A NMR guided approach for CsrA-RNA crystallization.
Structure determination of protein-nucleic acid complexes remains a challenging task. Here we present a simple method for generating crystals of a CsrA-nucleic acid complex, guided entirely by results from nuclear magnetic resonances spectroscopy (NMR) spectroscopy. Using a construct that lacks thirteen non-essential C-terminal residues, efficient binding to DNA could be demonstrated. One CsrA dimer interacts with two DNA oligonucleotides, similar to previous findings with RNA. Furthermore, the NMR study of the CsrA-DNA complex was the basis for successfully homing in on conditions that were suitable for obtaining crystals of the CsrA-DNA complex. Our results may be useful for those cases where RNA in protein-nucleic acid complexes may be replaced by DNA.
Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, BST3/Rm1050, Pittsburgh, PA 15260, USA.
Leonardus M I
LM
Koharudin
Rolf
R
Boelens
Rob
R
Kaptein
Angela M
AM
Gronenborn
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D055503:Protein Multimerization / Q000502:physiology; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D012313:RNA; D004247:DNA