The trp repressor of Escherichia coli (TR), although generally considered to be dimeric, has been shown by fluorescence anisotropy of extrinsically labeled protein to undergo oligomerization in solution at protein concentrations in the micromolar range (Fernando, T., and C. A. Royer 1992. Biochemistry. 31:3429-3441). Providing evidence that oligomerization is an intrinsic property of TR, the present studies using chemical cross-linking, analytical ultracentrifugation, and molecular sieve chromatography demonstrate that unmodified TR dimers form higher order aggregates. Tetramers and higher order species were observed in chemical cross-linking experiments at concentrations between 1 and 40 microM. Results from analytical ultracentrifugation and gel filtration chromatography were consistent with average molecular weight values between tetramer and dimer, although no plateaus in the association were evident over the concentration ranges studied, indicating that higher order species are populated. Analytical ultracentrifugation data in presence of corepressor imply that corepressor binding destabilizes the higher order aggregates, an observation that is consistent with the earlier fluorescence work. Through the investigation of the salt and pH dependence of oligomerization, the present studies have revealed an electrostatic component to the interactions between TR dimers.
Two samples of interlocked Xb2b5c DKA rings, Cl*tpC1 and the fraction containing a mixture of Cl*tpCltpCI* and C2*tpC1, were examined by electron mirroscopy.t Specmen grids were prepared by the protein film technique of Kleinschmidt and Zahn2 and stained with uranyl acetate as described by Wetmur et al. For the sample of Cl*tpCI, approximately 90% of the molecules viewed on the fluorescence screen were dimeric Xb2b5c DXA. For those molecules not excessively entangled or twisted, two rings of equal sizes with one or two junction points were easily discern ble. Three representative micrographs are depicted in Figure 1. The relative length of one ring to the other, from length measurements on ten of such dimeric molecules, is 1.00 f 0.02.For the sample containing both C1*tpCltpCl* and C2*tpC1, considerably more difficulty was encountered. The entanglement of molecules was more severe due to the larger size of the molecules, and it was sometimes difficult to discern unequivocally the individual rings. The large size of the molecules, plus the requirement that the specimen grid be sparsely populated to avoid accidental overlapping of molecules, made it difficult to score a statistically significant number of molecules. I t was certain, however, that over 70% of the molecules are trimeric Xb2b5c DKA. with some dimeric and monomeric molecules. Molecules with length longer than trimeric Xb2b5c DXA were rare. In Figure 2, two representative micrographs, one showing a dimeric and a monomeric ring topologically interlocked, and the other showing three monomeric rings linked in a chain, are depicted.We thank the Electron Microscope Laboratory of this campus for making their facilities available to us. R e thank Mr. Paul Omelich and Mr. Walter Baase for instructions on the use of the microscope and techniques for grid preparation.
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