Fluorescence studies of protein crystal nucleation

摘要

One of the most powerful and versatile methods for studying molecules in solution is fluorescence. Crystallization typically takes place in a concentrated solution environment, whereas fluorescence typcially has an upper concentration limit of approx 1 x 10~M. thus intrinsic fluorescence cannot be employed, but a fluorescent probe must be added to a sub population of the molecules. However the fluorescence cannot interfere with the selfassembly process. This can be achieved with macromolecules, where fluorescent probes can be covalently attached to a sub population of molecules that are subsequently used to track the system as a whole. We are using fluorescence rsonance energy transfer (FRET) to study the initial solution phase self-assembly process of etragonal lysozyme crystal nucleatiion, using covalent fluorescent derivatives which crystallize in the characteristic P4_32_12_1 space group. FRET studie4s are being carried out between N-terminal lysine bound Texas red as the donor and N-terminal lysine bound 5-(and-6)-carboxynaphthofluorescein as the acceptor. The estimated R_0 for this probe pair, the distance where 50