This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 11985–11997 11985 Cite this: Phys. Chem. Chem. Phys., 2011, 13, 11985–11997 Fluorescence quantum yield and photochemistry of bacteriophytochrome constructsw K. C. Toh,z a Emina A. Stojkovic´,y b Ivo H. M. van Stokkum, a Keith Moffat bc and John T. M. Kennis* a Received 6th January 2011, Accepted 5th May 2011 DOI: 10.1039/c1cp00050k Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS–GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804–807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS–GAF proteins and their P3 D216A, Y272F and P2 D202A PAS–GAF–PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS–GAF proteins was significantly longer than in their PAS–GAF–PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS–GAF–PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4–1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers. Introduction Phytochromes are red-light sensing proteins found in plants, bacteria, cyanobacteria and fungi that act as photochromic switches activated by distinct wavelengths in the red or far red regions. 1–8 Their light-sensing module comprises PAS, GAF and PHY domains and covalently binds a linear tetrapyrrole, phycochromobilin (PFB) in plant phytochromes, phycocyano- bilin (PCB) in cyanobacterial phytochromes, and biliverdin (BV) in bacteriophytochromes (BPh). They exist as dimers in which interfaces between the monomers are formed both by the N-terminal photosensory core and the C-terminal kinase domain. 9 In the dark, most phytochromes adopt a red-absorbing state known as Pr and upon light absorption convert to a far-red absorbing state known as Pfr. The light activation mechanism involves an isomerization process about the C15 Q C16 double bond of the linear tetrapyrrole, changing its configuration from 15Za to 15Ea. 10–12 Light-induced Z/E isomerization of the tetrapyrrole takes place on a timescale of tens to hundreds of ps. 13–18 The reaction then proceeds through several inter- mediate, spectroscopically-distinct states such as Lumi-R before the Pfr state is established. 19–22 The recent determination of crystal structures of various BPhs, the cyanobacterial phytochrome Cph1 and the NMR structure of the single domain, GAF-only cyanobacterial a Biophysics Section, Department of Physics and Astronomy, VU University, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands. E-mail: [email protected]b Department of Biochemistry and Molecular Biology, The University of Chicago, USA c Institute for Biophysical Dynamics, The University of Chicago, USA w Electronic supplementary information (ESI) available. See DOI: 10.1039/c1cp00050k z Current address: Division of Molecular Biosciences, Imperial College, London, UK. y Current address: Department of Biology, Northeastern Illinois University, Chicago, IL, USA. PCCP Dynamic Article Links www.rsc.org/pccp PAPER Downloaded by VRIJE UNIVERSITEIT on 16 June 2011 Published on 25 May 2011 on http://pubs.rsc.org | doi:10.1039/C1CP00050K View Online
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This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 11985–11997 11985
Fluorescence quantum yield and photochemistry of bacteriophytochrome
constructsw
K. C. Toh,za Emina A. Stojkovic,yb Ivo H. M. van Stokkum,aKeith Moffat
bcand
John T. M. Kennis*a
Received 6th January 2011, Accepted 5th May 2011
DOI: 10.1039/c1cp00050k
Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that
consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV)
to a conserved cysteine in the PAS domain. In a recent development, PAS–GAF variants were
engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and
co-workers, Science, 2009, 324, 804–807). Here, we report the fluorescence quantum yield and
photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3)
with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to
wild type P3 and P2 PAS–GAF proteins and their P3 D216A, Y272F and P2 D202A
PAS–GAF–PHY mutant proteins. In these mutants hydrogen-bond interactions between a
conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are
disrupted. The excited-state lifetime of the truncated P3 and P2 PAS–GAF proteins was
significantly longer than in their PAS–GAF–PHY counterparts that constitute the full
photosensory core. Mutation of the conserved Asp to Ala in the PAS–GAF–PHY protein had a
similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants
were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien
and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp
and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all
Bph variants studied here. H/D exchange resulted in a 1.4–1.7 fold increase of excited-state
lifetime. The results support a reaction model in which deactivation of the BV chromophore
proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone
of the conserved Asp or to a bound water. This work may aid in rational structure- and
mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR,
deep tissue, fluorescent markers.
Introduction
Phytochromes are red-light sensing proteins found in plants,
bacteria, cyanobacteria and fungi that act as photochromic
switches activated by distinct wavelengths in the red or far red
regions.1–8 Their light-sensing module comprises PAS, GAF
and PHY domains and covalently binds a linear tetrapyrrole,
phycochromobilin (PFB) in plant phytochromes, phycocyano-
bilin (PCB) in cyanobacterial phytochromes, and biliverdin
(BV) in bacteriophytochromes (BPh). They exist as dimers in
which interfaces between the monomers are formed both by
the N-terminal photosensory core and the C-terminal kinase
domain.9 In the dark, most phytochromes adopt a red-absorbing
state known as Pr and upon light absorption convert to a far-red
absorbing state known as Pfr. The light activation mechanism
involves an isomerization process about the C15QC16 double
bond of the linear tetrapyrrole, changing its configuration
from 15Za to 15Ea.10–12 Light-induced Z/E isomerization of
the tetrapyrrole takes place on a timescale of tens to hundreds
of ps.13–18 The reaction then proceeds through several inter-
mediate, spectroscopically-distinct states such as Lumi-R
before the Pfr state is established.19–22
The recent determination of crystal structures of various
BPhs, the cyanobacterial phytochrome Cph1 and the NMR
structure of the single domain, GAF-only cyanobacterial
a Biophysics Section, Department of Physics and Astronomy,VU University, De Boelelaan 1081, 1081HV Amsterdam,The Netherlands. E-mail: [email protected]
bDepartment of Biochemistry and Molecular Biology,The University of Chicago, USA
c Institute for Biophysical Dynamics, The University of Chicago, USAw Electronic supplementary information (ESI) available. See DOI:10.1039/c1cp00050kz Current address: Division of Molecular Biosciences, ImperialCollege, London, UK.y Current address: Department of Biology, Northeastern IllinoisUniversity, Chicago, IL, USA.
11996 Phys. Chem. Chem. Phys., 2011, 13, 11985–11997 This journal is c the Owner Societies 2011
configuration is significantly affected by the amino acid
identity at position 216 (P3 numbering), as shown earlier for
Agrobacterium Agp1 phytochrome.20
Abbreviations
BPh bacteriophytochrome
BV biliverdin
PCB phycocyanobilin
PFB phycochromobilin
KIE kinetic isotope effect
EADS evolution-associated difference spectrum
DADS decay-associated difference spectrum
DAS decay-associated spectrum
ESPT excited-state proton transfer
APC allophycocyanine
Acknowledgements
K.C.T. and J.T.M.K. were supported by the Earth and Life
Sciences Council of the Netherlands Foundation for Scientific
Research (NWO-ALW) through a VIDI grant to J.T.M.K.
E.A.S and K.M. were supported by NIH grant GM036452
to K.M.
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Thus the DADS are linear combinations of the EADS, and vice versa. Thus, the lth
EADS is a linear combination of the lth and following DADS. In particular, the first
EADS, which corresponds to the time zero difference spectrum, is the sum of all DADS;
and the final EADS is proportional to the final DADS.
In systems where photophysical and photochemical processes occur the sequential model
with increasing lifetimes provides a convenient way to visualize the evolution of the
(excited and intermediate) states of the system. Therefore, the EADS are shown in the
main text and the corresponding DADS are shown in the Supporting Information.
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