In Situ Formation of an Azo Bridge on Proteins Controllable by Visible Light

Article abstract of DOI:10.1021/jacs.5b06234

Optical modulation of proteins provides superior spatiotemporal resolution for understanding biological processes, and photoswitches built on light-sensitive proteins have been significantly advancing neuronal and cellular studies. Small molecule photoswitches could complement protein-based switches by mitigating potential interference and affording high specificity for modulation sites. However, genetic encodability and responsiveness to nonultraviolet light, two desired properties possessed by protein photoswitches, are challenging to be engineered into small molecule photoswitches. Here we developed a small molecule photoswitch that can be genetically installed onto proteins in situ and controlled by visible light. A pentafluoro azobenzene-based photoswitchable click amino acid (F-PSCaa) was designed to isomerize in response to visible light. After genetic incorporation into proteins via the expansion of the genetic code, F-PSCaa reacts with a nearby cysteine within the protein generating an azo bridge in situ. The resultant bridge is switchable by visible light and allows conformation and binding of CaM to be regulated by such light. This photoswitch should prove valuable in optobiology for its minimal interference, site flexibility, genetic encodability, and response to the more biocompatible visible light.

Full text of DOI:10.1021/jacs.5b06234

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In situ formation of an azo bridge on proteins controllable by visible light
Christian Hoppmann, Innokentiy Maslennikov, Senyon Choe, and Lei Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b06234 • Publication Date (Web): 24 Aug 2015
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In situ formation of an azo bridge on proteins controllable by visible
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‡
‡
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Christian Hoppmann, Innokentiy Maslennikov, Senyon Choe, Lei Wang *
†
Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, San
‡
Francisco, CA 94158. Structural Biology Laboratory, Qualcomm Institute, University of California San Diego, CA 92093, USA
Supporting Information Placeholder
amino acids (PSCaa), into proteins in E. coli and mammalian cells.5
ABSTRACT: Optical modulation of proteins provides superior
The PSCaa consists of an azobenzene side chain extended by a reactive
spatiotemporal resolution for understanding biological processes, and
group, which reacts with a nearby cysteine to form a photoswitchable
bridge on the protein in situ.5 A drawback of the initial generation of
PSCaas is that they isomerize in response to UV light (λ = 365 nm)
only, which cannot penetrate deep into tissues and may negatively
impact biological processes under study. Here we present the design
and genetic incorporation of a new PSCaa, F-PSCaa 1, which generates
a photoswitchable protein bridge in situ through a reaction mechanism
photoswitches built on light-sensitive proteins have been significantly
advancing neuronal and cellular studies. Small molecule photoswitches
could complement protein-based switches by mitigating potential
interference and affording high specificity for modulation sites.
However, genetic encodability and responsiveness to non-ultraviolet
light, two desired properties possessed by protein photoswitches, are
challenging to be engineered into small molecule photoswitches. Here
we developed a small molecule photoswitch that can be genetically
installed onto proteins in situ and controlled by visible light. A pen-
tafluoro azobenzene based photoswitchable click amino acid (F-
PSCaa) was designed to isomerize in response to visible light. After
genetic incorporation into proteins via the expansion of the genetic
code, F-PSCaa reacts with a nearby cysteine within the protein generat-
ing an azo bridge in situ. The resultant bridge built by F-PSCaa is
switchable by visible light and allows conformation and binding of
CaM to be regulated by such light. This photoswitch should prove
valuable in optobiology for its minimal interference, site flexibility,
genetic encodability, and response to the more biocompatible visible
light.
-6
7
unreported for proximity-enabled bioreactivity (Scheme 1). In addi-
tion, 1 enables photoswitching to be modulated by visible light. This
new photoswitch should prove valuable for optical modulation of
proteins with high specificity and biocompatibility in vivo.
