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Crystallization of canavalin as a function of pH
Elizabeth L Forsythe1, Sridhar Gorti, Marc L Pusey
1BAE Systems, Hunstville, AL 35812, USA.
Summary
Canavalin (CCAN) crystals, known to form as trimers, do not become monomers in solution. Instead, trimeric CCAN self-associates into larger species, driving crystal nucleation.
Area of Science:
- Protein crystallography
- Biochemistry
- Solution behavior of proteins
Background:
- Canavalin (CCAN), a protein known to crystallize as a trimer, is expected to become monomeric in weakly basic solutions.
- Previous studies suggested that CCAN exists as monomers under conditions where its crystals dissolve.
Purpose of the Study:
- To investigate the solution state of trypsin-treated canavalin (CCAN) across a pH range of 6.4-9.6.
- To determine the oligomeric state of CCAN and its role in crystal nucleation.
Main Methods:
- Crystallization experiments across a pH range.
- Light scattering measurements.
- Size-exclusion chromatography (gel filtration).
- Fluorescence anisotropy.
Main Results:
- CCAN crystallized in the canonical rhombohedral form across all tested pH values.
- No evidence of monomeric CCAN was found using light scattering and size-exclusion chromatography.
- Light scattering indicated that trimeric CCAN slowly self-associates into larger species.
- Fluorescence anisotropy, light scattering, and gel filtration confirmed that solutions primarily contain trimers, with self-association increasing with protein concentration.
Conclusions:
- CCAN does not exist as monomers in weakly basic solutions.
- Crystal nucleation of CCAN is driven by the self-association of trimers into larger oligomers, forming critical nuclei.