3.2 Pressure- and temperature-stabilized intermediates
To further characterize the pressure- and temperature-stabilized
intermediates, PRE effects on the 1H-transverse
relaxation rate constant, R 2 were measured for
the variant. Rather than directly measuring the relaxation rates from a
series of two-dimensional NMR experiments, we obtained the ratios of
peak heights for a paramagnetic (oxidized) sample to those of a
diamagnetic (reduced) sample, i.e.,I para/I dia (Eq.
2).22 Figure 4A shows the ratio for each amide
group in the E128C variant along with the residue number (i.e., 25–110)
at 0.1, and 250 MPa at 313 K. TheI para/I dia of residues
from 25 to 275 are shown in Figure S6. When the ratio was 1, no PRE
effects were observed. In contrast, a lower ratio indicated greater PRE
effects. Assuming an intrinsic line width at half-height (Δν(hertz) = R 2int/π) of 20 Hz and correlation time
of 12 ns for the folded OspA, distances between the paramagnetic probe
and nucleus of interest were calculated by a previously described method
(Figure S7, Eq. 3).22 Note that the
multiplicity of MTSL positions was not taken into account in the
calculation. At 0.1 MPa, ratios of less than 0.8, corresponding to a
distance of 15~20 Å from the paramagnetic probe, were
observed for residues 82, 83, 101, and 102, while values < 0.3
corresponded to a distance of 12~15 Å for residues
103–110. These results indicate that residues 103–110 are closer to
residue 128 than they are to residues 82, 83, 101, and 102, consistent
with the structure resolved by X-ray crystallography at atmospheric
pressure.6 At 250 MPa,I para/I dia showed both
increased and decreased values at several sites. For example, the ratios
for residues 82, 83, and 102 were increased by approximately 0.2,
indicating that they drifted away from residue 128. In contrast, a
remarkable decrease in the ratio for residue 59 indicated that it
shifted toward residue 128. Figure 5A shows the locations of residues
59, 62, 82, 83, and 128, while the pressure dependence details for the
ratios are shown in Figure 5B.
Figure 4B shows the intensity ratio,I para/I dia, for each amide
group of the E128C variant for each residue at 303 and 318 K at 0.1 MPa.
As the temperature increased, the ratios of residues 80, 82, and 83
increased by approximately 0.1–0.2, indicating they moved away from
residue 128. Alternatively, slight decreases in the ratios for residues
33, 36, 37, 59, 62, and 105 indicated that these residues shifted closer
to residue 128. Figure 5C shows the gradual increases and decreases inI para/I dia of residues 59,
62, 82, and 83 as temperature increases.
Different behaviors were observed at residues 36, 37, and 106 between
increasing pressure and increasing temperature, as shown in Figures 6
and S8. As pressure increased, split cross-peaks were observed (Figure
6A). The second (new) cross-peak increased at the expense of the first
(original) cross-peak with increasing pressure. In contrast, only a
single peak was observed as the temperature was increased from 303 to
318 K (Figure 6B). These observations indicate that these amide groups
have at least two folded conformations. Assuming a two-state exchange
process, split peaks can be observed if the exchange rate, namelyk ex, is much smaller than the chemical shift
difference (Δω ) between two states; however, a single averaged
peak is observed if k ex is much larger than
Δω . Figure 6C shows theI para/I dia values of
original and new peaks for residues 36 and 37, and 106 at 200 MPa, where
both original and new peaks were observed in the HSQC spectrum. For
residues 36 and 37, new peaks showed 0.2–0.4-fold smaller values than
the original peaks, indicating shorter distances in the intermediate
than in the native conformations (Figure S6). Conversely, the new peak
of residue 106 demonstrated a 0.6-fold larger value than the original
peak, indicating much longer distance in the intermediate than in the
native conformation. In the case of the averaged peak, the observed PRE
effect is the weighted population average of the PRE effects for the two
conformations, i.e., native and intermediate conformations. Since the
average I para/I dia values
of residues 36, 37, and 106 at 318 K and 0.1 MPa are similar to those of
their new peaks (200 MPa and 313 K) (Figure 6D), the second conformation
might be dominantly populated at 318 K.
