872.10/MMM38
Engineering opsins through computational, structure-guided protein
recombination to develop light-based sensors and actuators (Opsineering)
Claire N. Bedbrook1, Ken Y. Chan1, Nicholas C. Flytzanis1, Cheng Xiao1, Frances H. Arnold2, Viviana Gradinaru1
1. Dept. of Biology & Bioengineering, 2. Dept. of Chemistry and Chemical Engineering, California Institute of Technology,
Correspondence addressed to V.G. at viviana@caltech.edu
XFPs vs Non-functional Opsins as Controls for
Optogenetics
Opsin Diversification: Structure Based Recombination
Structure guided protein engineering enables
recombination of diversity from multiple opsin
parents. Parent proteins are divided into structural
blocks. Blocks are designed to minimize structural
disruption upon recombination. Machine learning
applied to data from a sample set allows prediction of
improved sequences. (left) Process of contiguous
recombination block design for Channelrhodopsin.
HEK:pLenti-mcherry
HEK::pLenti-ChR2
1. Structure
3. Library Design
2. Contact Map
ChR Recombination Library Construction
Rhodopsin Mechanism for Light Sensitivity
Re nal
O
p
s
in
hv
N
H
All-trans re nal
K to X
CamKIIa
Opsin
XFP
TS
Decreased light-induced
desensitization
Expression Localization of the Chimeras
0.5
Neuron:Chi3
20
40
60
Distance [pixels]
80
NpHR COp
1
1. High-throughput membrane localization assay by fluorescently
tagging only membrane localized opsin.
0.5
HEK:ArchT3.0
0
0
GFP-Binder-Tag-C1C2-mCherry
Acknowledgements
We thank the entire Gradinaru and Arnold labs for helpful discussions. We are grateful to
be supported in our research by awards from: Human Frontiers in Science Program, the
Beckman Institute, the Mallinckrodt Foundation, the Gordon and Betty Moore Foundation, the Pew
Charitable Trust, the Michael J. Fox Foundation, and the NIH / NINDS New Innovator (V.G.); the NIH
training grant (C.N.B., N.C.F & K.Y.C.)
0
-0.4
-0.8
Green light
0.5
0
Chi8
450
550
Wavelength [nm]
650
12
10
8
6
4
2
0
Chi10
Chi13
1800
1200
0
80
60
20
τdes
30
40
COp versions of ChR2, C1C2, Arch and eNpHR show very similar
membrane localization in both HEK cells and neurons. Whole cell
recordings of C1C2 (n = 9), C1C2 COp (n = 9), ChR2 (n=9), ChR2 Cop
(n=9), eNpHR (n=5) and eNpHR COp (n=9) shows significant
differences in light activated current produced by active vs COps.
C1C2(COp) and ChR2(COp) are activated with blue light, and
eNpHR(COp) is activated by green light. Scale bars 100 pA, 100 ms.
1.Initial work has been done to develop high-throughput assays for screening directed evolution libraries
2.Structure based recombination using SCHEMA results in a library of chimeras with a distribution of expression
localization and activation characteristics. Further work needs to be done to completely characterize the library
to make predictions of optimal chimeras.
3.We have built Control Opsins (COps) for the opsin tools most commonly used in optogenetics research (ChR2,
ReaCh, Arch3.0T, Arch3.0 and eNpHR). The COps appear to better mimic the membrane localization and
expression level of the active opsin than the currently used controls (FXPs) as tested in neuronal culture.
40
20
0
10
Neuron:ArchT3.0COp
COps current & recording noise
Conclusions
100
0
Neuron:ArchT3.0
-600
600
Ch
R2
Ch
i1
Ch
i2
Ch
i3
Ch
i4
Ch
i5
Ch
i
Ch 8
i1
Ch 0
i1
3
Cyan Light
ChR2
Chi2
Chi5
1
350
Chi4
Chi4
Ch
R2
Ch
i1
Ch
i2
Ch
i3
Ch
i4
Ch
i5
Ch
i
Ch 8
i1
Ch 0
i1
3
GCaMP6s
Ca2+ = 2 mM
(B) Blue light fluorescence taken of cells in
buffer with no extracellular Ca2+, and (C) cells
imaged 2 seconds after the addition of Ca2+ for
a final concentration of 2 mM Ca2+. Scale bar
20 μm.
