Lipid bilayer nanodisc platform for investigating
polyprenol-dependent enzyme interactions and activities
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Citation
Hartley, M. D., P. E. Schneggenburger, and B. Imperiali. “Lipid
Bilayer Nanodisc Platform for Investigating PolyprenolDependent Enzyme Interactions and Activities.” Proceedings of
the National Academy of Sciences 110, no. 52 (December 24,
2013): 20863–20870.
As Published
http://dx.doi.org/10.1073/pnas.1320852110
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Thu May 26 02:43:06 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/89087
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INAUGURAL ARTICLE
Lipid bilayer nanodisc platform for investigating
polyprenol-dependent enzyme interactions
and activities
Meredith D. Hartley1, Philipp E. Schneggenburger1, and Barbara Imperiali2
Department of Biology and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2010.
Membrane-bound polyprenol-dependent pathways are important
for the assembly of essential glycoconjugates in all domains of life.
However, despite their prevalence, the functional significance of
the extended linear polyprenyl groups in the interactions of the
glycan substrates, the biosynthetic enzymes that act upon them,
and the membrane bilayer in which they are embedded remains
a mystery. These interactions are investigated simultaneously and
uniquely through application of the nanodisc membrane technology. The Campylobacter jejuni N-linked glycosylation pathway has
been chosen as a model pathway in which all of the enzymes and
substrates are biochemically accessible. We present the functional
reconstitution of two enzymes responsible for the early membrane-committed steps in glycan assembly. Protein stoichiometry
analysis, fluorescence-based approaches, and biochemical activity
assays are used to demonstrate the colocalization of the two
enzymes in nanodiscs. Isotopic labeling of the substrates reveals
that undecaprenyl-phosphate is coincorporated into discs with the
two enzymes, and furthermore, that both enzymes are functionally reconstituted and can sequentially convert the coembedded
undecaprenyl-phosphate into undecaprenyl-diphosphate-linked disaccharide. These studies provide a proof-of-concept demonstrating
that the nanodisc model membrane system represents a promising
experimental platform for analyzing the multifaceted interactions
among the enzymes involved in polyprenol-dependent glycan assembly pathways, the membrane-associated substrates, and the
lipid bilayer. The stage is now set for exploration of the roles of
the conserved polyprenols in promoting protein–protein interactions
among pathway enzymes and processing of substrates through sequential steps in membrane-associated glycan assembly.
icance, particularly because extended polyprenols have no other
known role in cells (2, 4, 5).
The discovery (8) and biochemical investigation (9–11) of
N-linked protein glycosylation in the Gram-negative bacterium
Campylobacter jejuni has revealed a canonical polyprenol-dependent
glycan assembly pathway. The C. jejuni protein glycosylation (pgl)
pathway is an appealing subject for in-depth analysis because the
component enzymes can be heterologously expressed and purified
in good yields, and can be subjected to protein engineering
approaches for the introduction of tags and labels. As illustrated in
Fig. 1A, in the pgl pathway, a heptasaccharide is assembled onto
undecaprenyl-diphosphate (Und-P-P) by the sequential action of
five membrane proteins, designated as PglC, PglA, PglJ, PglH,
and PglI. Of these proteins, PglC and PglI are predicted to be
integral membrane proteins (12), whereas the other glycan assembly
enzymes (PglA, PglJ, and PglH) lack discrete transmembrane
domains (TMDs) and are peripheral membrane proteins. Both
the integral and peripheral membrane Pgl proteins form insoluble aggregates in the absence of detergent, which further
supports a model wherein these enzymes are recruited to the
membrane to collaborate in the sequential glycan assembly process. After biosynthesis of the undecaprenyl-diphosphate heptasaccharide is complete, a flippase (PglK) translocates the assembled
product to the periplasmic face of the inner membrane (13), where
the oligosaccharyltransferase (PglB) transfers the assembled glycan
to asparagine residues in selected acceptor proteins (10, 14). After
translocation through the outer membrane, glycosylated proteins
Significance
Linear polyprenols are recurring molecular components in the
biosynthetic pathways responsible for the assembly of essential glycoconjugates, including peptidoglycan and N-linked
glycoproteins. Despite their highly conserved presence in all
domains of life, the role of the extended linear polyprenyl
groups in the dynamics of membrane-bound glycan assembly
pathways remains a mystery. Here we apply the nanodisc
model membrane platform to simultaneously assess the interactions and activities of the polyprenyl-linked substrates,
enzymes, and lipid bilayer by investigating initial steps from
the Campylobacter jejuni N-linked glycosylation pathway. This
work represents a proof-of-concept demonstrating that
nanodiscs can be used for the precise manipulation and study
of polyprenol-dependent pathways.