The ability to control protein function with light provides excellent
temporal and spatial resolution for precise investigation in situ, and
thus is having significant impact on biological studies. Light-sensitive
proteins and domains have been exploited for optical modulation of
1
neuronal and cellular processes. The bulk of such domains and pro-
teins may interfere with the native function, location, and trafficking of
the target protein, limiting aspects of protein functions to be con-
trolled. Installation of photo responsiveness onto proteins using small
molecules can mitigate such interference. One elegant approach is to
chemically tether a photoswitchable ligand to receptors and channels.2
Unfortunately, ligand tethering has been limited to the extracellular
side of membrane proteins. Another approach links two appropriately
distanced cysteine residues using symmetric photoswitches bearing
Scheme 1. In situ formation of an azo bridge on proteins controllable
by visible light. The F-PSCaa 1, (S,E)-2-amino-3-(4-
3
two reactive groups, but such bivalent reactive units cannot be used in
(
(pentafluorophenyl)diazenyl)phenyl)propanoic acid, is genetically
vivo. In addition, while light-sensitive proteins and domains can be
genetically encoded providing high selectivity in targeting proteins,
cells and tissues, genetically encoding photoswitchable small molecules
incorporated into proteins in vivo through the expansion of the genetic
code. After incorporation, 1 reacts with a nearby cysteine residue via
4
-5
nucleophilic aromatic substitution (S Ar) to form an azo bridge in situ.
N
remains challenging.
Both the F-PSCaa and the resultant bridge isomerize in response to
visible light.
In order to selectively install small molecule photoswitches into pro-
teins in vivo for optical modulation, we recently genetically encoded a
new class of unnatural amino acids (Uaas), the PhotoSwitchable Click
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Substitution on azobenzene has been shown to shift the wavelengths
Notably, pentafluoro substitution resulted in distinct separation in
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of light driving the cis-trans isomerization. Bulky electron-rich substitu-
ents, when installed on the four ortho positions or the para position to
the azo group, result in azobenzene derivatives responding to green or
wavelength of both transition bands between the Z and E isomer, and
the separation at the n-π* transition band (Figure 1b insert) enabled
the use of green light for selective formation of the Z isomer as ob-
8
red light; fluorine atoms substituted at the four ortho positions also
served.
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shifts azobenzene absorbance to visible light. However, bulky substit-
To determine whether F-PSCaa (1) can undergo S Ar with thiols,
N
uents are not appropriate for PSCaa because resultant large side chain
may make it infeasible to evolve an aminoacyl-tRNA synthetase for
genetic incorporation of the PSCaa. We decided to use pentafluorina-
tion in designing F-PSCaa 1 based on the following considerations: 1)
fluorine has similar size to proton and does not significantly change the
bulk of the azobenzene unit; 2) perfluorination of a photoswitch unit
can modulate its photochromic performance and shift its absorbance,
but it has never been used before to generate a visible light-switchable
we incubated 1 with Boc-protected cysteine (2) at pH 7.5 and moni-
tored the reaction over time by HPLC-MS. After 2 h of incubation the
product 3 was formed (Figure S2). The reaction was also detectable by
UV-vis spectroscopy (Figure S3). The absorbance spectrum of the
product in E form shows a bathochromic shift as a result of the elec-
tron-donating sulfur in para position to the azo group (Figure S3). The
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π-π* transition band red-shifted from 326 nm (for E form of 1) to 340
nm (for E form of the product 3). Electron donating groups in para to
the azo unit can significantly decrease the thermal stability of the Z
1
0
azobenzene; 3) perfluoro-benzene allows nucleophilic aromatic
1
1
substitution (S
N
Ar), which may be harnessed for proximity-enabled
10b
state, however, we didn´t observe a decrease in thermal stability of
7
bioreactivity to generate a protein bridge in situ.
the Z form of 3. E-to-Z photoisomerization of the product 3 could also
be induced by green light illumination at λ = 540 nm, generating an
E/Z ratio of 23:77 (Figure S5).
