No abstract

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Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.

MScases causes problems during complementary SEM/EDS analyses when the sample is not transferred onto a different sample holder with regard to minimization of risk of its damage, loss or contamination. Therefore monocrystalline silicon plates with indicated conductivity of 5 Ω.cm -1 were tested in practice. This value is sufficient for SEM (based on measurements -carbon conductive strips specially intended for SEM have the conductivity of approx. 500 kΩ and higher). The plates with thickness of 300µm (±15µm) can also be used for subsequent direct FTIR analysis even in transmission mode, without the need of plate exchange. A method employing the image analysis system was introduced for precise sample adjustment for microdiffraction and the choice of the area to be measured. The trace of the primary beam on a fluorescent disc was scanned for different angles 2theta and subsequently saved as binary overlay images. A camera reads the live image of the sample through the focusing microscope and the operator can choose a spot for the analysis in ergonomic conditions (the second monitor was placed directly into the diffractometer chamber). This procedure is very convenient for heterogeneous samples, various abrasions, etc.The system is further used for quantitative drug analysis. Both Rietveld method or method with the scale factor and the RIR (Reference Intensity Ratio) values (also called I/Ic values) are used. The results are cross-checked by standard methods of organic analysis (GCMS, FTIR), XRD analysis, in addition, enables precise phase analysis of inorganic components.