Conformational control in a photoswitchable coiled coil

Article abstract of DOI:10.1039/d0cc08318f

The coiled coil is a common protein tertiary structure intimately involved in mediating protein recognition and function. Due to their structural simplicity, coiled coils have served as attractive scaffolds for the development of functional biomaterials. Herein we describe the design of conformationally-defined coiled coil photoswitches as potential environmentally-sensitive biomaterials.

Full text of DOI:10.1039/d0cc08318f

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Conformational control in a photoswitchable coiled coil
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Pages 1403–1528
Defined conformational photoswitching is exhibited in helical
coiled coils. Robust conformational control required precise
positioning of a small molecule chromophore across the
helical dyad to limit peptide fraying. This research suggests
that photoswitchable coiled coils may serve as attractive
scaffolds for the development of functional biomaterials.
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Conformational control in a photoswitchable
coiled coil†
Cite this: Chem. Commun., 2021,
7, 1442
5
Justin M. Torner and Paramjit S. Arora
*
Received 23rd December 2020,
Accepted 19th January 2021
DOI: 10.1039/d0cc08318f
rsc.li/chemcomm
2
1
The coiled coil is a common protein tertiary structure intimately manner. These minimal motifs, termed crosslinked helix
involved in mediating protein recognition and function. Due to their dimers or CHDs, have been shown to regulate protein–protein
2
4
structural simplicity, coiled coils have served as attractive scaffolds interactions in biochemical, cellular, and in vivo contexts.
for the development of functional biomaterials. Herein we describe Here, we apply the prior lessons in stabilizing helix dimers to
the design of conformationally-defined coiled coil photoswitches the development of switchable coiled coils.
2
5
as potential environmentally-sensitive biomaterials.
Small molecule chromophores, such as azobenzene which
adopt a cis or trans conformation upon irradiation with a
Reversibly controlled peptide assemblies are of interest as specific wavelength of light, have been employed to generate
1
–4
26
dynamic biological reagents and materials.
Methods photoswitchable protein secondary and tertiary structures.
5
,6
described in the literature have utilized redox chemistry, pH Although significant efforts to achieve defined conformational
7
,8
9–11
12,13
changes, metal coordination,
and photoisomerization
control have been described, photoswitching to control peptide
to achieve such control. Photoinduced conformational changes conformation in coiled coil motifs has been difficult to achieve.
in protein secondary structures, a-helices and b-hairpins, have The challenge is illustrated in Fig. 1. A stable coiled coil
1
2–16
been reproducibly demonstrated;
however, efforts to control assembly requires interhelical hydrophobic and salt bridge
conformation in protein tertiary structures remain in their interactions; once these interactions are in place to stabilize
infancy. The coiled coil motif represents the simplest helical the tertiary conformation, azobenzene switching often does not
tertiary conformation and attempts to create photoswitchable provide the sufficient energy to counteract these interactions.
coiled coils span two decades. The early efforts focused on Switching of a stabilized coiled coil, therefore, does not signifi-
1
7,19
photocontrol of bZIP transcription factors as a model protein– cantly disrupt the peptide helicity.
This result indicates the
1
7
DNA interaction. Other approaches have involved quaternary propensity of the peptide to adopt a conformation independent
1
8
19
structure formation and artificial enzyme modulation. However, of the geometry of the chromophore likely because the individual
robust conformational control, as defined by a significant loss of helices do not act as rigid rods and suffer from helical fraying. We
the helical signature in coiled coil upon irradiation, has not been hypothesized that precisely designed crosslinkers and judiciously
demonstrated and is the subject of the current study.
designed sequence may overcome this challenge and provide
We have recently focused on the design of minimal coiled defined peptide conformations that can be induced with light.
2
0,21
coils as potential inhibitors of protein–protein interactions.
Coiled coils are characterized by a repeating pattern of polar
Natural coiled coils span 430 residues on average and feature a and non-polar residues known as a heptad repeat (a, b, c, d, e, f, g);
hydrophobic interface which stabilizes the helical conforma- a and d positions are generally non-polar in natural coiled coils and
2
2,23
27
tion in individual segments.
