detection. However, the interference optical system is also sensitive to the redistribution of co-solvent molecules, which are not of primary experimental interest. In principle, their contribution can be eliminated by an exact geometric and compositional match of the sample solution and the reference solution, achieving the complete optical subtraction of unwanted buffer signals. Unfortunately, in practice, this can often not be perfectly achieved for various reasons, leading to signal offsets arising from unmatched sedimentation of solvent components. If unrecognized, this can lead to significant misfit, accompanied by significant errors in the macromolecular sedimentation parameters. In the present work, we describe an approach of computationally accounting for signals from sedimenting buffer components through explicitly modeling their redistribution with Lamm equation solutions, implemented in the software SEDFIT. We demonstrate how this can restore the SV analysis to yield a high quality fit of the data and to provide correct macromolecular sedimentation parameters.

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05–0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.

Proteins can perform completely distinct functions in response to the particular partners to which they bind. Consequently, determination of the mechanism of functional regulation in such systems requires elucidation of the mechanism switching between binding partners. The central protein of the Escherichia coli Biotin Regulatory System, BirA, switches between it’s function as a metabolic enzyme or a transcriptional repressor in response to binding either the Biotin Carboxyl Carrier Protein subunit of acetyl-CoA carboxylase or a second BirA monomer. These two protein:protein interactions are structurally mutually exclusive. Results of previous studies suggest that the system is regulated by kinetic partitioning between the two protein:protein interactions. In this work sedimentation velocity was employed to directly monitor the partitioning. Results of the measurements indicate similar equilibrium parameters governing formation of the two protein:protein interactions. Kinetic analysis of the sedimentation velocity data indicates that holoBirA dimerization is governed by very slow forward and reverse rate constants. The slow kinetics of holoBirA dimerization combined with fluctuations in the intracellular apoBCCP pool are critical determinants in partitioning of BirA between its distinct biological functions.

Gram-negative bacteria tightly regulate intracellular levels of iron, an essential nutrient. To ensure this tight regulation, some outer membrane TonB-dependent transporters (TBDTs) that are responsible for iron import stimulate their own transcription in response to extracellular binding by an iron-laden siderophore. This process is mediated by an inner membrane sigma regulator protein (an anti-sigma factor) that transduces an unknown periplasmic signal from the TBDT to release an intracellular sigma factor from the inner membrane, which ultimately upregulates TBDT transcription. Here we use the Pseudomonas putida ferric-pseudobactin BN7/BN8 sigma regulator, PupR, as a model system to understand the molecular mechanism of this conserved class of sigma regulators. We have determined the X-ray crystal structure of the cytoplasmic anti-sigma domain (ASD) of PupR to 2.0 Å. Size exclusion chromatography, small angle X-ray scattering, and sedimentation velocity analytical ultracentrifugation, all indicate that in contrast to other ASDs, the PupR-ASD exists as a dimer in solution. Mutagenesis of residues at the dimer interface identified from the crystal structure disrupts dimerization and protein stability, as determined by sedimentation velocity analytical ultracentrifugation and thermal denaturation circular dichroism spectroscopy. These combined results suggest that this type of inner membrane sigma regulator may utilize an unusual mechanism to sequester their cognate sigma factors and prevent transcription activation.

30 p.-14 fig. Zhao, Huaying, et alt., Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

The quality of fit of sedimentation velocity data is critical to judge the veracity of the sedimentation model and accuracy of the derived macromolecular parameters. Absolute statistical measures are usually complicated by the presence of characteristic systematic errors and run-to-run variation in the stochastic noise of data acquisition. We present a new graphical approach to visualize systematic deviations between data and model in the form of a histogram of residuals. In comparison with the ideally expected Gaussian distribution, it can provide a robust measure of fit quality and be used to flag poor models.

Sedimentation equilibrium (SE) analytical ultracentrifugation is a gold standard for the rigorous thermodynamic study of buoyant molecular weight and reversible interactions of macromolecules in solution. A significant drawback is the long experiment time, as it takes days to attain SE with standard solution columns. We have developed a new method for using a time-varying centrifugal field optimized such as to attain SE in significantly shorter time than usually required. Experimental data show that this permits long-column SE experiments to be carried out in times comparable to sedimentation velocity experiments, approximately fivefold shorter than standard SE. In contrast to the classical initial overspeeding method, which uses a single initial speed, we employ a freely varying rotor speed profile during an initial phase, for example, parameterized as a step-wise modulated exponential decay to the desired SE rotor speed. The rotor speed schedule is computationally optimized on the basis of numerical Lamm equation solutions for given macromolecular sedimentation parameter estimates, with the goal to provide a rapid attainment of equilibrium without the drawback of strong transient sample pre-concentration at the base of the solution column. The resulting rotor speed schedule frequently includes both over- and under-speeding sequences, and can be conveniently implemented on the Optima XLA/I analytical ultracentrifuge. We extended AUC data analysis models in SEDFIT to permit the analysis of concentration profiles in arbitrarily time-varying fields, to make it possible to exploit the migration in the initially high centrifugal field for estimates on macromolecular sedimentation parameters, which may be used in real-time to refine the prediction of the rotor speed schedule, so that the SE experiment can be optimized in both information content and time efficiency.

