Probing residual structure and backbone dynamics on the milli- to picosecond timescale in a urea-denatured fibronectin type III domain.

The energy landscape for the denatured state of a protein provides a key to understanding early folding events. We have attempted to map this landscape for the third fibronectin type III domain from human tenascin (TNfn3), a compact 9.5 kDa beta-sandwich protein, through measurement of 15N 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 were used to complete a full spectral assignment. Secondary chemical shifts, 3JHNHalpha coupling constants, amide proton temperature coefficients, interresidue nuclear Overhauser enhancement (NOE) intensities, R1 and R2 15N relaxation rates, and {1H-15N} steady-state NOE enhancements were analyzed at 11.74 T (500 MHz) and 303 K. Several parameters were also measured at 278 K. Off-resonance T1rho 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 persistant. Although increased sample viscosity dampens overall mobility at the lower temperature, the dynamic propensities of individual residues are temperature independent. Reduced mobility correlates to regions of extreme hydrophobicity or polarity. In these same regions, several other measures for random coil behavior are perturbed. Evidence for two nascent turn-like structures is reported. Otherwise, residual structure correlates more strongly to characteristics of individual residues than to structural elements of the native state.