Pressure and temperature effects onI para/I dia for each amide
group were also investigated for the variants D118C and A140C, as shown
in Figures S9 and S10, respectively. As pressure increased, theI para/I dia values of
residues 59–61 of the D118C variant increased by approximately 0.1–0.2
(Figure S9A), indicating that D118 moved away from the β3–β4 turn in
the pressure-stabilized intermediate. As the temperature increased, the
values of residues 57–58 and 93–94 of the variant increased by
approximately 0.2–0.3 (Figure S9B), indicating that residue 118 moved
away from the β3–β4 and β6–β7 turns in the temperature-stabilized
intermediate. Similarly, as pressure or temperature increased, theI para/I dia values of
residues 94–97 of the A140C variant increased by 0.2–0.3, indicating
that residue 140 moved away from the β7 in both of the intermediates
(Figure S10A and B).I para/I dia values of
residues 36, 37, and 106 for the A140C variant are summarized in Figure
S11. A pair of cross-peaks was only observed for residue 36. Unlike
E128C, the intensity ratio of the new residue 36 peak is similar to that
of the original one (Fig. S11A). The ratios at higher temperature were
significantly higher for residues 36 and 37, and slightly lower for
residue 106 (Fig. S11B), showing opposite tendencies with the data from
E128C. In addition, for the variants D118C and A140C, residues 33–39,
including the β1–β2 turn, maintained lowerI para/I dia values (i.e.,
0–0.6) in both intermediates.
These results indicate that the pressure- and temperature-stabilized
intermediates shared a common structural feature in which residue 128
moved away from the β5–β6 turn (consisting of residues 82 and 83) while
becoming more accessible to the β3–β4 turn region, including residues
59 and 62. Moreover, residues 118 and 140 moved away from the N-terminal
domain, yet remained accessible to the β1–β2 turn. As residues 118,
128, and 140 are in the turn region between β8–β9, β9–β10, and
β10–β11, respectively, these PRE results suggest that the central
β-sheet, i.e., β8–β10, is partially disordered, and thus is located a
longer average distance from the N-terminal domain in the intermediates
than it is in the native conformation (Figure 1). Alternatively, the
β-sheet region may retain weak specific contact with the β1–β2 turn,
owing to a twist of the central β-sheet and increased flexibility of the
polypeptide chain. Such specific long-range contact has also been
observed in intrinsically disordered proteins such as
α-synuclein.23,36
New peaks appeared in the central aspect of the spectrum, an area in
which disordered polypeptide chains are typically observed, as pressure
or temperature was increased. The original signals corresponding to the
N-terminal domain and the new NMR signals showed gradual decreases in
intensity after incubating the protein for a few days at 250 MPa, likely
owing to protein aggregation. Therefore, the assignment of new peaks
using the conventional triple-resonance NMR experiments has not been
completed. However, it is useful to analyze theI para/I dia ratios for new
peaks that likely correspond to amide groups in the central and
C-terminal domains. Figure 7A shows the frequency ofI para/I dia values for all
original cross-peaks that originated from the native protein
conformation, as the paramagnetic probe was attached at residue 140 in
the central domain. The ratios were widely distributed between 0 and 1;
however, were primarily above 0.8, indicating that a folded conformation
remained near the paramagnetic probe, and many amide groups were not
accessible to the probe. Figures 7B and C show the frequencies ofI para/I dia values of new
peaks in the pressure-stabilized and temperature-stabilized
intermediates, respectively, of the A140C variant. In the
temperature-stabilized intermediate (Figure 7C), the frequencyvs. I para/I dia plot
had a Gaussian shape, with a center value of approximately 0.7;
frequencies of less than 0.2 and more than 0.9 were not observed,
indicating an entirely different shape from the native conformation.
Since a Gaussian shape is typically obtained for random events, the PRE
results may have resulted from an ensemble of disordered conformations
of the polypeptide chain. The PRE provides 1/r 6ensemble-averaged distance information, and thus different conformations
may show different PRE effects. Even amide groups more than 25 Å, apart
from the paramagnetic probe in the native conformation, could
transiently access the probe in the intermediates due to the increased
flexibility of the polypeptide chain, while very few amide groups showed
consistently strong PRE effects. Although theI para/I dia-frequency
profile of the pressure-intermediate was similar to that of the
temperature, the Gaussian shape was not as apparent, and the strong and
weak I para/I dia ratios,
i.e., > 0.9 and < 0.2, were still observed
(Figure 7B). Since an intermediate population is retained in both
conditions (Figure S2 and Figure S3), the differences in theI para/I dia-frequency
profiles indicate that the pressure-intermediate has a more compact
conformation. Similar frequency profiles were observed for the D118C and
E128C variants, as shown in Figures S12 and S13, respectively.