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.