Electron paramagnetic resonance (EPR) spectroscopy using site-directed spin-labeling is an appropriate technique to analyze the structure and dynamics of flexible protein regions as well as protein-protein interactions under native conditions. The analysis of a set of protein mutants with consecutive spin-label positions leads to the identification of secondary and tertiary structure elements. In the first place, continuous-wave EPR spectra reflect the motional freedom of the spin-label specifically linked to a desired site within the protein. EPR spectra calculations based on molecular dynamics (MD) and stochastic dynamics simulations facilitate verification or refinement of predicted computer-aided models of local protein conformations. The presented spectra simulation algorithm implies a specialized in vacuo MD simulation at 600 K with additional restrictions to sample the entire accessible space of the bound spin-label without large temporal effort. It is shown that the distribution of spin-label orientations obtained from such MD simulations at 600 K agrees well with the extrapolated motion behavior during a long timescale MD at 300 K with explicit water. The following potential-dependent stochastic dynamics simulation combines the MD data about the site-specific orientation probabilities of the spin-label with a realistic rotational diffusion coefficient yielding a set of trajectories, each more than 700 ns long, essential to calculate the EPR spectrum. Analyses of a structural model of the loop between helices E and F of bacteriorhodopsin are illustrated to demonstrate the applicability and potentials of the reported simulation approach. Furthermore, effects on the motional freedom of bound spin-labels induced by solubilization of bacteriorhodopsin with Triton X-100 are examined.
If the backbone conformation of the binding site does not change, current docking programs can perform well by taking side-chain reorientations into account only. Future progress to account for full target flexibility in docking requires both accurate prediction of the essential modes of backbone motion and improvements in scoring to enhance selectivity. Thus, the scoring function should realistically cover energetic and particularly entropic contributions to binding, which would allow more realistic estimates of binding free energies.
A new strategy has been applied that combines molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy to study the structure and conformational dynamics of the spin-labeled photosynthetic reaction center (RC) of Rhodobacter sphaeroides. This protein serves here asa model system to demonstrate the applicability of this new methodology. The RC contains five native cysteines and EPR experiments show that only one cysteine, located on the H subunit, is accessible for spin labeling. The EPR spectra calculated from MD simulation trajectories of spin labels bound to the native cysteines C156 and C234 in subunit H reveal that only the spin label side chain at position 156 provides a spectrum which agrees with the experimental EPR spectrum.
AbstractA number of improvement proposals and corrections of the German Rili-BAEK (Guideline of the German Medical Association on Quality Assurance in Medical Laboratory Examinations) are discussed with special focus on the internal and external quality assurance (IQA/EQA) as well as reference intervals for quantitative results. Particular attention is paid to reconsider the retrospective analysis of control measurements. Such an analysis can be very useful to monitor establishing errors of measurement even before they become critical. The present method “Quadratischer Mittelwert der Messabweichung (QMMA)” has proved to be ineffective. Furthermore, the current idea of a common limit for single control measures and the retrospective statistics must be revised. As a more sophisticated concept, the novel Adaptive Retrospective Monitoring (ARM) has been developed. ARM is recommended as the new minimum requirement for the entire internal quality assurance. Further proposals to enhance clarity are given concerning the release decisions of medical devices and the EQA. Individualized medicine begins with a patient-specific interpretation of analytic results. This requires standardized subgroup-specific reference intervals with smooth age-related adaptations. Only large laboratories are able to ensure the desired specificity and a sufficient statistical significance of self-developed in-laboratory reference intervals. Hence, the need of a central database for harmonized reference intervals is discussed and recommended. Suitable and consistent reference intervals are also an essential prerequisite for unitless laboratory values like the zlog value.
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