The analytical ultracentrifugation (AUC) is a classic biophysical instrument to analyze protein interactions in solutions. A recently introduced fluorescence detection system (FDS) improves the specificity and sensitivity of the AUC, and offers the potential to analyze the protein interactions in biological fluids. We explore challenges posed by the application of FDS-AUC to the study of protein interactions in E.coli whole cell extracts, using fluorescent proteins (EGFPs) as model system. At experimentally feasible concentrations of cell extracts, we find no discernable effects of hydrodynamic nonideality on the sedimentation of EGFPs. However, at high concentrations E.coli whole cell extracts produce significant signals from auto fluorescence with complicated quenching patterns. Goal of this work is to establish detection limits and develop procedures to improve specificity.

Fluorescence detected sedimentation velocity (FDS-SV) analytical ultracentrifugation has emerged as a powerful technique for the study of macromolecular interactions, particularly high-affinity protein interactions, with hydrodynamic resolution exceeding that of diffusion-based techniques, and with sufficient sensitivity for binding studies at low picomolar concentrations. In order to fit the FDS data structure, in the quantitative analysis it is essential to adjust the conventional sedimentation models for detailed description of the sedimentation boundaries. A key consideration is the change in the macromolecular fluorescence intensity during the course of the experiment, caused by slow drifts of the excitation laser power, and/or by photophysical processes. In the present work we demonstrate that FDS-SV data have inherently a reference for the time-dependent macromolecular signal intensity, resting on a geometric link between boundary migration and plateau signal. We show how this new time-domain can be exploited to study molecules exhibiting photobleaching and photoactivation. This expands the application of FDS-SV to proteins tagged with photoswitchable fluorescent proteins, organic dyes, or nanoparticles, such as those recently introduced for sub-diffraction microscopy. At the same time, we find conventional fluorophores undergo minimal photobleaching under standard illumination in the FDS. These findings support the application of a high laser power density for the detection, which we demonstrate can further increase the data quality.

Epithelial cadherin is a transmembrane protein that is essential in calcium-dependent cell–cell recognition and adhesion. It contains five independently folded globular domains in its extracellular region. Each domain has a seven-strand β-sheet immunoglobulin fold. Short seven-residue peptide segments connect the globular domains and provide oxygens to chelate calcium ions at the interface between the domains (Nagar et al., Nature 1995;380:360–364). Recently, stability studies of ECAD2 (Prasad et al., Biochemistry 2004;43:8055–8066) were undertaken with the motivation that Domain 2 is a representative domain for this family of proteins. The definition of a domain boundary is somewhat arbitrary; hence, it was important to examine the effect of the adjoining linker regions that connect Domain 2 to the adjacent domains. Present studies employ temperature–denaturation and proteolytic susceptibility to provide insight into the impact of these linkers on Domain 2. The significant findings of our present study are threefold. First, the linker segments destabilize the core domain in the absence of calcium. Second, the destabilization due to addition of the linker segments can be partially reversed by the addition of calcium. Third, sodium chloride stabilizes all constructs. This result implies that electrostatic repulsion is a contributor to destabilization of the core domain by addition of the linkers. Thus, the context of Domain 2 within the whole molecule affects its thermodynamic characteristics. Proteins 2006. © 2005 Wiley-Liss, Inc.

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer–dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer–dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer–dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.

The molecular mechanism through which the interaction of a clonotypic αβ TCR with peptide/MHC (p/MHC) complexes leads to T cell activation is not yet fully understood. Here we exploit a high affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd) by combining crystallographic, NMR, and functional data. In addition to conformational changes in complementarity determining regions (CDR) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β chain dynamics reveals significant chemical shift effects in sites removed from the MHC binding site. In particular, a remodeling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests an allosteric mechanism for TCR signaling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T cell functional assays.

