Internal Mobility in the Partially Folded DNA Binding and Dimerization Domains of GAL4: NMR Analysis of the N−H Spectral Density Functions

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Abstract
The DNA binding domain (residues 1−65) of the yeast transcriptional activator GAL4 is only partially folded. While residues 10−41, the DNA recognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In order to describe the mobility of the protein, we have characterized the frequency spectrum of the motions of N−H bond vectors of GAL4(1−65) using a reduced spectral density mapping approach (an approximation of the full spectral density mapping technique) [Peng, J. W., & Wagner, G. (1992a) J. Magn. Reson. 98, 308−332; Peng, J. W., & Wagner, G. (1992b) Biochemistry 31, 8571−8586]. 15N spin−lattice relaxation [RN(Nz)], spin−spin relaxation [RN(Nx,y)], cross-relaxation [RN(Hz → Nz)], two-spin order [RNH(2HzNz)], and antiphase [RNH(2HzNx,y)] rates were determined for 52 of the 65 backbone amide groups at 10 °C and pH 6.5 at 11.74 T. Calculations of the spectral density functions using a reduced set of RN(Nz), RN(Nx,y), RN(Hz → Nz), and RNH(2HzNz) gave excellent agreement with those calculated using all six sets. The reduced method has the added advantage that the errant behavior seen at high field values is circumvented. A linear correlation was found between J(ωN) and J(0) with a limited and clearly defined range of J(0) values which defines the range of rates for internal motions in GAL4(1−65). It appears that all residues experience a combination of two movements: one of the overall tumbling (correlation time, 8.65 ns) and the other of fast internal fluctuations of the structure. The respective weights of these contributions vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(ωN) versus J(0) indicates the amount of angular averaging relative to the overall motion of the protein. A spectral density function for internal motions can be described.