C
ellular membranes accommodate abundant biological activities, including the transport of small molecules and proteins,
energy production, and multistep biosynthetic transformations.
These functions are crucial for cell viability; however, studying
these processes in biophysical and biochemical detail is challenging because of the complexity of working with both integral
and peripheral membrane proteins in lipid bilayer systems. An
important class of membrane-associated pathways involves the
assembly of complex glycoconjugates, which is dependent on
extended linear polyprenols (1–4). The products of these pathways are essential for cellular viability in all domains of life and,
intriguingly, the polyprenols that are used in glycan assembly
vary between organisms, with considerable differences in the
overall length and degrees of unsaturation (2, 5). For example,
bacteria generate peptidoglycan components on the membraneanchored undecaprenyl-diphosphate before cell wall assembly
(6), whereas eukaryotes produce glycans for N-linked protein
glycosylation via dolichyl-diphosphate–linked intermediates, which
feature polyprenols ranging from 14–25 isoprene units (1, 2, 7).
Despite the ubiquitous presence of linear polyprenols as glycan
carriers in these important biosynthetic pathways, it remains unclear why these structures have been so faithfully conserved
throughout evolution and what might be their functional signifwww.pnas.org/cgi/doi/10.1073/pnas.1320852110
Author contributions: M.D.H., P.E.S., and B.I. designed research; M.D.H. and P.E.S. performed research; M.D.H. and P.E.S. contributed new reagents/analytic tools; M.D.H., P.E.S.,
and B.I. analyzed data; and M.D.H., P.E.S., and B.I. wrote the paper.
The authors declare no conflict of interest.
See Profile, 10.1073/pnas.1321020110.
1
M.D.H. and P.E.S. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: imper@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1320852110/-/DCSupplemental.
PNAS Early Edition | 1 of 8
BIOCHEMISTRY
Contributed by Barbara Imperiali, November 5, 2013 (sent for review September 13, 2013)
H
J
PP
A
PP
C
PP
P
A
I
K
PP
B
P
PP
B
PglC
PglA
Fig. 1. Canonical reaction pathway of bacterial N-linked glycosylation in
C. jejuni. (A) Scheme of the membrane-bound enzymatic conversions that
produce the polyprenyl-diphosphate–linked glycan, which is used in protein
glycosylation. (B) The reactions of the first two enzymes in the C. jejuni
pathway, PglC and PglA, are shown.
are displayed on the cellular surface and appear to be involved in
adhesion, colonization, and host-cell invasion (15).
One of the major hurdles in deciphering the discrete biophysical and biochemical roles of polyprenols in membraneassociated pathways is implementing methods to simultaneously
investigate all three components involved in polyprenol action:
the lipid bilayer, membrane-associated proteins, and polyprenyllinked substrates. Prior studies on polyprenol-dependent pathways have principally focused on redacted experimental systems
representing two of the three key variables. For example, the
effect of varying the substrate polyprenol groups has been studied
using glycan assembly enzymes that have been solubilized from
the native lipid bilayer with detergent (16–18); and interactions
of polyprenyl-linked compounds with model membrane systems,
such as liposomes, in the absence of membrane-bound proteins
have been investigated using biophysical techniques, including
computational modeling and NMR spectroscopy (19–24).