The coupled transport behavior of macromolecular mixtures with rapidly reversible complex formation is of great interest in the study of protein interactions by many different methods. Complicated transport patterns arise even for simple bimolecular reactions, when all species exhibit different migration velocities, yet are coupled by mass action law. Although partial differential equations are available to describe the spatio-temporal evolution of such systems given particular initial conditions, a general overview of their behavior, for example, as a function of concentration, velocities, and binding constants, has not yet been reported. For two-component systems, based on the limit of non-diffusing species migrating under constant force, we have previously derived simple algebraic relationships describing the number, amplitude, and velocity of sedimentation boundaries, which are consistent with the principle that the time-average velocity of all co-sedimenting component fractions in each boundary must match. In combination, these can be considered ‘effective particles’, and their properties can be related to parameters extracted from experimental sedimentation profiles of mixtures of two interacting protein components. To support the study of three-component (and higher) interacting protein mixtures, here, we generalize this effective particle theory to N-component systems. These can have N-2 reaction boundaries of successively lower velocity and number of components. We report algebraic relationships for velocities and compositions. Considering the space of total component concentrations, we show it is subdivided into regions resulting in different boundary patterns, separated by discontinuous transitions along characteristic surfaces. These meet in a single point - a unique combination of component concentrations where the entire system can migrate as if it was a single species. The extension of the effective particle theory provides physical insights into the coupled co-migration processes, and we believe this will be useful to interpret experimentally robust parameters derived from measured sedimentation coefficient distributions of multi-component mixtures with rapidly reversible interactions.

Multisignal sedimentation velocity (MSSV) analysis is a powerful method for determining the stoichiometry of reversibly formed multi-protein complexes. For example, it has been used to unravel the number and stoichiometry of co-existing complexes, for example, in receptor-ligand interactions and in systems of adaptor proteins. By globally analyzing absorbance data acquired simultaneously at different wavelengths, differences in the component spectra can be exploited to obtain information on the composition of all hydrodynamically resolved species. For fluorescence data that are acquired only at a single excitation wavelength, it was recently shown that the characteristic quantum yield of photoswitching of reversibly photoswitchable fluorescent proteins leads to a temporal fluorescence signal change on the time-scale of sedimentation, which can substitute for spectral discrimination of components in an analysis analogous to MSSV. In the present work, we have embarked on combining the two concepts by considering the temporal extinction coefficient changes associated with reversible photoswitching in the context of spectral decomposition of components. To this end, we report pilot experiments, for the first time acquiring and analyzing multi-wavelength absorbance data with temporal modulation of extinction amplitudes. Theoretically, the combination of spectral and temporal signal domain is expected to greatly enhance the discrimination of sedimenting components, thereby facilitating the study of multi-protein complexes with a higher number of components in solution.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) is monitoring the migration of particles in free solution with real-time optical detection. It provides powerful methods for studying multi-component systems of biomacromolecules such as protein and DNA. It offers exquisite resolution regarding the size and shape of the components and population of co-existing complexes, and therefore binding energies and stoichiometries for reversible interacting systems can be resolved. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying molecules at low concentration because of the high sensitivity and selectivity of fluorescence. In order to make FDS-SV a robust and quantitative method for studying macromolecules, we have previously developed computational tools to specifically account for the unique data structure posed by the confocal design and other geometric factors. Such developments allow FDS-SV to be applied to high-affinity binding systems at concentrations well into the picomolar regime. Recently we extended FDS-SV to multi-dimensional signal detection by employing photoswitchable fluorescent proteins and taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching. Thus, FDS-SV is an excellent tool for the study of high-affinity protein interactions. We will show applications to protein-protein and protein-nucleic acid interactions with multiple states, quantitatively determining the binding affinity, stoichiometry and cooperativity.

Isothermal titration calorimetry experiments can provide significantly more detailed information about molecular interactions when combined in global analysis. For example, global analysis can improve the precision of binding affinity and enthalpy, and of possible linkage parameters, even for simple bimolecular interactions, and greatly facilitate the study of multi-site and multi-component systems with competition or cooperativity. A pre-requisite for global analysis is the departure from the traditional binding model, including an 'n'-value describing unphysical, non-integral numbers of sites. Instead, concentration correction factors can be introduced to account for either errors in the concentration determination or for the presence of inactive fractions of material. SEDPHAT is a computer program that embeds these ideas and provides a graphical user interface for the seamless combination of biophysical experiments to be globally modeled with a large number of different binding models. It offers statistical tools for the rigorous determination of parameter errors, correlations, as well as advanced statistical functions for global ITC (gITC) and global multi-method analysis (GMMA). SEDPHAT will also take full advantage of error bars of individual titration data points determined with the unbiased integration software NITPIC. The present communication reviews principles and strategies of global analysis for ITC and its extension to GMMA in SEDPHAT. We will also introduce a new graphical tool for aiding experimental design by surveying the concentration space and generating simulated data sets, which can be subsequently statistically examined for their information content. This procedure can replace the 'c'-value as an experimental design parameter, which ceases to be helpful for multi-site systems and in the context of gITC.

