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The pressure-induced changes in 15N
enriched HPr from Staphylococcus carnosus were
investigated by two-dimensional (2D) heteronuclear NMR
spectroscopy at pressures ranging from atmospheric pressure
up to 200 MPa. The NMR experiments allowed the simultaneous
observation of the backbone and side-chain amide protons
and nitrogens. Most of the resonances shift downfield with
increasing pressure indicating generalized pressure-induced
conformational changes. The average pressure-induced shifts
for amide protons and nitrogens are 0.285 ppm GPa−1
at 278 K and 2.20 ppm GPa−1, respectively.
At 298 K the corresponding values are 0.275 and 2.41 ppm
GPa−1. Proton and nitrogen pressure coefficients
show a significant but rather small correlation (0.31)
if determined for all amide resonances. When restricting
the analysis to amide groups in the β-pleated sheet,
the correlation between these coefficients is with 0.59
significantly higher. As already described for other proteins,
the amide proton pressure coefficients are strongly correlated
to the corresponding hydrogen bond distances, and thus
are indicators for the pressure-induced changes of the
hydrogen bond lengths. The nitrogen shift changes appear
to sense other physical phenomena such as changes of the
local backbone conformation as well. Interpretation of
the pressure-induced shifts in terms of structural changes
in the HPr protein suggests the following picture: the
four-stranded β-pleated sheet of HPr protein is the
least compressible part of the structure showing only small
pressure effects. The two long helices a and c show intermediary
effects that could be explained by a higher compressibility
and a concomitant bending of the helices. The largest pressure
coefficients are found in the active center region around
His15 and in the regulatory helix b which includes the
phosphorylation site Ser46 for the HPr kinase. This suggests
that this part of the structure occurs in a number of different
structural states whose equilibrium populations are shifted
by pressure. In contrast to the surrounding residues of
the active center loop that show large pressure effects,
Ile14 has a very small proton and nitrogen pressure coefficient.
It could represent some kind of anchoring point of the
active center loop that holds it in the right place in
space, whereas other parts of the loop adapt themselves
to changing external conditions.
A 20-residue peptide E5 containing five glutamates, an
analog of the fusion peptide of influenza virus hemagglutinin
(HA) exhibiting fusion activity at acidic pH lower than
6.0–6.5 was studied by circular dichroism (CD), Fourier
transform infrared, and 1H-NMR spectroscopy
in water, water/trifluoroethanol (TFE) mixtures, dodecylphosphocholine
(DPC) micelles, and phospholipid vesicles. E5 became structurally
ordered at pH ≤6 and the helical content in the peptide
increased in the row: water < water/TFE < DPC ∼
phospholipid vesicle while the amount of β-structure
was approximately reverse. 1H-NMR data and line-broadening
effect of 5-, 16-doxylstearates on proton resonances of
DPC bound peptide showed E5 forms amphiphilic α-helix
in residues 2–18, which is flexible in 11–18
part. The analysis of the proton chemical shifts of DPC
bound and CD intensity at 220 nm of phospholipid bound
E5 showed that the pH dependence of helical content is
characterized by the same pKa ≈5.6.
Only Glu11 and Glu15 in DPC bound peptide showed such elevated
pKas, presumably due to transient hydrogen
bond(s) Glu11 (Glu15) δCOO−(H+)
… HN Glu15 that dispose(s) the side chain of Glu11
(Glu15) residue(s) close to the micelle/water interface.
These glutamates are present in the HA-fusion peptide and
the experimental half-maximal pH of fusion for HA and E5
peptides is ∼5.6. Therefore, a specific anchorage of
these peptides onto membrane necessary for fusion is likely
driven by the protonation of the carboxylate group of Glu11
(Glu15) residue(s) participating in transient hydrogen
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