We synthesized 1, (S,E)-2-amino-3-(4-((pentafluoro phe-
nyl)diazenyl) phenyl) propanoic acid, by Mills coupling of the Boc-
protected 4-amino phenylalanine with the 1,2,3,4,5-pentafluoro-6-
nitrosobenzene, which was prepared from 1,2,3,4,5-pentafluoro-6-
aminobenzene using Oxone (Figure S1). Subsequent treatment with
trifluoroacetic acid gave the final pentafluoro azobenzene amino acid
F-PSCaa in acceptable yield and high purity over two steps (44%).
Figure 2. S
N
Ar reaction of F-PSCaa 1 (1 mM) with Boc-protected
19
cysteine 2 (3 mM) monitored by F-NMR spectroscopy in DMSO-d
6
.
The presence of fluorine on 1 enabled us to monitor the S
N
Ar reac-
12
19
tion of 1 with 2 in DMSO-d
6
using 19F-NMR (Figure 2). The
F
Figure 1. (a) LC profiles of the photo-equilibrium after illumination of
F-PSCaa 1 with green light (λ = 540 nm, top), UV light (λ = 365 nm,
middle) and blue light (λ = 405 nm, bottom), respectively. (b) UV-vis
spectra of the pure E photoisomer (black) and Z photoisomer (red) of
NMR spectrum of 1 showed three signals (Figure S11b): a triplet at -
55.25 ppm (1F, para), a quartet at -153.95 ppm (2F, ortho), and a
doublet of triplets at -164.99 ppm (2F, meta, not shown in the Figure
). After adding 2, as a result of the nucleophilic attack of cysteine’s
1
2
1
. Insert zooms in on the n-π* transition band.
thiol at the fluorine in para position to the azo unit, the intensity of the
triplet at -155.25 pm for the substituted para 1F decreased with time.
Meanwhile, signals for the meta and ortho 2F atoms also decreased with
a concomitant arising of two new quartet signals at -136.21 ppm (2F,
To investigate whether 1 is able to isomerize in response to visible
light, we illuminated the amino acid with green or blue light, and de-
termined the E/Z ratios of the corresponding photostationary state
meta, not shown in Figure 2) and -154.45 (2F, ortho), indicating com-
(
5
(
pss) by HPLC. As expected, illumination of 1 with green light (λ =
40 nm) results in E-to-Z isomerization yielding 78% of the Z isomer
Figure 1a, top LC profile). A similar ratio was achieved using common
UV activation (λ = 365 nm) (Figure 1a, middle LC profile). Illumina-
tion using blue light (λ = 405 nm) results in Z-to-E isomerization
restoring 73% of the E photoisomer (Figure 1a, bottom LC profile).
The UV-vis spectra of each pure photoisomer show typical features of
azobenzene: two absorbance peaks were observed representing π-π*
and n-π* transitions, respectively; upon E to Z isomerization the π-π*
absorption decreased while the n-π* absorption increased (Figure 1b).
19
plete conversion after ~6 h at 25 °C (Figure 2). The two quartet
F
signals correspond to symmetrical location of fluorines in ortho and
meta positions on the phenyl ring, confirming that the cysteine thiol
was substituted at the para position. Photoisomerization of the S
reaction product 3 was characterized with UV-vis, F NMR, and H
NMR spectroscopies (Figure S5-S7).
