Crystallization of canavalin as a function of pH.
The question was posed of what happens to a protein that is known to grow as an n-mer when it is placed in solution conditions where it is monomeric. The trypsin-treated or cut form of the protein canavalin (CCAN) has been shown to nucleate and grow crystals as a trimer from neutral to slightly acidic solutions. Under these conditions the solution is composed almost wholly of trimers. The crystalline protein can be readily dissolved by weakly basic solution, which has been proposed to result in a solution that is monomeric. Results are presented for crystallization experiments of CCAN over the pH 6.4-9.6 range. CCAN was found to crystallize in the canonical rhombohedral form at all pH values. Light scattering and size-exclusion chromatography data gave no indication of monomeric CCAN. The light-scattering data also showed that trimeric CCAN slowly self-associates to form larger species. Results are presented for crystallization experiments of CCAN over the pH 6.4-9.6 range. Fluorescence anisotropy, coupled with light-scattering and gel-filtration experiments, show that the solutions are primarily trimers, with association to form larger species occurring as a function of protein concentration. These results indicate that CCAN crystal nucleation occurs by the self-association of trimers to form progressively larger species in solution, which eventually become critically sized crystal nuclei.
Forsythe
Elizabeth L
EL
BAE Systems, Hunstville, AL 35812, USA.
Gorti
Sridhar
S
Pusey
Marc L
ML
D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification
D010940:Plant Proteins; C006882:canavalin protein, Canavalia ensiformis