To gain insight into the interactions among the C. jejuni glycan
assembly pathway enzymes and the specific roles of the polyprenyl-linked substrates, an ideal model system would allow for
reconstitution of the entire biosynthetic pathway in a native membrane environment. Importantly, the system should be flexible
enough to permit structure/activity studies of enzymes with different membrane components and polyprenyl-linked substrates
in a systematic manner. In addition, the system should be amenable to detailed biophysical analysis without the constraints
imposed by the application of detergent micelles or polydisperse
liposome structures.
With these parameters in mind, we have turned to nanodiscs,
a valuable and emerging membrane mimetic technology introduced by the Sligar laboratory (25). In nanodiscs, a membrane
scaffold protein (MSP), derived from the cholesterol transport
protein apolipoprotein A1 (ApoA-1), was engineered to produce
stable self-assembled structures. Two MSPs form a lariat, which
solubilizes the hydrophobic edge of a flat bilayer of lipids to
afford soluble assemblies with diameters ranging from 7 nm to
12 nm. Larger nanodiscs, with diameters ranging from 16–25 nm,
2 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1320852110
have also been prepared (26), although the current versions of
these discs have been shown to exhibit greater polydispersity. A
variety of synthetic lipids have been used for nanodisc preparation, including variants of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), as well as
Escherichia coli lipid extracts (25, 27–29). Diverse membrane
proteins have been incorporated into nanodiscs and studies on
these systems have revealed fascinating nuances of protein structure, protein oligomeric state, protein–protein interactions, substrate binding, and enzyme activity (30–36). In addition, particularly
noteworthy for the current study, the effects of lipid composition
on enzymes have been probed in a highly reproducible manner
relative to other membrane mimetic systems (37, 38).
To address the fundamental challenge of defining the physical
and biochemical roles of polyprenols in membrane-associated
pathways, we present the development and validation of an
experimental strategy to reconstitute the first two membranecommitted steps of the N-linked glycosylation pathway of C. jejuni
(9, 11) in membrane bilayer nanodiscs (28). The approach in this
study renders the simultaneous and controlled manipulation of
all three determining elements in polyprenol-dependent glycan
assembly at the membrane bilayer a tangible objective.
Results
Incorporation and Characterization of PglC and PglA in Membrane
Bilayer Nanodiscs. PglC is a phosphoglycosyltransferase contain-
ing one predicted N-terminal TMD (11). PglC catalyzes the
transfer of phospho-N,N′-diacetylbacillosamine from UDP-N,
N′-diacetylbacillosamine (UDP-diNAcBac) to undecaprenylphosphate (Und-P) (Fig. 1B). For these studies a GB1 (B1
domain of Ig binding protein G)–PglC fusion construct was
engineered in which a His6-tag was inserted N-terminal to the
GB1 domain (39). The GB1 domain vastly improved the expression and purification of PglC, which was critical for these
studies. The His6-GB1-PglC construct expresses well in E. coli (2–
5 mg/L cell culture) and can be solubilized from membrane lipids
with detergent and purified using Ni-NTA affinity chromatography for reconstitution in the nanodisc system (SI Text).
PglA is a glycosyltransferase that catalyzes the transfer of GalNAc
from UDP-GalNAc to form Und-P-P-diNAcBac-GalNAc (9) (Fig.
1B). Although there are no predicted TMDs for PglA (12), sizeexclusion chromatographic (SEC) analysis of the His8-tobacco etch
virus (TEV)-PglA construct in buffer without detergent reveals
that the protein aggregates. Based on this behavior and the fact
that PglA interacts with a membrane-bound substrate (Und-PPdiNAcBac), PglA is designated as a peripheral membrane protein.
Well-behaved monodisperse preparations of PglA can be achieved
in the presence of β-D-dodecylmaltoside at 0.05% (wt/vol). Typical
yields of PglA after purification and TEV protease treatment for
His8-tag removal range from 2 to 4 mg/L cell culture (SI Text).
Nanodisc preparation was carried out following literature
protocols (29) and is illustrated in Fig. 2A for the preparation of
nanodiscs involving PglC (PglC-NDs). A polar E. coli lipid extract with a ratio PE/PG/cardiolipin of 67:23:10 (wt %) was used
because the phospholipid composition of E. coli is expected to be
similar to that of C. jejuni, as both are Gram-negative bacteria.
