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Light intensity measurements are required by absorbance, fluorescence, turbidity and low-angle light scattering detectors in order to determine the concentration distributions encountered in analytical ultracentrifugation. By using four fast analog-to-digital converters operating in parallel, a data acquisition system has been developed for the analytical ultracentrifuge that can acquire light intensity readings from three detectors (e.g. photomultipliers, avalanche photodiodes, etc.) simultaneously. For each detector, up to forty thousand intensity readings are acquired during each rotor revolution, for up to ten revolutions. Software synchronizes data acquisition with the spinning rotor. The use of continuous light sources allows simultaneous acquisition of dozens of intensity readings from all of the samples. Data acquisition is fast, allowing rapid radial scanning of samples. This data acquisition scheme is used in the Aviv Biomedical AU-FDS fluorescence detection retrofit system for the Beckman XLI analytical ultracentrifuge. It also will be used for the updated XLI absorbance system, as well as the next generation of analytical ultracentrifuge.

The advantages of simultaneously detecting multiple wavelengths in ultracentrifugation experiments are obvious, especially for interacting systems. In addition, the detection of the wavelength dependence of turbidity opens up the possibility to obtain independent information on the particle size in addition to the usual sedimentation coefficient distribution for colloidal systems. We therefore made an effort to develop a fast UV/Vis detector, which is able to simultaneously detect the range from 200–800 nm. This is possible by the use of a modern CCD chip based generation of UV-Vis spectrometers, which translates the dispersed white light onto a CCD chip, where each pixel corresponds to a particular wavelength. In addition to the simultaneous detection of a large number of wavelengths in the range 200–800 nm, also with non integer values, these spectrometers are very fast. Current typical spectrum scan times with the necessary scan quality in the ultracentrifuge are in the range of 100 ms but this time can be significantly shortened down to 3 ms for higher light intensities and even down to 10 μs for a new generation of CCD chip based spectrometers.

The introduction of a fiber based UV-Vis optics into a preparative XL-80K ultracentrifuge with the associated hardware developments will be described as a first generation prototype. In this study, we use a wavelength dependent optical lens system instead of the necessary but more complex wavelength independent mirror optical system for a first check on possibilities and limitations of the optical system. First examples for biopolymers and latexes will be presented and compared to those obtained in the commercial XL-A ultracentrifuge. Already the fast detection enables completely new possibilities like the determination of a particle size distribution in a few minutes. Multiwavelength detection at constant position in dependence of time will be demonstrated, which is an important mode for the use of speed profiles for very polydisperse samples. Also, the use of radial multiwavelength scans will be demonstrated producing a three dimensional data space for monitoring the sedimentation via radial scans with multiwavelength detection. However, despite the advantages, the current problems with the detector will also be discussed including the main problem that much intensity is lost in the important UV range as a result of fiber coupling and bending.

We developed a system for acquisition and analysis of interference patterns from an analytical ultracentrifuge or a polarising diffusiometer. The system is the first of its kind to be based on highly sensitive Lebedev optical system. We also present a novel algorithm for automatic extraction of interference curves from photographs. The algorithm is fast, robust in the face of optical noise and may have potential applications in other domains.

The binding constants of self-associating proteins can be determined from sedimentation velocity runs using the numerical solution of the Lamm equation. This procedure requires good starting values of sedimentation and diffusion coefficients or their possible concentration dependencies. We found, based on fits of synthetic data files, a connection between the concentration dependence of sedimentation coefficients () and the estimated radius position at the cell base (). Deviation of the estimated value from the expected one is indicating the occurrence of that can be estimated by the program LammNum as a prerequisite for the calculation of reliable binding constants.

Sedimentation experiments can provide a large amount of information about the composition of a sample, and the properties of each component contained in the sample. To extract the details of the composition and the component properties, experimental data can be described by a mathematical model, which can then be fitted to the data. If the model is nonlinear in the parameters, the parameter adjustments are typically performed by a nonlinear least squares optimization algorithm. For models with many parameters, the error surface of this optimization often becomes very complex, the parameter solution tends to become trapped in a local minimum and the method may fail to converge. We introduce here a stochastic optimization approach for sedimentation velocity experiments utilizing genetic algorithms which is immune to such convergence traps and allows high-resolution fitting of nonlinear multi-component sedimentation models to yield distributions for sedimentation and diffusion coefficients, molecular weights, and partial concentrations.