N
1
Ar
19
We next explored whether 1 could be genetically incorporated into
proteins through the expansion of the genetic code with an orthogonal
tRNA/aminoacyl-tRNA synthetase pair to suppress the amber stop
codon TAG.13 We co-expressed in E. coli the orthogonal amber sup-
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Pyl
pressor tRNACUA–MmPSCaaRS pair with a calmodulin gene, which
encoded an amber TAG codon at position 76 for F-PSCaa incorpora-
tion (CaM_76TAG). The MmPSCaaRS is specific for Uaas containing
azobenzene and tolerant with different substituents at the meta posi-
or any other potential modification of 1 was detected, thus indicating
formation of the protein bridge through intramolecular S Ar crosslink-
ing in high efficiency. We note that the azobenzene bridge was formed
without any further treatment of the CaM protein following expression
and purification. The reaction described here between the F-PSCaa
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tion of the azo group. Because fluorine atoms do not significantly
change the bulk of the azobenzene, we thus expect the MmPSCaaRS
and cysteine represents the first S
N
Ar successfully employed for prox-
imity-enabled bioreactivity on proteins in live cells.7
,14
should also be able to incorporate 1. Indeed, F-PSCaa 1 was successful-
-
1
ly incorporated into CaM in E. coli to yield 1.0 mg L of CaM after
nickel-nitrilotriacetic acid (Ni-NTA) purification. In the absence of 1
in the culture, no full-length CaM was detected. Electrospray ioniza-
tion mass spectrometric (ESI-MS) analysis of the purified CaM protein
confirmed the incorporation of the intact 1 (Figure S8). An additional
peak corresponding to this CaM protein with glutathione (GSH)
added to 1 was also detected (Figure S8), indicating that 1 partly
reacted intermolecularly with endogenous GSH.
To determine whether the azobenzene bridge built onto CaM could
be directed by visible light, we detected the isomerization process of
the purified bridged CaM using UV-vis spectroscopy. Before light
illumination, the bridged CaM showed a strong absorbance at 336 nm
and a weak band at 445 nm, representing π-π* and n-π* transitions,
respectively (black line, Figure 4a). This pattern is characteristic of E
azobenzene. After green light illumination at λ = 540 nm, the absorb-
ance at 336 nm decreased substantially, indicating a clear E-to-Z pho-
toisomerization of the azobenzene bridge (red line, Figure 4a). Subse-
quent illumination with blue light resulted in Z-to-E photoisomeriza-
tion (blue line, Figure 4a) yielding the photostationary state (pss) of
the E state. Successive illumination with either green or blue light
allowed for reversible transformation between the two states without
showing fatigue of the azo bridge on the protein (Figure 4b). Moreo-
ver, photoisomerization of the azo bridge allowed conformational
changes of CaM to be driven reversibly as detected by circular dichro-
ism (CD). The recorded spectra are typical for α-helical CaM proteins
with a band at 222 nm and 208 nm, respectively (Figure 4c). Upon E-
to-Z photosiomerization the intensity of the n-π* transition band at
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08 nm decreased (red line, Figure 4c), with magnitude comparable to
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+
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that of wild-type CaM before and after Ca binding, suggesting a
clear conformational change of the CaM protein. Notably, the confor-
mation of the ground E state was restored by Z-to-E photoisomeriza-
tion using blue light subsequently (blue line, Figure 4c).
Figure 3. Incorporation of 1 into CaM forming an azobenzene bridge
with cysteine in situ. (a) SDS-PAGE stained with Coomassie shows
Pyl
that tRNACUA–MmPSCaaRS specifically incorporated 1 into CaM in E.
coli. Samples were normalized for constant cell numbers for each lane.
The arrow indicates the position of the full-length CaM protein. (b)
High resolution ESI-FTMS analysis of intact CaM expressed in the
To test whether the binding function of CaM could be photo-
modulated by the azo bridge, we measured the binding of CaM to the
CaM binding domain of the neuronal nitric oxide synthase (NOS-I).
Upon binding CaM changes its conformation and wraps around the
NOS-I peptide,16 which has been reliably monitored with fluorescence
Pyl
presence of tRNACUA–MmPSCaaRS supplemented with 1 mM of 1.
