FASTDXL: A Generalized Screen to Trap Disulfide-Stabilized Complexes for Use in Structural Studies

Structure. 2007 Jul;15(7):773-80. doi: 10.1016/j.str.2007.05.006.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Cross-Linking Reagents / chemistry
  • Crystallography
  • Cysteine / chemistry
  • DNA Primase / chemistry*
  • DNA Primase / genetics
  • DNA, Single-Stranded / chemistry*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • Disulfides / chemistry*
  • Mutation
  • Protein Binding
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Bacterial Proteins
  • Cross-Linking Reagents
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Disulfides
  • DNA Primase
  • Cysteine