Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI.
We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.
Niemann
Hartmut H
HH
Division of Structural Biology, German Research Centre for Biotechnology (GBF) Mascheroder Weg 1, D-38124 Braunschweig, Germany. hartmut.niemann@gbf.de
Schmoldt
Hans-Ulrich
HU
Wentzel
Alexander
A
Kolmar
Harald
H
Heinz
Dirk W
DW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029775:Momordica / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004220:Disulfides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease