A size filtration approach to purify low affinity complexes for crystallization.
Low affinity protein complexes are difficult to isolate and handle in crystallization experiments. Size-exclusion chromatography often does not allow purification of the homogeneous complex. Here we used a size-filtration approach for the purification and concentration of the 19 microM affinity complex of yeast Rab-GTPase and its guanine nucleotide disassociation inhibitor (GDI). The homogeneous protein complex solution was crystallized and the structure was solved using the molecular replacement method. The resulting model of the low affinity unprenylated Rab-GDI complex should reflect a transient Rab-GDI complex when GDI is bound to the membrane-anchored Rab protein and is poised to extract Rab to cytosol.
Ignatev
Alexander
A
Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Piatkov
Konstantin
K
Pylypenko
Olena
O
Rak
Alexey
A
D003460:Crystallization; D005374:Filtration; D005656:Fungal Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry; D010316:Particle Size; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D020691:rab GTP-Binding Proteins / Q000737:chemistry
D005656:Fungal Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D011506:Proteins; D020691:rab GTP-Binding Proteins