Polypeptide chain dynamics
We next measured 15N spin-lattice/spin-spin relaxation rates as well as heteronuclear NOEs for the mHIV-1-PR1-95 at 25°C, at a field strength of 17.6 Tesla (750 Mhz). These relaxation parameters are sensitive towards motions on the sub-nanosecond timescale. In addition, the R2relaxation rate provides insights into motions on the millisecond to microsecond timescale. Full sets of relaxation data could be extracted for a total of 79 (5 °C), 82 (4 M urea), 85 (8 M urea), 87 (1 M GdmCl) and 88 (25% acetic acid) residues.
For all five denatured states, the R1 values remained more or less constant throughout the sequence, with average values of 1.44 ± 0.04 (5 °C), 1.48 ± 0.01 (4 M urea), 1.50 ± 0.01 (8 M urea), 1.56 ± 0.03 (1 M GdmCl) and 1.36 ± 0.02 ms-1(25% acetic acid), respectively, Fig. 5. The N- and the C-termini showed lower R1 compared to the rest of the protein, consistent with faster timescale movements usually experienced for chain termini. In all five profiles, we observed a stretch (V77-V82) of significantly lower values followed by a stretch (I84-L89) of significantly increased values. The average R1rates for the acetic acid and for the cold denatured states were clearly reduced compared to those associated with the other two denatured states.
Measurements of the heteronuclear steady-state NOEs showed mostly positive values apart from those associated with the N- and C-terminal regions. The profile of the heteronuclear NOE did not agree with a fully unfolded state, but rather followed a profile of four arcs for all four denatured states.
The R 2 value is usually the most informative parameter for denatured proteins as it can reveal regions that undergo chemical exchange. For a fully extended protein where chain dynamics is dominated by unrestrained segmental motion, this profile usually adopts the shape of an inverted U, with a plateau along the chain and steep drops at the N- and C-terminal ends62. For all five denatured states, the R 2 profiles deviated from an inverted U-shape. Instead, they displayed a four-arcs-like pattern distributed almost evenly over the sequence, and covering R8-L24, V32-G48, V56-G68 and G78-A95 (cf. Fig. S7 in the SI). This unusual pattern of R 2 rates persisted at 8 M urea where the unfolded mHIV-1-PR1-95 showed a more elongated conformation, as testified by the correspondingRh value (Table 1).
The probability function of finding motions at a given angular frequencyω can be described by the spectral density function J(ω) . As unfolded states cannot be described in terms of an overall rotational correlation time, we instead chose to describe the relaxation data by reduced spectral density mapping63. We used the values of R1 , R2 and the heteronuclear NOE recorded at 17.6 Tesla to derive the spectral density function at three frequencies (0, ωH and ωN, cf. Fig. 5). Neither J(ωH)nor J(ωN) showed large variation in their profiles when plotted against the sequence, in agreement with the related profiles for the hetNOE and the R1values, respectively. Instead, the J(0) values displayed the same pattern of four arcs as described for R2 . Of importance we note that the arches mostly revolve around prolines.