EVIDENCE FOR A DIMERIC INTERMEDIATE ON THE CRYSTALLIZATION PATHWAY OFRIBONUCLEASE-A

Early steps in the crystallization process of pancreatic ribonuclease have been investigated by time-dependent fluorescence anisotropy, usin g a labeled protein as a fluorescent probe. Previous experiments have shown that steady-state fluorescence anisotropy is sensitive to protei n-protein interactions and can be used to find new crystallization con ditions. The present work describes an attempt, by means of time-resol ved experiments, to detect and characterize species appearing in the e arly stages of the crystallization pathway. Fluorescence anisotropy de cay was measured with synchrotron radiation as a light source under a variety of conditions where it is known that the solutions tend toward s crystallization; the decay was analyzed by a maximum-entropy method that calculates a rotational correlation-time distribution. Fluorescen ce anisotropy originates in the Brownian rotatory motion of macromolec ules and the values of the correlation times are related to the size a nd shape of different species present in the solution. In the presence of high salt concentrations, a bimodal distribution is always observe d. Whereas a peak of protein monomer is still present, a second peak a ppears as a stable intermediate in the crystallization pathway. The co rrelation time of this new species varies between two and three times the correlation time of the monomer. The second peak is possibly the s ymmetrical dimer of the ribonuclease molecules commonly observed in al l the high-salt crystal forms.