Average and monoisotopic (indicated by MI) masses are labeled. The
1
7
S
N
Ar crosslinking reaction of 1 with cysteine results in the loss of HF.
of dyes labeled onto CaM. We labeled the azo bridge-containing CaM
+
2+
CaM containing the covalent azobenzene bridge: [M-HF+H] , ex-
pected 18116.33 Da, measured 18116.31 Da; [M-Met-HF+H] , ex-
with dansyl chloride in the presence of Ca and recorded dansyl fluo-
+
rescence upon binding of the NOS-I peptide. After addition of NOS-I
peptide (10 µM) to the E form of the bridged CaM/Ca2+ (0.1 µM), the
fluorescence intensity markedly increased by 215% and the emission
peak slightly shifted to shorter wavelength (from 517 to 492 nm),
which is indicative of the NOS-I peptide binding to CaM (green line,
Figure 4d). In contrast, the change of fluorescence intensity upon
pected 17985.31 Da, measured 17985.28 Da. CaM with non-
+
crosslinked F-PSCaa and cysteine: [M+H] , expected 18136.32 Da,
+
not detected; [M-Met+H] , expected 18005.28 Da, not detected. The
peak at 18331.43 did not correspond to CaM with F-PSCaa modified
by any known potential intracellular reactants such as Cys, GSH or
imidazole. In addition, in control protein CaM(F-PSCaa at 76 and Ala
at 83), no mass peak corresponding to this 18331.43 peak was detected
2+
addition of NOS-I to the Z pss of CaM/Ca is much less (by 50%,
green line, Figure 4e), indicating that photoswitching the azo bridge to
Z form decreased CaM binding to the peptide. Illumination of the
CaM/Ca2+ in the absence of peptide neither influenced the emission
wavelength nor intensity (black and red line, Figure 4e).
In summary, we have developed and genetically encoded the pen-
tafluoro photoswitchable click amino acid F-PSCaa 1, which isomeriz-
es upon visible light and enables in situ formation of a visible light-
(
Figure S8), suggesting that the 18331.43 peak was not related to F-
PSCaa modification.
We then investigated whether the genetically encoded F-PSCaa 1
could react with a cysteine at an appropriate distance via proximity-
enabled bioreactivity to build an azo bridge on proteins in situ. We co-
Pyl
expressed in E. coli the tRNACUA–MmPSCaaRS pair with a calmodulin
controllable bridge onto proteins through intramolecular S Ar reaction
N
gene containing the TAG codon at position 76 and a cysteine at posi-
tion 83 ([Cys83]-CaM_76TAG). As expected, full-length CaM was
produced in the presence of 1 (Figure 3a). Analysis of the Ni-NTA
purified CaM using high resolution electrospray ionization Fourier
transform ion trap mass spectrometry (ESI-FTMS) confirmed the
incorporation of 1 and, more importantly, the formation of a protein
bridge through 1 reacting with Cys83 (Figure 3b). The mass peak
detected at 18116.31 Da corresponds to CaM bearing the covalent
azobenzene bridge; a second peak measured at 17985.28 Da corre-
sponds to CaM containing the covalent bridge and lacking the initiat-
ing Met. No peak corresponding to non-crosslinked F-PSCaa/cysteine
with a cysteine. The F-PSCaa and its bridge formed in situ represent
the first genetically encoded small molecule photoswitch that can be
modulated by visible light. In comparison with protein-based pho-
toswitches, 1 can be selectively incorporated into any site of protein in
vivo, providing greater flexibility in target site selection and expanding
the scope of proteins addressable by light. In addition, a light-
controllable bridge generated onto proteins in situ makes it possible to
optically modulate protein secondary structures and domains spanning
multiple residues in addition to a single residue. Moreover, responsive-
ness to visible light avoids negative effects induced by UV light and
makes 1 and its bridge more compatible with in vivo usage. We thus
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expect that F-PSCaa and its bridge will find broad applications in the
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emerging field of optobiology for its minimal interference, site-
specificity, genetic encodability, and response to the more biocompati-
ble visible light.