Our results revealed the chal- drive interhelix knob into hole packing. Extensive reengineering of
lenge in inducing the helical conformations in short peptides the azobenzene-linked peptide dimer was required to achieve
that do not have the benefit of extensive interhelical contacts. conformationally-defined photoswitching. Our acquired under-
We learned that judiciously designed crosslinkers can stabilize standing in designing minimal coiled coil peptides stabilized with
parallel and antiparallel helical dimers in a predetermined a crosslinker suggests that a minimum of three-four a/d heptad
residues are needed to enable sufficient knob-into-hole packing for
helix stability; a higher number of a/d contacts would over-stabilize
a coiled coil yet a lower number would likely lead to conforma-
Department of Chemistry, New York University, 100 Washington Square East,
New York, NY, 10003, USA. E-mail: arora@nyu.edu
tionally unstable constructs. Our study reveals linker trajectory
to be a critical factor that must be controlled to afford precise
†
Electronic supplementary information (ESI) available: Synthesis and character-
ization of coiled coil peptides. See DOI: 10.1039/d0cc08318f
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Fig. 1 (A) We developed azobenzene-based coiled coil photoswitches.
In our designs, trans-azobenzene promotes alignment of hydro-
phobic residues (grey) and helical assembly while the photo-switched
conformation reduces hydrophobic packing. (B) A challenge in developing
conformationally-defined coiled coil switches is that individual helices may
act as elastic rods and unfold upon conformational switching (left) or
undergo partial helix unravelling (right).
Fig. 2 (A) Potential orientations of azobenzene linkages (yellow) relative
to putative helical axes. (B) List of peptide chain sequences aligned by
heptad repeat position. (C) Chemical structure of azobenzene linker
connected to two peptide strands.
switching. The directionality of the linker vis a vis the helix axis is
illustrated in Fig. 2A and Fig. S4 (ESI†).
for knobs-into-holes packing for coiled coil formation followed by
We based the designs of the parallel coiled coil photo- disruption of the helical interface upon photoswitching. The
switches on tropomyosin – a well-characterized cytoskeletal correspondence between the linker length and attachment point
2
8,29
coiled coil protein.
We placed the azobenzene linker at is likely to be a factor favouring some designs but our analysis
the N-terminus of two peptides to generate parallel coiled coils suggests that a second key factor is likely the potential fraying of
that span four a/d heptad residues each. We tested four the peptide strand away from the inner hydrophobic packing
different crosslinking positions at different spots around the (Fig. 1B and Fig. S4, ESI†). In an ideal scenario, the individual
helical wheel (Fig. 2B). Peptides 1 through 4 are listed in a-helices would act like elastic rods that form a dimer or dissociate
decreasing N-terminus distances in a putative coiled coil with into monomers in response to the mechanical pressure generated
linkage sites ranging from across the entire structure to directly from photoswitching. However, the force may instead lead to
adjacent (Fig. 2A and B). The N-termini distances in Peptides fraying of the termini or unravelling of a-turns; we hypothesized
1
–3 are nearly the same and optimal for the designed azoben- that this fraying is the key reason why some coiled coil designs may
3
0
17,19
zene linker based on our own prior studies and designed helix not display the intended conformational switching.
loop helices. Residues at a and d positions were mutated to tide designs test this hypothesis. We conjectured that attachment
Ile and Leu, respectively, for optimal parallel coiled coil hydro- of the azobenzene dye at the g/g heptad positions (Peptide 3), is
phobic packing,
Our pep-
3
1
0
2
2,32–34
and N-terminal residues were mutated likely not to lead to a conformational change upon photoswitching
to Ala to maintain consistent and minimal steric bulk in the because the g residues may easily fray; while, attachment of the
0
0
space adjacent to the chromophore’s isomerization. The pep- crosslinker at the c/c or f/f positions (Peptide 1 and 2) may allow
tides were synthesized as described in the ESI.† We installed a the individual helices to dissociate upon photoswitching because
mercaptopropionic acid residue at the N-terminus of each the presence of two-three residues beyond the helical interface
0
peptide to afford a thiol group for facile alkylation with 4,4 -dibro- likely provides sufficient stability against helix fraying to engender
moacetamido-azobenzene (Fig. 2C and ESI†).
rod-like behaviour. Using similar logic, we predicted that Peptide 4,
In designing variants 1–4, we hypothesized that certain cross- with adjacent crosslinking and a mismatched linker length will not
linking sites may appropriately align the a and d position residues switch conformations.
