The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.
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HIV؉ subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination.
Barrier‐to‐autointegration factor (BAF) is a nuclear protein conserved in metazoans. BAF is necessary for nuclear assembly, chromatin organization, and gene expression. BAF forms dimers that bind to the LEM domain proteins of the inner nuclear membrane. Although several cellular functions of BAF are known, some of the binding sites on the surface of the BAF dimer are not well defined. We are interested in determining the regions of the BAF dimer involved in contacting other proteins. The results from our studies will aid in understanding BAF protein interactions at the molecular level. This will help define how these interactions regulate BAF's diverse cellular functions. For our experiments, we over‐expressed human BAF in E. coli and purified the protein by affinity and size‐exclusion chromatography. We generated BAF crystals for structural studies to help map the sites of protein interaction on BAF. We crystallized BAF using 0.01 Tris pH 8.5 and 20% ethanol as precipitant and collected a diffraction data set to 1.45A resolution. We used the previously published crystal structure (PDB code info:x-wiley/pdb/1CI4) for phasing with the molecular replacement method. We plan to use multiple solvent crystal structures, (a technique in which we soak the protein crystals in different organic solvents) and computational techniques to study the details of the binding surface of BAF.
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