EVIDENCE OF ASSOCIATED STATES DURING REFOLDING OF CYTOCHROME c. Dan Segel¹, Sangita Seshadri², Hirotsugu Tsuruta³, Anthony L. Fink², Keith O. Hodgson^3,4, Sebastian Doniach^5. Department of ¹Physics, ⁴Chemistry, ⁵Applied Physics, Stanford University, Stanford, CA 94305; Department of ²Chemistry, University of California, Santa Cruz, CA 95064; ³SSRL, Stanford University, Stanford, CA 94309

Preliminary time-resolved small angle x-ray scattering (SAXS) studies of cytochrome c refolding show association of the protein sample to form possibly dimers at protein concentrations as low as 3.5mg/ml. These studies extend earlier results¹.

In these experiments SAXS data were taken upon 7-fold stopped-flow dilution from protein in 3.5M GdnHCl solution at pH7. We found the forward scattering intensity to decrease by a factor of 1.3 over a 21s observation period, indicating dissociation of dimers or higher order associates. Longer term kinetics studies of cytochrome c refolding were also performed on a manually mixed solution. Beginning 10min. after mixing, SAXS data showed a steady forward scattering intensity which is nearly twice that measured for the native protein dissolved in buffer at pH7. This suggests the formation of a long-lived, fully dimerized state.

The time-resolved data was analyzed in the Guinier region to obtain the time course of the radius of gyration. R_g in 3.5M GdnHCl at pH7 was found to be about 34.5A, falling to 22.5A on 7-fold dilution. R_g for the native solution is about 14A. Thus the observed R_g of the refolded sample is consistent with dimer formation. Extraction of p(r), pair-distance distribution function, from the higher angle data is also qualitatively consistent with that generated using a simple dimer model.

¹Transient association during the folding of cytochrome c, D.Eliezer, H.Tsuruta, H.Kihara, S.Doniach and K.O.Hodgson, submitted for publication.