AUTHOR INFORMATION
Corresponding Author
Lei.Wang2@ucsf.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
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C.H. gratefully acknowledges a fellowship by the Deutsche For-
schungsgemeinschaft DFG (HO 4778/1-1) and the Pioneer Founda-
tion. S.C. acknowledges support from U.S. National Institute of Health
(
GM098630). L.W. acknowledges support from U.S. National Insti-
tutes of Health (1DP2OD004744).
REFERENCES
(
1) (a) Fenno, L.; Yizhar, O.; Deisseroth, K. Annu. Rev. Neurosci. 2011, 34,
89-412. (b) Bernstein, J. G.; Boyden, E. S. Trends Cogn. Sci. 2011, 15, 592-600.
c) Yazawa, M.; Sadaghiani, A. M.; Hsueh, B.; Dolmetsch, R. E. Nat. Biotechnol.
009, 27, 941-945. (d) Wu, Y. I.; Frey, D.; Lungu, O. I.; Jaehrig, A.; Schlichting,
3
(
2
I.; Kuhlman, B.; Hahn, K. M. Nature 2009, 461, 104-108. (e) Kennedy, M. J.;
Hughes, R. M.; Peteya, L. A.; Schwartz, J. W.; Ehlers, M. D.; Tucker, C. L. Nat
Methods 2010, 7, 973-975. (f) Levskaya, A.; Weiner, O. D.; Lim, W. A.; Voigt,
C. A. Nature 2009, 461, 997-1001. (g) Crefcoeur, R. P.; Yin, R.; Ulm, R.;
Halazonetis, T. D. Nat. Commun. 2013, 4, 1779. (h) Zhou, X. X.; Chung, H. K.;
Lam, A. J.; Lin, M. Z. Science 2012, 338, 810-814.
(2) (a) Szobota, S.; Isacoff, E. Y. Annu. Rev. Biophys. 2010, 39, 329-348. (b)
Fehrentz, T.; Schonberger, M.; Trauner, D. Angew. Chem. Int. Ed. Engl. 2011,
Figure 4. The azo bridge built on CaM controls CaM conformation and
binding in response to visible light. (a) UV-vis spectra of CaM bearing
the azobenzene bridge formed by F-PSCaa76 reacting with Cys83.
Green light illumination (λ = 540 nm, 5 min) resulted in E-to-Z pho-
toisomerization of the built-in azo bridge indicative by the substantial
decrease of the band at 336 nm (black line, before illumination; red
line, after green light illumination). Subsequent blue light illumination
(λ = 470 nm, 5 min) induced Z-to-E photoisomerization (blue line).
(b) Successive green and blue light illumination does not show fatigue
of the azo bridge on CaM. The first cycle was from the E form to the Z
pss state and thus had larger change than subsequent cycles switching
between the E pss and Z pss states. (c) Circular dichroism (CD) spec-
tra of the E form (black), the Z pss (red) and the E pss (blue) of CaM
bearing the azo bridge in PBS buffer (protein conc. 5 µM). After green
light activation to the Z pss, the band at 208 nm changed significantly
indicating conformational changes within CaM. The conformation of
the ground state can be recovered by blue light. (d) Binding of NOS-I
5
0, 12156-12182.
(3) (a) Schierling, B.; Noel, A. J.; Wende, W.; Hien le, T.; Volkov, E.;
Kubareva, E.; Oretskaya, T.; Kokkinidis, M.; Rompp, A.; Spengler, B.; Pingoud,
A. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1361-1366. (b) Zhang, F.; Zarrine-
Afsar, A.; Al-Abdul-Wahid, M. S.; Prosser, R. S.; Davidson, A. R.; Woolley, G. A.
J. Am. Chem. Soc. 2009, 131, 2283-2289.
(
4) Bose, M.; Groff, D.; Xie, J.; Brustad, E.; Schultz, P. G. J. Am. Chem. Soc.
