Structure−Dynamics of Homopolymeric Model Peptides
A R T I C L E S
5-mm 1H{13C/15N} z-axis gradient probe, a 700 MHz spectrometer
with a 5-mm 1H{13C/15N} z-axis gradient cryogenic probe, a 800 MHz
spectrometer with a 5-mm 1H{13C/15N} z-axis gradient cryogenic probe
and a 900 MHz spectrometer with a 5-mm 1H{13C/15N} z-axis gradient
cryogenic probe. The NMR data were processed with Bruker XWIN-
NMR 3.5 and TopSpin 1.3 programs and analyzed with either Bruker
programs or Felix2000 (Accelrys). An automated routine32 was used
for extracting the J-coupling constants from the E.COSY pattern in
Felix2000. The temperature was calibrated by methanol or glycol
thermometer33 for each spectrometer. 1H chemical shifts were referenced
to the methyl resonance of internal DSS (3-(trimethylsilyl)-1-propane-
sulfonic acid sodium salt). 13C and 15N chemical shifts were referenced
indirectly to the 1H standard using published34 conversion factors.
Spectral resonance assignment was done with a combination of standard
HSQC and HMBC experiments up to Ala5. For Ala6 and Ala7, a
semiconstant time version of the HNN35 experiment was applied. The
HEWL-peptides were assigned following the standard method36 utilizing
the program CARA.37 Chemical shift values are listed in the Supporting
Information. Chemical exchange was measured with phase-sensitive38
1H,1H NOESY experiments with excitation sculpting39 for water
suppression. The forward and reverse rate constants were obtained from
the experimental peak volumes by solving eq 26 in the review article
of Perrin and Dwyer40 using the program EXSYCalc (Mestrelab
Research). The given error is the standard deviation of the obtained
rate constants for different mixing times.
of the second kind on the coupling constant determination are very
small and can safely be neglected.
The temperature dependence of 3J(HN,HR) coupling constant of Ala3
were measured on AAA, AA#A, and AAA# (A# ) 15N isotopic labeled).
Coupling constants have been measured both from low to high
temperature and from high to low temperature to ensure proper thermal
equilibration of the NMR setup.
3J(HN,C’), 3J(HR,C’), 3J(C’,C’), 3J(HN,Câ), 3J(HN,CR), 1J(N,CR), and
2J(N,CR) were measured with soft HNCA-COSY,42 CO-coupled (H)-
NCAHA,43 (HN)CO(CO)NH,44,45 HNHB[CB] E.COSY,46 HNCO[CA]
E.COSY,47 and J-modulated 1H, 15N HSQC’s,48 respectively. The signal
overlap in the (HN)CO(CO)NH experiment was so severe that it was
only possible to measure the 3J(C′,C′) coupling constant in a few cases.
For the other experiments, the signal overlap of intra- and interresidual
correlations was avoided either by measuring at higher magnetic fields
or by the applied isotope-labeling schemes. The given statistical error
is obtained from at least two measurements. Examples for the excellent
quality of the experimental data and the summary of the used acquisition
and processing parameters are found in Figure 1 and in the Supporting
Information.
CD Spectroscopy. CD spectra were recorded on a Jasco J-810
spectropolarimeter equipped with a Jasco PTC-423S temperature control
unit using quartz cuvettes with 0.2-mm pathlengths. Data were collected
at 0.5-nm increments from 260 to 185 nm with a scanning speed of
100 nm/min. For the measurements the same solvents as for the NMR
measurements were used, and the peptide concentrations were the
following: c(Ala3) ) 1.6 mmol/L, c(Ala7) ) 0.9 mmol/L, c(Val3) )
2.6 mmol/L, c(HEWL-9mer) ) 0.6 mmol/L, and c(HEWL-19mer) )
0.1 mmol/L. Ten scans were averaged (HEWL-19mer 20 scans), and
the solvent baseline was subtracted, but no line smoothing was applied.
Molecular Dynamics Simulation. We used the GROMOS96 force
field 43a149 to model the peptides and the SPC water model50 to describe
the solvent. The peptides Alan were placed in cubic boxes containing
650, 807, 874, 1096, and 1243 water molecules for n ) 3, 4, 5, 6, and
7, respectively. The simulation boxes of the Gly3 and Val3 were similar
to that of Ala3. In all simulations, the GROMACS program suite51,52
was employed. The equations of motion were integrated by using a
leapfrog algorithm with a time step of 2 fs. Covalent bond lengths were
constrained via the SHAKE53 procedure with a relative geometric
tolerance of 10-4. We used the particle-mesh Ewald method to treat
the long-range electrostatics interactions.54 The nonbonded interaction
pair-lists were updated every 5 fs, using a cutoff of 1.2 nm. The systems
were minimized using the conjugate gradient method. Subsequently,
the solvated systems were equilibrated for 100 ps at constant pressure
(1 atm) and temperature (T ) 300 K), respectively, using the Berendsen
coupling procedure.55 Each system was then run for 100 ns, and the
data were collected every 0.2 ps.
3
1
It was found that the J(HN,HR) coupling constant measured in H
one-dimensional (1D) spectra depends on the exact NMR pulse
sequences which implement different water suppression schemes. For
gradient-based suppression methods such as excitation sculpting,
homonuclear scalar coupling evolves during the suppresion schemes
which leads to a significant larger coupling constant. Thus, the
1
3J(HN,HR) coupling constants were measured in 1D H spectra with
presaturation for water suppression. There, the time during which
homonuclear coupling can evolve is kept to a minimum and can be
safely corrected for by linear phase correction. Direct determination
of the 3J(HN,HR) coupling constants from the splitting of amide protons
underestimates the true coupling constant value whenever the doublet
components are not resolved to baseline. This seems to be the case for
the reported8 values of Ala3. However, deconvolution of the spectrum
by fitting a Lorentzian function to the peaks or apodization of the FID
with a Lorentz-to-Gauss transformation prior to Fourier transformation
recovers true J-coupling constants. In most cases, the average value
derived by both methods is given, except for residue A3, A5 of Ala5,
A11 of HEWL-9mer (same numbering as for HEWL-19mer for easy
comparison), and A9 to A11 of HEWL-19mer where we only applied
the apodization due to partial overlap of the signals. When applying
the deconvolution routine it did not matter if we used a prior Lorentzian
broadening factor for the window function between 0 and 1 Hz for
spectra processing. In the case of Gly3, the 3J(HN,HR) coupling constant
was measured on the HR protons because of line broadening of the
amides signals. Note also that for the peptides with low molecular
weight, systematic errors as reported by Harbison41 due to relaxation
The calculation of the J-coupling constants from the MD simulations
is based on Karplus relations of the type J(æ) ) A cos2(æ + θ) + B
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