Influence of divalent cations in protein crystallization.
We have tested the effect of several cations in attempts to crystallize the ligand-bound forms of the leucine/isoleucine/valine-binding protein (LIVBP) (M(r) = 36,700) and leucine-specific binding protein (LBP) (M(r) = 37,000), which act as initial periplasmic receptors for the high-affinity osmotic-shock-sensitive active transport system in bacterial cells. Success was achieved with Cd2+ promoting the most dramatic improvement in crystal size, morphology, and diffraction quality. This comes about 15 years after the ligand-free proteins were crystallized. Nine other different divalent cations were tried as additives in the crystallization of LIVBP with polyethylene glycol 8000 as precipitant, and each showed different effects on the crystal quality and morphology. Cd2+ produced large hexagonal prism crystals of LIVBP, whereas a majority of the cations resulted in less desirable needle-shaped crystals. Zn2+ gave crystals that are long rods with hexagonal cross sections, a shape intermediate between the hexagonal prism and needle forms. The concentration of Cd2+ is critical. The best crystals of the LIVBP were obtained in the presence of 1 mM CdCl2, whereas those of LBP, with trigonal prism morphology, were obtained at a much higher concentration of 100 mM. Both crystals diffract to at least 1.7 A resolution using a conventional X-ray source.
Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
S
S
Trakhanov
F A
FA
Quiocho
D001426:Bacterial Proteins; D002104:Cadmium / Q000494:pharmacology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D007532:Isoleucine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D011092:Polyethylene Glycols; D014633:Valine / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; C488923:LivJ protein, E coli; C024913:LivK protein, E coli; D033902:Periplasmic Binding Proteins; C028332:leucine-isoleucine-valine binding protein, bacteria; D002104:Cadmium; D007532:Isoleucine; D011092:Polyethylene Glycols; D007930:Leucine; D014633:Valine