2
006, 128, 388-389.
(5) Hoppmann, C.; Lacey, V. K.; Louie, G. V.; Wei, J.; Noel, J. P.; Wang, L.
Angew. Chem. Int. Ed. Engl. 2014, 53, 3932-3936.
(6) Hoppmann, C.; Schmieder, P.; Heinrich, N.; Beyermann, M.
ChemBioChem 2011, 12, 2555-2559.
(
7) Xiang, Z.; Ren, H.; Hu, Y. S.; Coin, I.; Wei, J.; Cang, H.; Wang, L. Nat.
Methods 2013, 10, 885-888.
(8) (a) Beharry, A. A.; Sadovski, O.; Woolley, G. A. J. Am. Chem. Soc. 2011,
133, 19684-19687. (b) Samanta, S.; Beharry, A. A.; Sadovski, O.; McCormick,
T. M.; Babalhavaeji, A.; Tropepe, V.; Woolley, G. A. J. Am. Chem. Soc. 2013,
135, 9777-9784. (c) Kienzler, M. A.; Reiner, A.; Trautman, E.; Yoo, S.; Trauner,
D.; Isacoff, E. Y. J. Am. Chem. Soc. 2013, 135, 17683-17686.
(9) Bleger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S. J. Am. Chem. Soc. 2012,
134, 20597-20600.
2+
peptide to the E state of CaM/Ca studied by fluorescence. Upon
addition of NOS-I to the E form (black line), the fluorescence intensity
significantly increased (green line, increased by 215%) indicative of
peptide binding. (e) CaM/Ca2+ in the E state (black line) was illumi-
(10) (a) Irie, M. Chem. Rev. 2000, 100, 1685-1716. (b) Matsui, M.;
Funabiki, K.; Shibata, K. Bull. Chem. Soc. Jpn. 2002, 75, 531-536.
nated with green light generating the Z pss state (red line). Subsequent
addition of NOS-I peptide to the Z pss state (green line, increased by
50%) showed less increase in intensity in comparison to the E state
(
11) Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y. S.; Pentelute, B. L.
J. Am. Chem. Soc. 2013, 135, 5946-5949.
12) Marsh, E. N.; Suzuki, Y. ACS Chem. Biol. 2014, 9, 1242-1250.
(13) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292,
498-500.
(
(
green line, Figure 4d). The data obtained suggest that the remaining E
form present in the Z pss state binds to the peptide resulting in the
slight increase of the intensity. The Z pss state of F-PSCaa 1 consists of
(14) (a) Xiang, Z.; Lacey, V. K.; Ren, H.; Xu, J.; Burban, D. J.; Jennings, P.
A.; Wang, L. Angew. Chem. Int. Ed. Engl. 2014, 53, 2190-2193. (b) Chen, X.-H.;
Xiang, Z.; Hu, Y. S.; Lacey, V. K.; Cang, H.; Wang, L. ACS Chem. Biol. 2014, 9,
1956-1961.
2
2% E form (Figure 1a).
ASSOCIATED CONTENT
Supporting Information
Synthesis of F-PSCaa 1, experimental details, additional H- and 19F-
NMR spectra, and Figures S1-S11 are provided as supporting infor-
mation. This material is available free of charge via the Internet at
(
15) Shifman, J. M.; Choi, M. H.; Mihalas, S.; Mayo, S. L.; Kennedy, M. B.
Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 13968-13973.
16) Valentine, K. G.; Ng, H. L.; Schneeweis, L.; Kranz, J. K.; Frederick, K.
(
1
K.; Alber, T.; Wand, A. J. 2006, PDB code 2O60
(17) (a) Censarek, P.; Beyermann, M.; Koch, K. W. Biochemistry 2002, 41,
8598-8604. (b) Spratt, D. E.; Taiakina, V.; Guillemette, J. G. Biochim. Biophys.
Acta 2007, 1774, 1351-1358.
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