Reversible photocontrol of peptide conformation with a rhodopsin-like photoswitch

Article abstract of DOI:10.1021/ja301868p

Reversible photocontrol of biomolecules requires chromophores that can efficiently undergo large conformational changes upon exposure to wavelengths of light that are compatible with living systems. We designed a benzylidene-pyrroline chromophore that mimics the Schiff base of rhodopsin and can be used to introduce light-switchable intramolecular cross-links in peptides and proteins. This new class of photoswitch undergoes an ～10 A change in end-to-end distance upon isomerization and can be used to control the conformation of a target peptide efficiently and reversibly using, alternately, violet (400 nm) and blue (446 nm) light.

Full text of DOI:10.1021/ja301868p

Communication
pubs.acs.org/JACS
Reversible Photocontrol of Peptide Conformation with a Rhodopsin-
like Photoswitch
Marina Blanco-Lomas,†,§ Subhas Samanta,‡,§ Pedro J. Campos,† G. Andrew Woolley,*,‡
and Diego Sampedro*,†
†Departamento de Química, Unidad Asociada al C.S.I.C., Universidad de La Rioja, Madre de Dios 51, E-26006 Logrono, La Rioja,
̃
Spain
‡Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6
S
* Supporting Information
test of the ability of this class of molecules to function as
biomolecular photoswitches.
We report here the experimental photocontrol of peptide
conformation by the newly designed rhodopsin-like photo-
switch 1 (Scheme 1). We based our design on the previously
ABSTRACT: Reversible photocontrol of biomolecules
requires chromophores that can efficiently undergo large
conformational changes upon exposure to wavelengths of
light that are compatible with living systems. We designed
a benzylidene−pyrroline chromophore that mimics the
Schiff base of rhodopsin and can be used to introduce
light-switchable intramolecular cross-links in peptides and
proteins. This new class of photoswitch undergoes an ∼10
Å change in end-to-end distance upon isomerization and
can be used to control the conformation of a target peptide
efficiently and reversibly using, alternately, violet (400 nm)
and blue (446 nm) light.
Scheme 1. Synthesis of the Photoswitch (E Form)
olecular switches based on E/Z photoisomerization have
M
been used in a variety of contexts to control diverse
properties.1 Biomolecular photocontrol places a number of
demands on photoswitches. In particular, (i) they must
undergo isomerization efficiently at wavelengths compatible
with biological systems, and (ii) they must undergo substantial
conformational change so that they can drive functional change
in a biomolecule.2 The discovery of new or alternative
photoswitch types could expand the applicability of the switch
concept to different and increasingly complex molecular
environments.
described benzylidene−pyrroline (BP) and N-protonated BP
(NHBP) systems.6 The synthetic route permits the synthesis of
a family of switches by modification of the starting aldehyde.
Placing amino groups at both ends of the switch unit allowed
for the synthesis of thiol-reactive chloroacetamide derivative 2,
which was suitable for site-specific introduction into a Cys-
containing peptide as an intramolecular cross-link. The
photochemical behavior of this new photoswitch was computed
using the multiconfiguration complete active space second-
order perturbation theory//complete active space self-consis-
tent field (CASPT2//CASSCF)6,14 strategy. The amido
substituents, together with the added phenyl ring, were
predicted to lead to longer-wavelength absorption and a higher
isomerization quantum yield relative to the parent benzyli-
dene−pyrroline (see the Supporting Information).
The protonated retinal Schiff base chromophore of
rhodopsins3 constitutes an example of a very efficient E/Z
photoswitch shaped by biological evolution. In bovine
rhodopsin, selective photoisomerization of the 11-cis chromo-
phore occurs rapidly (150 fs) via evolution of a single π → π*
excited state (S1), providing the all-trans ground state (S0)
product with high efficiency (Φ = 67%).3 While rhodopsin
itself can be engineered as a photoswitch,4 the limited
information on the conformational changes that occur hampers
the broad applicability of this approach. When removed from
the opsin protein, the isolated retinal chromophore exhibits
complex photochemistry, including isomerization at any of
three double bonds.5 However, a number of analogues of the
rhodopsin chromophore designed to act as photoswitches in
isolation have been explored both computationally and
experimentally.6−11 Computational work has shown that
some of these molecules may work as efficient photoswitches
for peptides,12,13 but to date there has been no experimental
The thiol-reactive photoswitch 2 was coupled to the peptide
SS-11 (see below), which confers aqueous solubility to the
Received: February 24, 2012
Published: April 6, 2012
© 2012 American Chemical Society
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molecule. The UV/vis spectrum of the photoswitch in aqueous
solution in the dark-adapted E form is shown in Figure 1a. The
Figure 2. Multiple rounds of photoswitching of the SS-11 peptide
cross-linked using 2 with alternating violet (400 nm) and blue (446
nm) light in aqueous solution at 20 °C.
Figure 1. (a) UV−vis spectra of the SS-11 peptide cross-linked with 2
in the dark-adapted E form in aqueous solution at different pHs (as
indicated) at 20 °C. (b) Photoisomerization of the SS-11 peptide
cross-linked with 2 at pH 5.0. Spectra of the pure E and Z forms of the
cross-linked peptide (isolated by HPLC) are shown. Photostationary
