SNAP-tag-reactive lipid anchors enable targeted and spatiotemporally controlled localization of proteins to phospholipid membranes

Article abstract of DOI:10.1021/jacs.5b00040

The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single

Full text of DOI:10.1021/jacs.5b00040

Communication
pubs.acs.org/JACS
SNAP-Tag-Reactive Lipid Anchors Enable Targeted and
Spatiotemporally Controlled Localization of Proteins to Phospholipid
Membranes
Andrew K. Rudd, Joan M. Valls Cuevas, and Neal K. Devaraj*
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States
S
* Supporting Information
membrane curvature, where efficient and localized protein
attachment is required to elicit specific responses.1,2 Further-
ABSTRACT: The natural mechanisms that direct pro-
teins to membranes are typically complex, requiring
multiple steps and accessory components. It would be
advantageous to develop simplified methods to direct
proteins of interest to phospholipid membranes in a single
step. Here we report a modular method for membrane
localization of proteins by using chemically modified
phospholipid anchors capable of covalent attachment to
O6-methylguanine DNA methyltransferase (SNAP-tag)
fusion proteins. To our knowledge, this is the first use of
SNAP-tag reactions to modify benzylguanine-function-
alized lipid membranes. We demonstrate that photocaged
lipid precursors enable light-triggered spatial and temporal
control over protein localization. The anchoring system is
compatible with cell-free expression, allowing for genetic
targeting of proteins to lipid membranes of giant
unilamellar vesicles. This technique can be used to control
membrane curvature effects, similar to what has been
previously observed with certain membrane-bound
proteins. This work addresses a current need in synthetic
biology for simplified and robust methods to control
membrane localization of expressed proteins and shows
promise as a general tool for protein targeting to lipid
vesicles and cellular membranes.
more, such tools would be capable of guiding in situ synthesized
proteins to membranes for synthetic biology applications. Here
we present a simplified approach to membrane localization that
results in the selective and covalent linkage of proteins to the lipid
bilayer. This approach utilizes benzylguanine (BG)-modified
lipid anchors and the well-established mutant O6-methylguanine-
DNA methyltransferase, SNAP-tag,15 to tether proteins to
phospholipid membranes. Additionally, it allows for photo-
activation of protein to defined vesicles or portions of a single
vesicle membrane with spatial resolution on the order of
micrometers. To our knowledge, this is the first demonstration of
the use of the SNAP-tag reaction to anchor proteins to BG-
functionalized phospholipid membranes.
SNAP-tag technology has seen increasing use in biomolecular
imaging due to its several attractive features, which include the
ability to access a broad range of bright and stable synthetic
fluorophores, the availability of fluorogenic probes, and the
relatively small size of the reactive protein (∼20 kDa).16−19 The
remarkable versatility of SNAP-tag has enabled numerous
applications beyond imaging, such as the detection of drug−
protein interactions,20 conjugation of biomolecules,21,22 prox-
imity ligation of oligonucleotides23 and immobilization of
protein on self-assembled monolayers.24 However, despite
these past results, SNAP-tags have not been explored for lipid
membrane modification. We thus set out to investigate whether
reactive lipid anchors on phospholipid membranes could capture
SNAP-tag proteins.
To determine if SNAP-tags could be used for lipid membrane
modification, we synthesized several SNAP-tag reactive BG lipid
anchors. Interestingly, initial experiments without a linker and
with a short 7-member poly(ethylene glycol) (PEG) linker
between the BG and lipid headgroup failed to promote
localization of SNAP-tag to model phospholipid membranes
(compounds 7 and 9, Supporting Information). Reasoning that
the phospholipid membrane-bound BG group may have
hindered access to the active site of the SNAP-tag protein,25
we chose to explore longer and more flexible linkers and found
that 17- and 45-member PEG linkers (DOPE-PEG17-BG and
DSPE-PEG45-BG) were able to maintain reactivity of
membrane-bound BG with SNAP-tag proteins (Figure S1). To
confirm the covalent reactivity of DSPE-PEG45-BG, the lipid
embrane-bound proteins are critical for regulating
M
signaling, transport, binding interactions, and curva-
ture.1−3 Thus, controlled localization of proteins to discrete
regions of the cellular membrane is essential for all organisms.
