排序方式: 共有125条查询结果,搜索用时 296 毫秒
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Krystal Cole Christian G. Roessler Elizabeth A. Mulé Emma J. Benson-Xu Jeffrey D. Mullen Benjamin A. Le Alanna M. Tieman Claire Birone Maria Brown Jesus Hernandez Sherry Neff Daniel Williams Marc Allaire Allen M. Orville Robert M. Sweet Alexei S. Soares 《PloS one》2014,9(7)
High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size. 相似文献
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Gregory P. Owens Alanna Ritchie Andrea Rossi Kristin Schaller Scott Wemlinger Hannah Schumann Andrew Shearer Alan S. Verkman Jeffrey L. Bennett 《The Journal of biological chemistry》2015,290(19):12123-12134
Neuromyelitis optica-immunoglobulin G (NMO-IgG) binds to aquaporin-4 (AQP4) water channels in the central nervous system leading to immune-mediated injury. We have previously demonstrated that a high proportion of CSF plasma cells of NMO patients produce antibody to the extracellular domains of the AQP4 protein and that recombinant IgG (rAb) derived from these cells recapitulate pathogenic features of disease. We performed a comprehensive mutational analysis of the three extracellular loops of the M23 isoform of human AQP4 using both serial and single point mutations, and we evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C (137TP138 and Val150) and loop E (230HW231) amino acids for binding, two broad patterns of NMO-IgG recognition could be distinguished based on differential sensitivity to loop A amino acid changes. Pattern 1 NMO rAbs were insensitive to loop A mutations and could be further discriminated by differential sensitivity to amino acid changes in loop C (148TM149 and His151) and loop E (Asn226 and Glu228). Alternatively, pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A (63EKP65 and Asp69) and loop C (Val141, His151, and Leu154). Amino acid substitutions at 137TP138 altered loop C conformation and abolished the binding of all NMO rAbs and NMO-IgG, indicating the global importance of loop C conformation to the recognition of AQP4 by pathogenic NMO Abs. The generation of human NMO rAbs has allowed the first high resolution mapping of extracellular loop amino acids critical for NMO-IgG binding and identified regions of AQP4 extracellular structure that may represent prime targets for drug therapy. 相似文献
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