τdes
Chi3
τoff [ms]
GCaMP6s
Ca2+ = 0 mM
C
sensors
Ipeak
τoff
Chi2
Peak and Ssteady state
[pA]
2. Medium-throughput opsin activity assay using
ISS
Time to peak [ms]
Green fluorescence =
Membrane localized C1C2
expression
Chi1
Photocurrent [norm]
ChR2
Ca2+
-0.42
0.4
80
Electrophysiology of the Chimeras
Binder-GFP
B
20
40
60
Distance [pixels]
-200
-400
HEK:ArchT3.0-COp
-0.14
-0.33
-1.2
Tag-C1C2-mCherry
A
Neuron:C1C2
-0.32
Noise
0
HEK:Chi13
0
C1C2 COp
eNpHR
Intensity
1
Noise
200
0
HEK:Chi4
HEK:hChR2-COp
Blue light
Intensity
CHIMERAS
PARENTS
HEK:VChR1
plenti-CamKIIa-eArch-YFP COp
HEK:hChR2
Neuron:ChR2
HEK:Chi2
HEK:Chi1
plenti-CamKIIa-ReaCh-mcherry COp
ChR2
HEK:ChR2
plenti-CamKIIa-eArchT-YFP COp
ER export
TS
Ca2+
plenti-CamKIIa-C1C2-mcherry COp
Localization & Electrophysiology of the COps
WPRE
mcherry
plenti-CamKIIa-eNpHR-YFP COp
NpHR
NpHR COp
Chimera
Inhibitory
C1C2
CamKIIa
ER export
plenti-CamKIIa-hChR-mcherry COp
O
p
s
in
To eliminate different rhodopsin’s light
sensitivity we targeted the Lysine (K) residue
that covalently binds retinal. We built a
number of different point mutants to alter the
lysine residue. The mutants were then screened
and selected based on how well they mimic the
wild-type expression level and expression
localization properties.
WPRE
ChR2
ChR2 COp
Increased ion current
Increased ion-selectivity
Red fluorescence =
Total C1C2 expression
13-cis re nal
Construction of Control Opsins (COps)
Excitatory
Assay Development for Directed Evolution of
ChRs
Lysine of C1C2 covalently
binds retinal. Retinal is
responsible for rhodopsin’s
light sensitivity.
N
H
K+
Na+ Ca2+
Lysine
Structure based recombination library of ChR2, VChR1 and ChR1. The ChR structure was broken into 8
blocks using SCHEMA design. Single ‘block-swaps’ into the ChR2 backbone resulted in a set of 16
recombination variants for the initial test-set. We have cloned these chimeras and started to characterize
their different properties.
Shifted activation wave-length
Protein
Engineering
HEK:pLenti-ChR2-COp
C1C2
C1C2 COp
Optogenetics is genetically encoded, optically induced, control of cells through transgenic
expression of microbial opsins in electrically excitable cells. When opsins are expressed in
a cell-type specific manner and light-activated, they provide separated stimulation or
inhibition to neurons in living animals. The optogenetics tools currently available operate
on the order of milliseconds, a time scale relevant to neuronal activity, and can be
expressed in the membrane of distinct cell-types with high temporal precision in welldefined brain regions. These tools significantly advanced our understanding of neuronal
circuits underlying various animal behaviors. To facilitate neuronal circuit interrogation,
we aim to advance the repertoire of current optogenetic tools, with focus on diversifying
light-wavelength selectivity, activation kinetics and ion specificity. We have addresses
these limitations through directed evolution and structure-guided protein recombination.
We have developed two high-throughput screens. One screen focuses on selection of
channelrhodopsins (ChR) based on membrane localization and the second screen is based
on their Ca2+. We also designed a recombination library from three ChR parents: two
distinct opsins from Clamydomonas reinhardtii (ChR1 & ChR2) and one opsin from
Volvox carteri (VChR1). In this design the three parents are divided into eight structural
blocks, which are then recombined to build novel chimeric ChRs. In a proof-of-concept,
sixteen of these chimeras have been built and show robust membrane expression in
mammalian cells (HEK and cultured hippocampal pyramidal neurons). We used
electrophysiology to characterize in detail each of the sixteen chimeras for ion selectivity,
activation kinetics, reversibility, and shifted spectral sensitivity. Four of the chimeras have
been assayed and showed significantly different responses to various colors of light. These
new channel proteins will have applications for probing the brain’s circuitry to better
understand and model healthy and non-healthy brain function as a foundation for
controlling and diagnosing neurological disorders.
Current [pA]
Abstract
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