Cells accomplish this task through several transport and
lipidation pathways, often requiring enzymes specific to the
target protein.4,5 Current techniques for incorporating mem-
brane proteins into phospholipid membranes involve the growth
of isolated cellular membranes6 or require the use of
proteoliposomes and detergent,7,8 making in situ protein
localization difficult. Approaches to facilitate membrane local-
ization of proteins of interest include the use of transmembrane
domain and isoprenylation sequence fusions,9−11 membrane-
localizing ligands,12 optogenetics,13 or supramolecular inter-
actions.14
These techniques, while useful, have several drawbacks,
including a lack of covalent membrane attachment and the
inability to control interactions in a spatiotemporal manner. A
tool that overcomes these hurdles would allow for controlled
studies on topics such as protein signaling and induced
Received: July 25, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b00040
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Journal of the American Chemical Society
Communication
was incubated with a purified SNAP-tag protein at 37 °C
overnight. SDS-PAGE gel analysis of the completed reaction
showed an expected increase in protein mass, indicating covalent
linkage of SNAP-tag and the lipid anchor (Figure S2). The
sensitivity of the membrane reaction to linker structure offers a
possible explanation for why past attempts to extend SNAP-tags
to phospholipid membrane modification have not met with
success.26 PEG linkers are an attractive solution to this problem
because they increase the flexibility of the reactive substrate while
still maintaining a short end-to-end distance. Modeling experi-
ments suggest that 18-member poly(ethylene oxide) molecules,
very similar in length and structure to the linker region of DOPE-
PEG17-BG, have an average length of 25.3 Å.27
serve as a valuable tool for studying the effect of lipid domains on
the function and structure of embedded proteins.
A major advantage of using SNAP-tag reactive lipids is the
potential to spatiotemporally control membrane protein
modification. Elegant work has established that photocaged
alkylguanosines can be activated with light to trigger binding with
SNAP-tagged proteins.31 With this in mind, we explored the
feasibility of light-activated protein immobilization using a
photocaged lipid anchor (DSPE-PEG45-BG-NPE) (Figure
1A). Kinetic studies showed rapid uncaging of the lipid in
vesicles (t1/2 = 80 s) upon irradiation with 360 nm light. To test
the reactivity of the DSPE-PEG45-BG-NPE vesicles to SNAP-
tag, DOPC GUVs were synthesized containing 5 mol % DSPE-
PEG45-BG-NPE. Obtained vesicles were irradiated with 360 nm
light for 10 min and then incubated with AF488-labeled SNAP-
tag protein for 1 h (see Supporting Information for full
experimental details). The UV activated vesicles showed marked
localization of protein to the membrane compared to non-
activated vesicles, which lacked any discernible binding even after
2 h of incubation with SNAP-tag proteins (Figure S5). An
exciting feature of this technique is its compatibility with laser
scanning confocal microscopy. By controlling the region of light
exposure with a 405 nm diode laser, we were able to activate a
selected vesicle of a population without triggering protein
localization in neighboring vesicles. Activation was rapidly
accomplished using 4.5 s of light exposure. After deprotection,
SNAP-tag protein localization occurred within minutes (Figure
2B). We next synthesized DPPC GUVs containing 5 mol %
DSPE-PEG45-BG-NPE using a solvent evaporation method.32
By employing DPPC (Tm = 41 °C) as the main component of the
phospholipid membrane, we were able to produce vesicles
possessing a gel phase membrane at ambient temperature.
Because the vesicle membranes are in the gel phase, lateral
diffusion of constituent phospholipids is extremely slow, thus
preventing lipid anchors from translating throughout the
membrane.33 This characteristic allowed us to photo-uncage
lipid anchors on only a portion of the membrane. Vesicles were
incubated with 230 μM SNAP-tag GFP and then irradiated for 2
s on one side of a vesicle membrane. After exposure, protein
localization was observed within 90 s and after 10 min no further
binding was evident. This unique capability may allow for the
controlled decoration of membranes with several different fusion
proteins at specific locations on a vesicle, an area that is of interest
for drug delivery applications.34,35
Recently there has been increasing interest in the use of vesicle
encapsulated cell free expression systems as cell mimics.