Analytical ultracentrifugation (AUC) offers substantial advantages for studying protein complex formation, providing information on size, shape and binding energies for reversible systems from analysis of the sedimentation profiles of molecular mixtures in free solution. By virtue of the hydrodynamic resolution achieved in sedimentation velocity experiments, multiple co-existing complexes can be identified, even in the presence of impurities and aggregates, but until recently the limited sensitivity of AUC has prevented accurate analysis of high affinity interactions. A recently introduced fluorescence optical detection system (FDS) for AUC offers specific advantages for studying high-affinity protein interactions due to the high sensitivity of fluorescence, but the unique characteristics of the optical system in this instrument require new analytical tools. We have developed computational approaches for analysis of FDS data that provide excellent fits to the observed sedimentation boundaries, correcting for effects intrinsic to the use of confocal laser optics. With these tools we have explored the detection limit of FDS, and have now recorded sedimentation signals with hydrodynamic resolution at low picomolar concentrations. This allows us to analyze sedimentation isotherms for interacting systems with Kds in the pM to nM range. Using different systems with high-affinity interaction we will demonstrate how the FDS-AUC system can be reliably applied to study high-affinity protein self- and hetero-associations at orders of magnitude lower concentrations and with much higher accuracy than previously possible.

γ-crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ-crystallins have high contents of sulfur-containing methionine and cysteine, but this reaches extremes in fish γM-crystallins with up to 15% Met. In addition, fish γM-crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ-crystallins and in the related β-crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ-crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β-sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full-length and truncated versions of human αB-crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ-crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ-crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ-crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ-crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B1 could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure-based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure-based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.

Significant progress in the interpretation of analytical ultracentrifugation (AUC) data in the last decade has led to profound changes in the practice of AUC, both for sedimentation velocity (SV) and sedimentation equilibrium (SE). Modern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in SV size-distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of AUC, such as the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology. New global multi-signal modeling and mass conservation approaches in SV and SE, in conjunction with the effective-particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multi-protein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current commentary is to supplement previous AUC protocols, Current Protocols in Protein Science 20.3 (1999) and 20.7 (2003), and 7.12 (2008), and provide an update describing the current tools for the study of soluble proteins, detergent-solubilized membrane proteins and their interactions by SV and SE.

Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.

Multi-component protein complex formation is of great interest in physiological and biochemical studies as it is ubiquitous in biological systems. Analytical ultracentrifugation (AUC) provides powerful methods for studying such systems. It offers information on size, shape and binding energies for interacting systems from the analysis of sedimentation profiles of molecular mixtures in free solution. By virtue of the superb hydrodynamic resolution achieved in sedimentation velocity, co-existing complexes can be identified, even in the presence of impurities and aggregates. A recently introduced fluorescence optical detection system (FDS) for AUC substantially extends capabilities for studying high-affinity protein interactions because of the selectivity of fluorescence high sensitivity, allowing studies at picomolar concentrations. To overcome limitations posed by a single excitation wavelength, in the current study, we employed photoswitchable fluorescent proteins (FP) as probes in FDS-AUC. Taking advantage of their characteristic time-dependent fluorescent signal change due to photoswitching during FDS-AUC, a new dimension of detection is created that allows resolving specific FPs in multi-component mixtures. We have developed a computational framework for the analysis of sedimentation data exploiting the new temporal dimension. Experimentally we demonstrated this approach using mixtures of FPs commonly used in imaging, which could be readily distinguished from experimental FDS data. This approach extends FDS-AUC to more complicated protein system with multiple components and allows for quantitative characterization of protein complexes with regard to the binding mechanism and stoichiometry under very low macromolecular concentrations.

Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)–like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I–like immunoevasin/MHC-I–like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.

Cooperativity, multi-site and multi-component interactions are hallmarks of biological systems of interacting macromolecules. Their thermodynamic characterization is often very challenging due to the notoriously low information content of binding isotherms. We introduce a strategy for the global multi-method analysis of data from multiple techniques (GMMA) that exploits enhanced information content emerging from the mutual constraints of the simultaneous modeling of orthogonal observables from calorimetric, spectroscopic, hydrodynamic, biosensing, or other thermodynamic binding experiments. We describe new approaches to address statistical problems that arise in the analysis of dissimilar data sets. The GMMA approach can significantly increase the complexity of interacting systems that can be accurately thermodynamically characterized.

