FASTDXL: a generalized screen to trap disulfide-stabilized complexes for use in structural studies.
Structural studies of macromolecular complexes have produced extraordinary insights into a wide variety of biological processes. Unfortunately, as structural biologists pursue larger and more challenging assemblies, weakly stable and/or nonspecific interactions can become significant roadblocks to structure determination. We have developed a rapid and effective pool-based screen, termed FASTDXL (focused array screening technique for disulfide X-linking), to produce and identify disulfide-stabilized protein-nucleic acid assemblies. A significant strength of FASTDXL is that it can take advantage of prior structural knowledge about molecular interactions, but does not necessarily rely upon it. A detailed application of the approach to the difficult problem of trapping a bacterial primase-ssDNA complex is described, validating the method as a route toward obtaining diffracting crystals suitable for structure determination.
Corn
Jacob E
JE
Department of Molecular and Cell Biology, University of California, Berkeley, 327B Hildebrand Hall, Berkeley, CA 94720, USA.
Berger
James M
JM
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D003461:Crystallography; D003545:Cysteine / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004220:Disulfides; D019915:DNA Primase; D003545:Cysteine