Applications include fundamental studies on the essential
elements for cellular function, minimal cells for synthetic
biology, and drug delivery.36,37 Seminal work in this area has
demonstrated the efficient expression of protein in GUVs that
encapsulate either E. coli lysate or the recombinant PURE
system.10,38 A challenge in this area has been to target in situ-
synthesized proteins to the GUV membrane. Having this
capability is essential for the reconstitution of natural membrane
protein function and the design of new artificial systems in self-
contained minimal cells. While there have been elegant
demonstrations using spontaneous incorporation of certain
integral membrane proteins,12 and reconstitution of the Sec
translocon to create lipid/protein membranes,39 the use of
SNAP-tag reactive lipids could offer a simple method to
genetically target a much wider range of proteins to the
surrounding lipid bilayer (Figure 3A). We therefore examined
whether DSPE-PEG45-BG lipids could be utilized in con-
To investigate the utility of these anchors for phospholipid
membrane modification (Figure 1B), dioleoylphosphatidyl-
Figure 1. (A) DSPE-PEG lipid anchor with benzylguanine (BG) and
photocaged benzylguanine (BG-NPE) head groups. (B) Proposed
reaction of SNAP-tag protein with a membrane embedded DSPE-PEG
lipid-anchored BG substrate. Transfer of the benzyl group to the active
site of the SNAP-tag results in covalent attachment of the protein to the
phospholipid membrane. (C) Visualizing membrane localization of a
fluorescently tagged (AF-488) SNAP-tag protein on a giant unilamellar
vesicle. The vesicle was formed with 5 mol % DSPE-PEG45-BG lipid
anchors, which captures the SNAP-tag protein (scale bar 10 μm).
choline (DOPC) giant unilamellar vesicles (GUVs) containing
5 mol % DSPE-PEG45-BG were formed by adapting a previously
published inverse emulsion technique.28 Formed vesicles were
incubated with 3 μM Alexa Fluor 488 (AF488) dye-labeled
SNAP-tag for 1 h at 37 °C. Fluorescent microscopy showed
excellent membrane localization of SNAP-tag to DSPE-PEG45-
BG containing membranes (Figure 1C). To confirm that the
localization we observed was due to reaction with the lipid
anchor and not other nonspecific interactions, we incubated
AF488-labeled bovine serum albumin with DSPE-PEG45-BG
vesicles and observed no membrane localization. Additionally,
AF488-SNAP-tag incubated with DOPC vesicles lacking the
reactive lipid anchor showed no localization to the membrane,
indicating that the fluorescent SNAP-tag protein does not bind
due to inherent phospholipid membrane affinity (Figure S3).
Having determined that DSPE-PEG45-BG is capable of
reacting with SNAP-tag protein, we next explored whether the
lipids could be used to target specific microdomains formed on
mixed phospholipid vesicles. Previous studies have shown that
mixtures of saturated and unsaturated phospholipids, along with
cholesterol, can produce vesicles with two distinct lipid
phases.29,30 Using a 1:1:1 mixture of DOPC:distearoylphospha-
tidylcholine (DSPC):cholesterol (Chol) with 5 mol % DSPE-
PEG45-BG, we electroformed GUVs with two lipid phases. After
introduction of AF488-labeled SNAP-tag, specific labeling of the
less ordered lipid domains occurred, evidenced by colocalization
with the fluorescent lipid Texas Red DHPE (Figure S4 and
Movie M1). These results suggest that BG-modified lipids could
B
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Communication
emulsion method. After protein expression at 37 °C for 1 h,
vesicles containing lipid anchor showed excellent localization of
expressed fusion protein to the vesicle membrane (Figure 3B).
Control experiments with vesicles lacking reactive lipid or using a
control GFP resulted in no localization of membrane protein.
This result demonstrates the ability of the SNAP-tag reactive
lipid system to effectively emulate cellular protein trafficking and
allow for genetically targeted control over membrane local-
ization.
