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 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.