The MSP1E3 variant used in these studies forms nanodiscs with
a Stokes hydrodynamic diameter of 12.1 nm, as measured previously by SEC, or a diameter of 12.9 nm, as measured by smallangle X-ray scattering (25). The His6-TEV-MSP1E3 construct
was expressed, purified, and subjected to TEV proteolysis to
afford high yields of purified protein (60 mg/L cell culture). For
the PglC-ND preparation, a 10:1 MSP:PglC (5:1 ND:PglC) ratio
afforded a discrete population of discs, which could be purified
away from empty discs and the remaining nanodisc assembly
components via Ni-NTA chromatography using the unique His6tag on PglC (Fig. 2B). To estimate the number of PglCs per disc,
gel densitometry by quantitative Coomassie staining was carried
out and revealed the presence of one PglC species per nanodisc
assembly (Fig. 3A and Fig. S1). In addition, SEC analysis of the
Hartley et al.
B
PglC-NDs
P
kDa
50
36
lipids
Und-P
22
incubation
detergent
withdrawal
His6-PglC
PglA
Ni-NTA
purification PglC/PglA-NDs
of PglC-NDs
Nanodisc assembly mixture
FT W1 W2 W3 E1 E2 E3 E4
PglC
MSP
PglC-NDs
kDa
50
36
MSP
22
purified Nanodiscs
INAUGURAL ARTICLE
A
PglC/PglA-NDs
PglA
PglC
MSP
labels (Fig. 4A) to probe the cofacial organization of the two
reconstituted proteins in the nanodisc assembly using FRET.
Based on estimated distances in the PglC/PglA-ND (Fig. 4B), the
known tetramethylrhodamine (TAMRA) and Cyanine5 (Cy5)
FRET-pair (R0 ∼ 6.5 nm) was selected. For fluorophore labeling,
PglC lacks native Cys residues and therefore single Cys mutations were introduced to generate two mutants for site-specific
thiol-directed modification using a Cy5-maleimide derivative (SI
Text and Fig. S3). The labeling sites were selected such that the
Cy5 would modify either the globular domain of PglC [PglC(Cy5globular)] or the N terminus [PglC(Cy5-terminal)], which would
be localized on the distal side of the membrane bilayer (Fig. 4B
and Fig. S3). The chemical labeling proceeded to ∼50% conversion and the precise labeling efficiency was quantified following reconstitution of the PglC mutants into nanodiscs. With
respect to PglA, the native sequence includes six Cys residues,
and therefore an alternate labeling strategy was adopted. Labeling of PglA (Fig. 4A) with the TAMRA fluorophore was
achieved by sortase-mediated ligation (40); this method guaranteed quantitative labeling of the donor species, which is
beneficial in a static ensemble FRET experiment. For this
process, a PglA variant with a C-terminal sortase A recognition
sequence (LPETG) and a TAMRA-labeled, sortase-active peptide [GGGYK(TAMRA)KG] were reacted to yield the modified
PglA(TAMRA). The C terminus was chosen for labeling because
addition of an N-terminal peptide tag had been found to disrupt
the association of PglA with PglC-NDs (Fig. S4). Fluorescence
labeling was carried out as summarized in Fig. 4A and presented
in the SI Text.
Fluorescently labeled proteins were reconstituted into nanodiscs
using the approaches developed for the unlabeled species. Fluorescent PglC/PglA-NDs were adjusted to the same concentration
Ni-NTA–purified nanodiscs showed an elution profile consistent
with a PglC-ND assembly (Fig. 3B and Fig. S2).
Nanodiscs were also prepared in the presence of PglC and PglA
at an MSP:PglC:PglA ratio of 10:1:1 (5:1:1 ND/PglC/PglA) and
subjected to affinity chromatography on Ni-NTA. In this case, the
only nanodisc species binding to the stationary phase are those
containing His6-GB1-PglC, because the affinity tags of all other
components were removed previously. At this stage, two possible
outcomes were anticipated: Either there would be a statistical
distribution of PglA among all of the discs, and therefore only
20% of the discs isolated after Ni-NTA purification would contain
both PglC and PglA; or alternatively, a positive interaction between PglC and PglA in the discs would bias the distribution.