Having demonstrated the wide range of capabilities and
control offered by SNAP-tag reactive lipid anchors, we wished to
explore if this technique could be used to recreate membrane
effects caused by natural phospholipid-bound proteins. Recently
it has been shown that some natural membrane-bound proteins
such as epsin1 are able to induce membrane curvature changes
due to crowding effects alone and that even tethering GFP to the
membrane at high concentration can result in membrane
tubulation. We therefore reasoned that our membrane anchor
could be used to induce membrane curvature via crowding by
tethering protein near the membrane in high concentration.40
Electroformed vesicles possessing disordered microdomains
were synthesized containing 5 mol % DSPE-PEG45-BG. Upon
incubation with 185 μM SNAP-GFP, membrane tubulation and
deformation was observed within 90 s (Figure 4 and Movie M2),
Figure 2. (A) Exposure of DSPE-PEG-BG-NPE containing vesicles to a
405 nm laser results in the loss of the NPE caging group and
spatiotemporally activates binding of SNAP-tag proteins to the
membrane. (B) Left: 20:1 DOPC:DSPE-PEG-BG-NPE vesicles
incubated with dye (AF488)-labeled SNAP-tag before photoactivation
(scale bar 30 μm).: A single vesicle of the population was irradiated with
the 405 laser line for 4.5 s (white box). Right: After 10 min, protein has
bound specifically to the uncaged vesicle. (C) Left: 20:1 DPPC:DSPE-
PEG45-BG-NPE vesicles incubated with SNAP-tag GFP fusion before
photoactivation (scale bar 2 μm). A portion of the vesicle was exposed to
the 405 laser line (white arrow) for 2 s. Right: after 10 min protein
binding is observed only on the selected portion of the gel phase
membrane.
Figure 4. Introduction of an excess of SNAP-tag GFP fusion protein to
microdomain containing GUVs results in significant membrane
deformation. Images show a representative GUV, labeled with 0.1 mol
% Texas Red DHPE, undergoing tubulation and blebbing events on the
minute time scale. Time noted in seconds after introduction of protein
(scale bar 5 μm).
Figure 3. (A) Expression by E. coli lysate encapsulated in 5 mol % DSPE-
PEG-BG vesicles leads to subsequent membrane localization of SNAP-
tag GFP fusion protein. (B) GUVs expressing fluorescent protein (green
channel, bottom row). Left: DOPC GUVs with no DSPE-PEG-BG
expressing SNAP-tag GFP fusion (scale bar 15 μm). Middle: 5 mol %
DSPE-PEG45-BG vesicles expressing GFP (scale bar 10 μm). Right: 5
mol % DSPE-PEG45-BG vesicles expressing SNAP-tag GFP fusion
(scale bar 25 μm).
while controls lacking membrane anchor displayed no noticeable
change in membrane morphology (Figure S6). Membrane
curvature phenomenon could also be observed with lower
concentrations of SNAP-GFP, down to 10 μM (Figure S7).
These results demonstrate that the protein is anchored close
enough to the membrane to allow for collective protein behavior
to be transmitted to the lipid anchor and result in a dramatic
change in membrane curvature.
While this technology offers several opportunities for
modifying model phospholipid membranes, there is also
tremendous interest in directing proteins to living cellular
membranes for applications ranging from live cell imaging, drug
junction with cell free expression systems to genetically trigger
membrane localization of a SNAP-tag fusion protein. Plasmid
encoding for a SNAP-tag GFP fusion protein and an S30 T7
protein expression system were encapsulated in GUVs
containing 5 mol % DSPE-PEG45-BG using a modified inverse
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Journal of the American Chemical Society
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* Supporting Information
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Supplementary data, experimental procedures, and character-
ization data; Movie M1, showing localization of AF488-labeled
SNAP-tag to DSPE-PEG-BG-containing membrane micro-
domains; Movie M2, showing membrane deformation upon
introduction of a high concentration of SNAP-tag GFP fusion
protein to microdomain containing GUVs. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*ndevaraj@ucsd.edu
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors gratefully acknowledge helpful discussions with
Prof. Simpson Joseph. This work was supported by the U.S.
Department of Defense Army Research Office contract
W911NF-13-1-0383.
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