Cloning, expression, crystallization and preliminary crystallographic analysis of a pentapeptide-repeat protein (Rfr23) from the bacterium Cyanothece 51142.
A unique feature of cyanobacteria genomes is the abundance of genes that code for hypothetical proteins containing tandem pentapeptide repeats approximately described by the consensus motif A(N/D)LXX. To date, the structures of two pentapeptide-repeat proteins (PRPs) have been determined, with the tandem pentapeptide-repeat sequences observed to adopt a novel type of right-handed quadrilateral beta-helix, or Rfr-fold, in both structures. One structure, Mycobacterium tuberculosis MfpA, is a 183-residue protein that contains 30 consecutive pentapeptide repeats and appears to offer antibiotic resistance by acting as a DNA mimic. The other structure, Cyanothece 51142 Rfr32, is a 167-residue protein that contains 21 consecutive pentapeptide repeats. The function of Rfr32, like the other 35 hypothetical PRPs identified in the genome of Cyanothece, is unknown. In an effort to understand the role of PRPs in cyanobacteria and to better characterize the structural properties of Rfr-folds with different amino-acid sequences, a second PRP from Cyanothece 51142, Rfr23, has been cloned, expressed and purified. Selenomethione-substituted protein was crystallized by vapor diffusion in hanging drops. Nearly complete SAD and native diffraction data sets were collected from these crystals to 2.5 and 2.1 A resolution, respectively, using synchrotron radiation. The crystals belonged to space group I4(1), with unit-cell parameters a = b = 106.61, c = 53.37 A, and one molecule per asymmetric unit. Preliminary analysis of the electron-density map from the SAD data shows that Rfr23 contains an Rfr-fold.
Buchko
Garry W
GW
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. garry.buchko@pnl.gov
Robinson
Howard
H
Ni
Shuisong
S
Pakrasi
Himadri B
HB
Kennedy
Michael A
MA
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D046889:Cyanothece / Q000737:chemistry*; D018895:Microsatellite Repeats; D017510:Protein Folding
D001426:Bacterial Proteins