48
2 Synthesis of In-Tether Chiral Center Peptides …
previously. The final helical content presented as relative to peptide 2b, as fixed the
peptide 2b as 100% helicity. Thermal disruption curves were acquired by monitoring
the signal at 222 nm while the temperature was increased at 5 ºC intervals with an
equilibration time of 10 min between temperature increases. The data were fit to a
two-state folding model using Origin Pro 9.0.
2.4.4 NMR Spectroscopy
NMR data were recorded on a Bruker AVANCE III 400 (or 500) MHz spectrometer. DMSO-d
6 was used for
1 H NMR to characterize the peptides. NMR data were
processed using Topspin 3.0. Temperature coefficients were used as tools to characterize the propensity for exchangeable protons to form intramolecular hydrogen
bonds (IMHBs). For this experiment, the peptides were dissolved in 9:1 H 2 O:
D 2 O. The protection of the IMHBs decreases the temperature dependence of the
chemical shift of the exchangeable protons. This ultimately results in a smaller
value of compared to non-IMHB donors. Generally, the cutoff value of
for IMHBs is solvent dependent. In aqueous solution, values of more than
-4 ppb/K usually indicate hydrogen bonding. 2D NMR data were collected on
a Bruker Avance III 500 MHz spectrometer with a TXI probe. Watergate pulse
sequence with gradients were used for water suppression in 1D and 2D 1H spectrum. 2D 1H-1H TOCSY and NOESY spectra were acquired with mixing time of
100 ms and 300 ms, respectively. The TOCSY and NOESY spectra were acquired
with a width of 10 ppm and 13C spectra width of 100 ppm, and size of 1024 ×
400 complex points. All the 2D NMR spectra were processed by Topspin
to
final 2048 × 1024 complex points, and analyzed by CCPNMR software.
3 J (NH-Ha)
couplings were measured from 1D-1H spectrum. Temperature dependence for amide
NH chemical shifts was measured from 2D TOCSY spectra recorded at temperature
ranges from 288 K to 313 K with 5 K interval. At each temperature, the sample
was allowed to equilibrate for 15 min, and the chemical shifts were calibrated with
standard 4,4-dimethyl-4-silapantane-1-sulfonic acid (DSS).
2.4.5 Crystallization and Data Collection
Peptide 10b. Ac-(cyclo-1,5)-[CAAIS 5 (2-Me)]-NH 2 was dissolved in 50% CH 3 OH
at 10 mg/ml and crystallized at 25°C using sitting drop vapor diffusion method
against reservoir solution of 50% CH 3 OH. Crystals are flash frozen in liquid nitrogen
and cryo-protected by 30% glycerol in mother liquor. The crystals were screened
and collected at 100 K by in-house X-ray diffraction system equipped with highintensity sealed copper tube X-ray generator (Rigaku® MicroMax-002 +), an AFC11
goniometer, a Saturn 944 + CCD detector (Rigaku®), and an Oxford Cryo-system.
2 Synthesis of In-Tether Chiral Center Peptides …
previously. The final helical content presented as relative to peptide 2b, as fixed the
peptide 2b as 100% helicity. Thermal disruption curves were acquired by monitoring
the signal at 222 nm while the temperature was increased at 5 ºC intervals with an
equilibration time of 10 min between temperature increases. The data were fit to a
two-state folding model using Origin Pro 9.0.
2.4.4 NMR Spectroscopy
NMR data were recorded on a Bruker AVANCE III 400 (or 500) MHz spectrometer. DMSO-d
6 was used for
1 H NMR to characterize the peptides. NMR data were
processed using Topspin 3.0. Temperature coefficients were used as tools to characterize the propensity for exchangeable protons to form intramolecular hydrogen
bonds (IMHBs). For this experiment, the peptides were dissolved in 9:1 H 2 O:
D 2 O. The protection of the IMHBs decreases the temperature dependence of the
chemical shift of the exchangeable protons. This ultimately results in a smaller
value of compared to non-IMHB donors. Generally, the cutoff value of
for IMHBs is solvent dependent. In aqueous solution, values of more than
-4 ppb/K usually indicate hydrogen bonding. 2D NMR data were collected on
a Bruker Avance III 500 MHz spectrometer with a TXI probe. Watergate pulse
sequence with gradients were used for water suppression in 1D and 2D 1H spectrum. 2D 1H-1H TOCSY and NOESY spectra were acquired with mixing time of
100 ms and 300 ms, respectively. The TOCSY and NOESY spectra were acquired
with a width of 10 ppm and 13C spectra width of 100 ppm, and size of 1024 ×
400 complex points. All the 2D NMR spectra were processed by Topspin
to
final 2048 × 1024 complex points, and analyzed by CCPNMR software.
3 J (NH-Ha)
couplings were measured from 1D-1H spectrum. Temperature dependence for amide
NH chemical shifts was measured from 2D TOCSY spectra recorded at temperature
ranges from 288 K to 313 K with 5 K interval. At each temperature, the sample
was allowed to equilibrate for 15 min, and the chemical shifts were calibrated with
standard 4,4-dimethyl-4-silapantane-1-sulfonic acid (DSS).
2.4.5 Crystallization and Data Collection
Peptide 10b. Ac-(cyclo-1,5)-[CAAIS 5 (2-Me)]-NH 2 was dissolved in 50% CH 3 OH
at 10 mg/ml and crystallized at 25°C using sitting drop vapor diffusion method
against reservoir solution of 50% CH 3 OH. Crystals are flash frozen in liquid nitrogen
and cryo-protected by 30% glycerol in mother liquor. The crystals were screened
and collected at 100 K by in-house X-ray diffraction system equipped with highintensity sealed copper tube X-ray generator (Rigaku® MicroMax-002 +), an AFC11
goniometer, a Saturn 944 + CCD detector (Rigaku®), and an Oxford Cryo-system.