3.1 Pressure- and temperature-induced changes in1H-15N HSQC spectra
1H/15N HSQC spectra of the three
variants were measured with the oxidized and reduced forms of MTSL at
0.1, 200, and 250 MPa for 313 K, using an AVANCE3 950 MHz spectrometer.1H/15N HSQC cross-peaks in the
variant proteins were assigned to individual amino-acid residues with
reference to the assignments of WT* OspA (BMRB code:
4076).9 To evaluate the effects of residue
substitution and the MTSL-tag, the 1H and15N chemical shifts of individual cross-peaks in the
HSQC spectra were compared between the WT* and MTSL-tagged cysteine
variants (D118C, E128C, and A140C) at 313 K and 0.1 MPa. Chemical shift
changes (i.e. ((Δδ H)2 + (ΔδN/5)2)0.5) are shown in Figure S1.
Since chemical shift changes larger than 0.2 ppm were only observed at
residues spatially proximal to the substituted residue, they were likely
the result of local changes in magnetic shielding, and thus, the effects
of residue substitution, and the MTSL-tag on the structure and
conformational dynamics of the protein are expectedly limited.
Pressure effects on the 1H/15N-HSQC
spectra of the E128C variant reduced form are shown in Figure 2. The
HSQC cross-peaks corresponding to the folded conformation were
well-dispersed, and the chemical shifts of the backbone amide proton and
nitrogen were similar to those of WT* (Figure S1B); thus, the folded
conformation of the variant was similar to that of WT*. Pressure-induced
changes in the intensities (i.e., volumes) of the cross-peaks present in
the 0.1 MPa spectrum (the original cross-peaks) are plotted in Figure
S2. As pressure was increased to 150 MPa, the intensities of original
cross-peaks showed minimal changes. However, several peaks corresponding
to amide groups in the central and C-terminal regions preferentially and
gradually decreased when pressure exceeded 150 MPa, after which relative
intensities reached values of approximately 0.2. Additionally, many new
peaks appeared in the central portion of the spectrum, where disordered
polypeptide chains are typically observed (Figure 2). These results
indicate that the locally disordered, intermediate conformation was
partially stabilized under high pressure and occurred within 80% of the
total protein.
Moreover, peak intensities (i.e., volumes) relative to those at
atmospheric pressure are plotted along with the residue number at 250
MPa (Figure 3A), indicating that pressure-induced conformational changes
occur at residues β8 to the C-terminus. A few peak also showed
mountain-like intensity profiles (initial gain followed by a decrease in
intensity with pressure) similar to those of WT* (Figure S2). As
discussed previously14 , conformational dynamics
on the microsecond-to-millisecond time scale may correlate with
broadening and missing of the cross-peaks, even at 0.1 MPa.
When the temperature was increased from 303 to 318 K at 0.1 MPa,
preferential decreases in cross-peaks were observed for residues between
β8 and the C-terminus (Figure 3B), and relative intensities reached
values of approximately 0.2 (Figure S3). Besides, many new peaks
appeared in the central aspect of the spectrum (Figure S4), as observed
in the pressure experiments.
Similar pressure- and temperature-induced decreases in peak intensities
were observed in the 1H NMR spectra for L109 and V199
methyl protons (Figure S5), which are located within the central β-sheet
and C-terminal domain of the variant (data not shown). The transitions
of the two side-chain signals, occurring in the same pressure and
temperature ranges as those of backbone amides, serve as direct evidence
for the cooperative transition of the central β-sheet and the C-terminal
domain into the intermediate state. Based on pressure-induced changes in
peak intensities of the methyl groups, the Gibbs free energy difference
(ΔG 0) and the partial molar volume difference
(ΔV 0) between the two conformers at 0.1 MPa and
313 K were estimated to be 17 ± 3 kJ/mol and −80 ± 12 mL/mol,
respectively, which are slightly smaller than those of
WT*.14 As discussed previously, the large
volume decrease observed by this transition may be the result of
collapse and hydration of the large hydrophobic cavity in the C-terminal
domain (Fig. 1).14,35
An interesting observation is that cross-peak intensities of several
amide groups in the N-terminal domain were relatively increased when the
central and C-terminal domains were denatured under higher pressure
(above 150 MPa) or temperature (higher than 313 K) (Figures S2 and S3).
Although we did not delve deeper into the issue, a rotational motion of
the folded N-terminal domain may have been enhanced, thereby producing
more intense signals when the central and C-terminal domains were
denatured.