Jf. Lefevre et al., INTERNAL MOBILITY IN THE PARTIALLY FOLDED DNA-BINDING AND DIMERIZATION DOMAINS OF GAL4 - NMR ANALYSIS OF THE N-H SPECTRAL DENSITY-FUNCTIONS, Biochemistry, 35(8), 1996, pp. 2674-2686
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.