γ-crystallins constitute the major protein component in the nucleus of the vertebrate eye lens. Present at very high concentrations, they exhibit extreme solubility and thermodynamic stability to prevent scattering of light and the formation of cataracts. However, functions beyond this structural role have remained mostly unclear. Here, we calculate molecular refractive index increments of crystallins. We show that all lens γ-crystallins have evolved a significantly elevated molecular refractive index increment, which is far above those of most proteins, including non-lens members of the βγ-crystallin family from different species. The same trait has evolved in parallel in crystallins of different phyla, including in the S-crystallins of cephalopods. A high refractive index increment can lower the crystallin concentration required to achieve a suitable refractive power of the lens, and thereby reduce their propensity to aggregate and form cataract. To produce a significant increase of the refractive index increment, a substantial global shift in the amino acid composition is required, which can naturally explain the highly unusual amino acid composition of γ-crystallins and their functional homologues. This function provides a new perspective for interpreting their molecular structure.

Sedimentation velocity (SV) experiments of heterogeneous interacting systems exhibit characteristic boundary structures that can usually be very easily recognized and quantified. For slowly interacting systems, the boundaries represent concentrations of macromolecular species sedimenting at different rates, and they can be interpreted directly with population models based solely on the mass action law. For fast reactions, migration and chemical reactions are coupled, and different, but equally easily discernable boundary structures appear. However, these features have not been commonly utilized for data analysis, for the lack of an intuitive and computationally simple model. The recently introduced effective particle theory (EPT) provides a suitable framework. Here, we review the motivation and theoretical basis of EPT, and explore practical aspects for its application. We introduce an EPT-based design tool for SV experiments of heterogeneous interactions in the software SEDPHAT. As a practical tool for the first step of data analysis, we describe how the boundary resolution of the sedimentation coefficient distribution c(s) can be further improved with a Bayesian adjustment of maximum entropy regularization to the case of heterogeneous interactions between molecules that have been previously studied separately. This can facilitate extracting the characteristic boundary features by integration of c(s). In a second step, these are assembled into isotherms as a function of total loading concentrations and fitted with EPT. Methods for addressing concentration errors in isotherms are discussed. Finally, in an experimental model system of alpha-chymotrypsin interacting with soybean trypsin inhibitor, we show that EPT provides an excellent description of the experimental sedimentation boundary structure of fast interacting systems.

National Institute for Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, USA.Studies on protein-protein interactions are of considerable interest in the fields of macromolecular recognition, signal transduction and cellular regulation. With the introduction of modern instrumentation and computational methods, sedimentation velocity (SV) analytical ultracentrifugation has reemerged in the last decade as a powerful technique for characterizing binding equilibria. For rapidly reversible systems of interacting macromolecules when the lifetime of a complex is short relative to the transport time of a SV experiment, complicated transport patterns arise even for simple bimolecular reactions, when all species migrate at different velocities in the solution. Their physical origin of these patterns has been poorly understood, and this has limited fully exploiting all aspects of SV for rapid protein interactions. Recently, we have reported new solutions (effective particle theory, EPT) to the transport equations for rapidly reacting systems, which describe the average sedimentation coefficients and the composition of undisturbed and reaction boundaries with simple analytical expressions and provide a physical picture of the phenomenon of combined transport and reacting processes. In this work, we apply EPT to characterize several protein-protein interactions and demonstrate how the prediction of the transport patterns helps to quantify the assembly energetic interacting systems.Key words: protein interactions; transport; sedimentation; signal transduction

When studying macromolecular interactions, the thermodynamics and stoichiometry of binding are of considerable interest because they indicate the physical-chemical nature of the biological mechanism. Since a single biophysical technique is limited in the number of observable properties and may provide only insufficient information for more complex systems, one promising approach is the simultaneous consideration of data from multiple biophysical methods. In the past, we have developed a robust computational framework (SEDPHAT) for this purpose which has been widely used in the biophysical community. However, the best strategy for assembling individual data sets into a global analysis has not been explored. It requires understanding of the limitations and consideration of possible systematic errors for each method. In this work, we have performed experiments on a model system (α-chymotrypsin binding to soybean trypsin inhibitor) to study the detailed compatibility of data from calorimetry (ITC), surface binding (SPR), sedimentation (SV) and fluorescence anisotropy. The significance of each data set from the different techniques has been explored through both individual and global analysis with detailed error surface projection using the program, SEDPHAT. We propose a rational strategy for global analysis that deviates from the purely statistical point of view, by rescaling the weights of each data set such that all techniques can make significant contributions. This allows a more detailed picture of the interaction to emerge.