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. 2006 Dec 1;62(Pt 12):1251-4.
doi: 10.1107/S174430910604663X. Epub 2006 Nov 30.

Cloning, expression, crystallization and preliminary crystallographic analysis of a pentapeptide-repeat protein (Rfr23) from the bacterium Cyanothece 51142

Garry W Buchko  1 , Howard Robinson, Shuisong Ni, Himadri B Pakrasi, Michael A Kennedy
Affiliations

Cloning, expression, crystallization and preliminary crystallographic analysis of a pentapeptide-repeat protein (Rfr23) from the bacterium Cyanothece 51142

Garry W Buchko et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

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.

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Figures

Figure 1
Figure 1
Stylized representation of a complete coil in an Rfr-fold, the higher order structural unit adopted by four tandem pentapeptide repeats. Each pentapeptide repeat is colored differently and is labeled relative to the central residue i. The coils stack on top of each other with a rise of ∼4.8 Å.
Figure 2
Figure 2
Crystals of SeMet-substituted Rfr23 from Cyanothece 51142 grown by mixing 2 µl protein solution (∼8 mg ml−1) with 2 µl reservoir buffer. The reservoir buffer used for the crystals in the main figure contained 0.192 M magnesium acetate, 0.096 M sodium cacodylate, 10%(w/v) polyethylene glycol 8000, 10%(v/v) glycerol pH 6.5. The reservoir buffer used for the crystal in the inset contained 0.185 M ammonium sulfate, 0.0925 M sodium cacodylate, 20.8%(w/v) polyethylene glycol 8000, 7.5% glycerol pH 6.5. SAD and native data sets reported here were collected from crystals of the type shown in the inset.
Figure 3
Figure 3
The F o electron-density map for Rfr23 contoured at 1.9σ. One significant heavy-atom site per molecule was identified by the program SOLVE and is shown in red for two horizontally orientated molecules that pack end-to-end in the crystal lattice. Six complete or partial coils, numbered 1–6, characteristic of Rfr-folds are clearly visible for both molecules. At both corners of the figure splices through two molecules packed perpendicularly to the two horizontal molecules are shown. These splices illustrate the characteristic four-faced nature of the Rfr-fold. For the splice on the left, the central bulky side chain on each face is indicated by an ‘i’.
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