INTERNAL MOBILITY IN THE PARTIALLY FOLDED DNA-BINDING AND DIMERIZATION DOMAINS OF GAL4 - NMR ANALYSIS OF THE N-H SPECTRAL DENSITY-FUNCTIONS

The DNA binding domain (residues 1-65) of the yeast transcriptional ac tivator GAL4 is only partially folded. While residues 10-41, the DNA r ecognition domain, form a well-defined structure in the free protein, the whole polypeptide folds up and dimerizes upon binding DNA. In orde r to describe the mobility of the protein, we have characterized the f requency spectrum of the motions of N-H bond vectors of GAL4(1-65) usi ng a reduced spectral density mapping approach (an approximation of th e 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]. N-15 spin-lattice relaxation [R(N)(N-z)] , spin-spin relaxation [R(N)(N-x,N-y)], cross-relaxation [R(N)(H-z --> N-z)], two-spin order [R(NH)(2H(z)N(z))], and antiphase [R(NH)(2H(z)N (x,y))] rates were determined for 52 of the 65 backbone amide groups a t 10 degrees C anti pH 6.5 at 11.74 T. Calculations of the spectral de nsity functions using a reduced set of R(N)(N-z), R(N)(N-x,N-y), R(N)( H-z --> N-z), and R(NH)(2H(z)N(z)) gave excellent agreement with those calculated using all six sets. The reduced method has the added advan tage that the errant behavior seen at high field values is circumvente d, A linear correlation was found between J(omega(N)) and J(0) with a limited and clearly defined range of J(0) values which defines the ran ge of rates for internal motions in GAL4(1-65). It appears that all re sidues experience a combination of two movements: one of the overall t umbling (correlation time, 8.65 ns) and the other of fast internal flu ctuations of the structure. The respective weights of these contributi ons vary with the primary sequence and faithfully mirror the secondary and tertiary elements of the protein. The position on the correlation line of J(omega(N)) versus J(0) indicates the amount of angular avera ging relative to the overall motion of the protein. A spectral density function for internal motions can be described.