states (PSSs) at 400 and 446 nm are 7/93 E/Z and 78/22 E/Z,
respectively.
with a half-life of ∼10 h at 50 °C (see the Supporting
Information). This slow relaxation permitted separation of the
E and Z isomers by HPLC (see the Supporting Information).
The spectra of the pure E and Z forms at pH 5.0 are shown in
Figure 1b.
The change in end-to-end distance of 2 upon isomerization
was estimated using molecular dynamics simulations (see the
Supporting Information). Figure 3 shows that E/Z isomer-
ization produces a large (∼10 Å) change in the mean end-to-
end distance with very little overlap between isomers. The size
of this conformational change is significantly greater than those
in many azobenzene-based photoswitches.2 Azobenzene
derivatives specifically designed to undergo large changes in
end-to-end distance upon photoisomerization have exhibited
lower photoisomerization efficiencies or more complex
conformational distributions than those exhibited by 2 (Figure
3a).21,22
Photocontrol of peptide α-helical structure can be achieved
by matching the end-to-end distance of a photoswitchable
cross-linker in one isomeric state with the distance between
cross-linker attachment points in the peptide.23,24 The end-to-
end distance of the E isomer matches well with the distance
between Cys side chains with an i, i + 11 sequence spacing in a
peptide α-helix (Figure 3a), whereas the Z isomer is too short
to permit normal helix formation with this Cys side-chain
spacing. The peptide SS-11, studied previously as an ideal helix-
forming sequence in water,25 was chosen as target for
photocontrol by 2. The peptide is long enough to confer
aqueous solubility on the relatively hydrophobic cross-linker.
Since 2 is not a symmetrical molecule, two distinct species
result when it reacts with a peptide to form an intramolecular
cross-link. Since the end-to-end distance change caused by
isomerization is the same for both species, we did not attempt
to characterize these isomers separately for conformational
analysis (see the Supporting Information).
chromophore (in both isomers) is predicted to have a pKa of
7.4
0.2 (ACD/iLabs; see the Supporting Information), a
value significantly lower than that of the Schiff base in
rhodopsin.15 Lowering the pH to 5.0 caused a red shift in the
spectrum consistent with the protonation of the Schiff base
moiety,15−17 while this longer-wavelength band appeared only
partially at pH 7.0. (Figure 1a). Presumably, the pKa of the
photoswitch could be raised so that the Schiff base would be
protonated at physiological pH by the appropriate introduction
of electron-donating substituents. Alternatively, the amino
group could be quaternized to maintain a permanent positive
charge, as has been done previously with Schiff base
analogues.6,17 The molar extinction coefficient was calculated
to be 24 700 M−1 cm−1 at 375 nm, a value that compares well
to those of azobenzene photoswitches, which typically are ∼20
000 M−1 cm−1.18
Irradiation with violet light (400 nm) efficiently produces a
photostationary state (PSS) of the cross-linked peptide that is
93/7 Z/E. (Figure 1b). The Z isomer has decreased absorbance
at 375 nm and exhibits a long-wavelength tail. This long-
wavelength tail enables photoswitching of the chromophore
back to the E isomer with blue light (446 nm). Reversible
photoswitching was also possible at pH 7.0 (see the Supporting
Information), although the process was less efficient since the
photoswitch is only partially protonated at this pH. Multiple
rounds of photoisomerization gave no evidence of photo-
bleaching/photooxidation (Figure 2).
The E-to-Z isomerization quantum yield of 1 was measured
to be ∼0.6 for the unprotonated chromophore alone in
acetonitrile and ∼0.24 for the protonated chromophore
attached to a peptide in water using trans-azobenzene or a
trans-azobenzene-modified peptide19 as an actinometer (see the
Supporting Information). The value for the quantum yield of
the isolated chromophore is significantly larger than those
reported for some biarylidenes (0.07−0.55).20 The overall
spectroscopic behavior of 1 compares well with predictions
from the CASPT2//CASSCF theory (see the Supporting
Information for details).6
Cross-linking the SS-11 peptide with the E form of 2 leads to
stabilization of the α-helical content, as judged by circular
dichroism (CD) spectroscopy (see the Supporting Informa-
tion), and the peptide is essentially fully helical at 20 °C.
Irradiation with 400 nm light to produce the 7/93 E/Z PSS
leads to a marked decrease in helicity, as predicted (Figure 3b).
Since the cross-linker is near the N-terminus of the peptide,
conformational distortion is likely to be focused there, with the
rest of the peptide remaining helical (Figure 3d,e). It is unlikely
that the photoswitch itself contributes significantly to the
observed CD change, since no induced CD signal was seen at
Slow thermal relaxation of the Z isomer of the cross-linked
peptide to the thermodynamically more stable E form was seen
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ASSOCIATED CONTENT
■
S
* Supporting Information
Computational details, synthetic procedures, UV/vis and CD
data, and HPLC characterization of peptides. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
awoolley@chem.utoronto.ca; diego.sampedro@unirioja.es
Author Contributions
§M.B.-L. and S.S. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the Natural Sciences and
Engineering Research Council of Canada and the Spanish
MICINN (CTQ2011-24800). M.B.-L. thanks the Spanish
MEC for her grant.
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