Ae. Meekhof et Smv. Freund, Probing residual structure and backbone dynamics on the milli- to picosecond timescale in a urea-denatured fibronectin type III domain, J MOL BIOL, 286(2), 1999, pp. 579-592
The energy landscape for the denatured state of a protein provides a key to
understanding early folding events. We have attempted to map this landscap
e for the third fibronectin type III domain from human tenascin (TNfn3), a
compact 9.5 kDa beta-sandwich protein, through measurement of N-15 backbone
dynamics on the milli- to picosecond timescale and a number of structural
parameters. TNfn3 was fully denatured with 5 M urea and buffered at pH 4.9
with 50 mM acetate. Under these conditions, multinuclear NMR experiments we
re used to complete a full spectral assignment. Secondary chemical shifts,
(3)J(HNH alpha) coupling constants, amide proton temperature coefficients,
interresidue nuclear Overhauser enhancement (NOE) intensities, R-1 and R-2
N-15 relaxation rates, and {H-1-N-15} steady-state NOE enhancements were an
alyzed at 11.74 T (500 MHz) and 303 K. Several parameters were also measure
d at 278 K. Off-resonance T-1 rho experiments at 14.1 T (600 MHz) and 278 K
reveal a lack of motion on the milli- to microsecond timescale, indicating
that no element of residual structure in the denatured domain is persistan
t. Although increased sample viscosity dampens overall mobility at the lowe
r temperature, the dynamic propensities of individual residues are temperat
ure independent. Reduced mobility correlates to regions of extreme hydropho
bicity or polarity. In these same regions, several other measures for rando
m coil behavior are perturbed. Evidence for two nascent turn-like structure
s is reported. Otherwise, residual structure correlates more strongly to ch
aracteristics of individual residues than to structural elements of the nat
ive state. (C) 1999 Academic Press.