photokinetic experiments used the latter arrangement. Irradiation
of the sample (at 90◦ to the light source and detector used for
the absorbance measurements) was carried out using a xenon
lamp (450 W, Osram 450XBO/2 OFR) coupled to a double
monochromator. Rates of thermal cis-to-trans isomerization were
measured for a series of temperatures by monitoring absorbance
at 450 nm after irradiation to convert a percentage of the solution
to the cis isomer. The light used for the absorbance measurement
was of sufficiently low intensity to cause negligible isomerization.
Solution conditions are described in the figure legends.
Molecular modeling
Models of cis and trans cross-linkers were built using HyperChem
(v.8, Hypercube Inc.), with the linker terminated with methyl
groups representing theb carbon of Cys in the cross-linked peptide,
and minimized using the Amber99 forcefield. Restraints were
added to the azo bond for the cis conformation (force constant
16). Molecular dynamics runs were performed in vacuo essentially
as described previously19 with a distant dependent dielectric, and
1–4 scale factors of 0.833 for electrostatic and 0.5 for van der
Waals interactions, a step size of 1 fs and 300 K as the simulation
temperature. Trajectories were analyzed to verify that numerous
torsion angle changes occurred for all single bonds during the
course of the simulation to ensure that conformational space was
adequately sampled. All points histograms were then produced for
the S–S distance during the full set of simulations for each isomer.
Additional conformational searches were performed using the
conformational search algorithm of HyperChem (v.8, Hypercube
Inc.) and the AM1 method. The ranges for acyclic torsions and
ring torsion flexing were 60 to 180◦ and 30 to 120◦, respectively.
A usage directed search algorithm was employed, with structures
A
molar extinction coefficient for the cross-linker of
16 800 M-1 cm-1 at 450 nm in pH 7.0 phosphate buffer was
determined by comparing absorbance at 450 nm vs. 280 nm for
the cross-linked peptide FK-11W. An extinction coefficient of
5625 M-1 cm-1 at 280 nm from the Trp residue at the N-terminus
of the cross-linked FK-11W was used. The UV–Vis spectrum of
cross-linked GSH (which does not have a Trp residue) was used
to correct for contributions of the cross-linker to the measured
absorbance at 280 nm.
˚
with non-H atom positions that varied by less than 0.25 A RMS or
Circular dichroism measurements
with torsion angle changes less than 5◦ considered as duplicates.
Structures less than 6 kcal mol-1 in energy above the lowest found
were retained.
Circular dichroism (CD) measurements were performed on a Jasco
Model J-710 spectropolarimeter. Tris(carboxyethyl)phosphine
(1 mM) was present in the uncross-linked peptide samples to
ensure that cysteine residues were in their reduced form. Peptide
concentrations were determined using a molar extinction coeffi-
cient for the cross-linker of 16 800 M-1 cm-1 at 450 nm in pH 7.0
phosphate buffer. For fast CD measurements, the instrument was
set to time-drive mode. Samples were dissolved in 10 mM pH 7.0
sodium phosphate buffer and/or 50% methanol in 10 mM pH 7.0
sodium phosphate buffer. Temperatures were measured using a
microprobe directly in the sample cell. The nitrogen flow rate
was increased to minimize condensation on the cuvette when the
temperature of the sample was 6 ◦C. Samples in cuvettes were
irradiated for 1 minute with a metal halide lamp (150 W, Osram
HQI-SE150/NDX (unfiltered), then measured immediately.
Acknowledgements
We are grateful to the Natural Sciences and Engineering Research
Council of Canada for financial support.
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The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 4323–4332 | 4331
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