Early steps in the crystallization process of proteins: An approach by fluorescence anisotropy

An investigation of the early steps occurring in the crystallization process of a protein has been attempted by means of fluorescence anisotropy using ribonuclease A with ethanol as precipitating agent. It is found that the apparent fluorescence anisotropy is sensitive to protein-protein interactions and increases linearly with protein concentration. The virial coefficient for rotational diffusion appears to increase sharply beyond 40% ethanol, the concentration which corresponds to crystallization conditions of the protein. The results reported here demonstrate that fluorescence anisotropy can be used to monitor directly the prenucleation phase of the crystallization process.