1443 | Chem. Commun., 2021, 57, 1442ꢀ1445
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We modelled several variations and found plausible linker
configurations that could accommodate a coiled coil structure
envisioned in Fig. 1A and proceeded to assess the designs
experimentally. The conformation of the four coiled coil
designs was analysed by circular dichroism (CD) spectroscopy.
CD spectra of peptides linked to both the trans- and the cis-
azobenzene chromophores were obtained. The trans-azobenzene
state predominates in the dark; conversion to the cis-state was
achieved by irradiating the sample with 370 nm light. Peptide 1
exhibited the greatest change beginning with a strong helical
signature in the ground state and loss of structure upon irradia-
tion. The magnitude of the change in the helical confor-
mation was probed by the mean residual ellipticity at 222 nm –
a signature for a-helices – and a 50% reduction in helicity was
observed upon photoswitching (Fig. 3A). To our knowledge, this is
the highest amount of photoswitching observed in a coiled coil
model. The irradiated peptide reverts back to its higher helical
state upon placement of the sample in the dark for 110 minutes at
room temperature. As a control, a CD spectrum was also collected
for the peptide chain alone without the azobenzene linkage
(Fig. S8, ESI†). The control peptide 1c was less helical highlighting
the role of the crosslinker in enforcing the tertiary structure
conformation. Peptides 2 and 4 failed to demonstrate a minimal
helical signature for either isomer states. In keeping with our
predictions, Peptide 3 showed robust helicity but failed to
adequately shift conformationally (Fig. 3A and Fig. S7, ESI†).
The difference in the behaviour of Peptides 1 and 3 supports the
hypothesis that linker heptad position relative to the interface
has a significant impact on photoswitching. Specifically, the
absence of switching in 3 suggests that helix fraying may be a
culprit although it is difficult to directly support helix fraying
with NMR structural analysis due to the short lifetime of the
cis-azobenzene state.
Successful photoisomerization of the azobenzene-linked
peptide 1 was confirmed by UV-vis spectrophotometry. Irradia-
tion with 370 nm light led to the disappearance of the trans-
azobenzene peaks and subsequent onset of those associated with
the cis state (Fig. 3B). Thermal relaxation in the dark gradually
returned the azobenzene to the trans state after 110 minutes. To
determine the extent of photoisomerization in 1, we used reversed-
phase HPLC and measured areas under the curves for the dark-
adapted sample and upon UV-irradiation (Fig. 3C). Data suggest that
roughly 90% of the azobenzene-linked peptide undergoes the
desired conformational change upon irradiation, which is con-
sistent with previous reports of similarly substituted azobenzene
16
photoswitches.
We examined the oligomerization state of peptide 1 by size
exclusion chromatography (Fig. 3D). Lysozyme, aprotinin, and
vitamin B12 were analysed as reference standards along with a
monomeric crosslinked helix dimer (AB-4) with a solved NMR
Fig. 3 (A) CD spectra of peptides 1 (top) and 2 (bottom). Black curves
2
1
structure. Peptide 1 eluted with a retention time corresponding are dark-adapted samples and blue curves are after 370 nm irradiation.
(
B) UV-Vis spectrum of peptide 1. Dashed black curve is dark-adapted
to three times the expected molecular weight based on the
controls suggesting it exists as a trimer. The high oligomeriza-
tion state of 1 was corroborated by the concentration-dependent
observation of larger particles by dynamic light scattering
sample, blue curve is after 370 nm irradiation, and green curve is after
allowing photoswitch to revert back in the dark. (C) RP-HPLC chromato-
gram of peptide 1 before (black) and after (blue) photoswitching. (D) SEC
chromatograms of peptide 1 (yellow) along with reference standards.
(Fig. S10, ESI†). No large particle was observed at the Theoretical chromatogram of 1 as a monomer is shown as a dashed line.
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concentration (30 mM) used for the CD studies, while a particle size 10 A. J. Doerr and G. L. McLendon, Inorg. Chem., 2004, 43,
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oligomer at higher concentrations. Further studies are required to
understand the nature of the apparent multimerization.
In conclusion, we report a short, coiled coil peptide con-
struct whose structure can be controlled by irradiation with
1
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