For modeling the low-resolution shape of the dodecameric subunit of hemoglobin, experimental small-angle X-ray scattering (SAXS) data and ab initio modeling approaches using a genetic algorithm or simulated annealing have been applied. In addition to the use of strict ab initio approaches, procedures which additionally include available structural information concerning symmetry and shape in the form of constraints or templates have been employed to improve the results. Templates for the subunit were preferably derived from SAXS-based models for the native hexagonal bilayer (HBL) complex that were biased by electron microscopic reconstructions. The obtained subunit models were carefully examined by variation of different selection and averaging methods and other checks such as surface renderings of the models. The findings were quantified by prediction of scattering profiles, () and (), and structural and hydrodynamic parameters (, ,   , , ). The best matching models for the subunit were also scrutinized by comparing them to a model derived from currently available crystallographic data. The following results could be obtained: (i) The obtained parameter predictions for the dodecameric subunit are satisfactory, if compared to the SAXS data (consensus model, profiles and molecular parameters) or the results from hydrodynamic studies. (ii) The comparison between solution and crystal data of the dodecameric subunit, however, unequivocally proves a different behavior of the subunit in solution and the crystalline state.

The joint use of small-angle X-ray scattering (SAXS) and hydrodynamic data permits biologically useful reconstructions of protein structures to be determined. Low-resolution shapes of proteins can be obtained by SAXS-based modeling approaches, among them the ab initio approaches being the most recent and challenging ones. The programs DAMMIN and GASBOR have been applied to starch phosphorylase in a case study, to test in a systematic manner the principles governing the evaluation strategies of the approaches applied. Therefore, emphasis was laid on the elaboration of modeling aspects rather than on biological details. Optimum results concerning the predictions of particle shapes and molecule properties have been obtained by utilizing tight constraints for modeling, such as symmetry and anisometry information. The use of pure ab initio conditions yields rather moderate shape and parameter predictions. Application of erroneous constraints generally leads to unrealistic particle shapes, although the parameter predictions may be satisfactory. The usage of the program DAMMIN turned out to be superior to application of the program GASBOR, whether the latter approach was used in the reciprocal- or real-space version. For hydrodynamic modeling, a modified version of the program HYDRO was adopted. By recourse to known crystallographic 3D structures for phosphorylases from other sources, SAXS profiles of anhydrous proteins can be modeled. Procedures for the addition of individual water molecules to anhydrous protein envelopes based on the atomic coordinates yield biologically relevant models for hydrated phosphorylases. This requires the usage of advanced surface calculation programs such as SIMS and of appropriate hydration algorithms such as those implemented in our programs HYDCRYST and HYDMODEL. The resulting SAXS profiles and structural and hydrodynamic parameters of the hydrated proteins can be compared with the data obtained by solution scattering.

We have characterized the hydrodynamic behaviour in and DO of two detergents, dodecyl-β-D-maltoside (DDM) and octaethylene glycol monododecyl ether (CE), by analysing sedimentation velocity profiles obtained with interference optics in terms of continuous particle distribution and non-interacting species. The analysis in H0 provides values for the sedimentation and diffusion coefficients that give aggregation numbers of 130 and 115 for DDM and CE, respectively, close to that given in the literature. The analysis of the number of interference fringes as a function of detergent concentration gave values for the critical micelle concentration and refractive index increments that are also in good agreement with published values. The values of the partial specific volumes of DDM and CE, obtained by sedimentation in HO and DO – where CE floats – are identical to those obtained from density measurements. These results indicate that sedimentation velocity has good potential for proper characterisation of detergent-solubilized proteins.

Self-association of phosphorylase kinase (PhK) has been studied using analytical ultracentrifugation and dynamic light scattering under the conditions of molecular crowding arising from the presence of high concentrations of osmolyte. Sedimentation velocity analysis shows that in accordance with the predictions of molecular crowding theory, trimethylamine N-oxide (TMAO) greatly favours self-association of PhK induced by α and α . On the contrary, proline suppresses this process, probably, due to its specific interaction with PhK. We have also established that α-crystallin, a protein possessing chaperone-like activity, counteracts the self-association of PhK under molecular crowding conditions. Using dynamic light scattering we have shown that the increase in the light scattering intensity accompanying self-association of PhK is due to the formation of particles having hydrodynamic radius of hundreds of nanometers. The hydrodynamic radius of the start associates (seeds of association) was found to be approximately 80 nm. TMAO facilitates the formation of the associates of larger size whereas proline and α-crystallin suppress self-association of PhK.

This study considers the oligomeric of TBP1 from the extreme halophilic archaeaon Haloferax volcanii. This protein is in the first step of the cascade of binding events leading to transcription initiation. Sedimentation velocity shows that at 3M KCl, the protein appears to be in several oligomeric forms.