When the purified nanodiscs were analyzed by gel densitometry,
the PglC:PglA ratio was 1.0:0.7 (Fig. 3A). This result implies that
PglA is colocalized with PglC in ∼70% of the population.
Dynamic light scattering (DLS) was applied to estimate the
hydrodynamic radii of the various nanodisc assemblies (Fig. 3C).
The measurements show particles with average hydrodynamic
particle radii of 6.6 nm for empty nanodiscs, 12.9 nm for PglCNDs, and 15.4 nm PglC/PglA-NDs, which is consistent with the
effect of systematically adding proteins to the nanodisc assemblies. As a control, PglC and MSP in the absence of phospholipids did not afford detectable particles, and it is likely that the
protein components precipitated under detergent withdrawal
conditions. We note that the measured polydispersities are
somewhat high, which may indicate the presence of a population
of aggregates, which has been documented previously with DLS
measurements on nanodisc assemblies (33).
Fluorescence Labeling of PglC and PglA and FRET Analysis. PglC and
PglA were engineered to incorporate site-specific fluorescent
12
PglC (std.)*
2
5
16
* all values in (pmoles)
8
11
ND
PglC
MSP
MSP (std.)*
PglC (std.)*
9 13.5 18
7
11 15
PglA (std.)*
4
7
10
ND
B 1.6
PglC NDs
1.2
Abs . (a . u . )
7
PglA
PglC
MSP
0.8
void
0.4
0.0
0
40 r = 12.9 nm (99.9 % mass,
47 % Pd)
30
30
20
10
10
0
0.1
20
1
10
100
Diameter (nm)
Hartley et al.
10
3
0
0.1
1
100
10
Diameter (nm)
103
5
10
40 r = 15.4 nm,
(100 % mass,
30 49 % Pd)
80
% mass
50 r = 6.6 nm (99.9 % mass,
67 % Pd)
40
% mass
% mass
C
3
% mass
PglC/PglA NDs
PglC NDs
A MSP (std.)
20
10
0
0.1
15
V (mL)
20
25
r = 0.1 nm (99.5 % mass,
8 % Pd)
60
40
20
1
10 100
Diameter (nm)
10
3
0
0.1
1
10
100
Diameter (nm)
10
3
Fig. 3. Characterization of nanodiscs. (A) Quantification of PglC-NDs and PglC/PglA-NDs by gel
densitometry using quantified protein standards
of MSP, PglC, and PglA. The estimated ratios were
MSP/PglC = 11.4/6.1 pmoles and MSP/PglC/PglA =
14.0/8.9/6.1 pmoles. (B) SEC analysis of PglC-NDs.
The void volume is indicated. (C) DLS analysis of
(left to right): empty nanodiscs, PglC-NDs, PglC/
PglA-NDs, and a nanodisc preparation mixture
(PglC-NDs) lacking the lipid components (negative
control). The average particle radius (r), the percentage of the total analyzed mass, and the
polydispersity (Pd) are displayed in each panel.
PNAS Early Edition | 3 of 8
BIOCHEMISTRY
Fig. 2. Protein reconstitution in nanodiscs. (A) Nanodisc self-assembly is initiated by removal of detergent. Nanodiscs are purified using Ni-NTA chromatography, which binds the unique His6-tag on PglC-NDs. (B) SDS/PAGE analysis of Ni-NTA fractions (E, elution fractions; FT, flow through; W, wash) from
a PglC-NDs preparation (Upper) and a PglC/PglA-NDs preparation (Lower).
S145C
Gb1 (7-61)
271
TMD
(81-103)
TEV
His6
C7
A 1 insert
PglC
Cy5 globular
Cy5 terminal
TAMRA
B
LPET/GG-HHHHHH
C
Cy5 globular
2R0 13 nm
TAMRA
lA
Pg
5.5 nm
Fex 515nm (a.u.)
PglC
4x10
6
3x10
6
2x10
6
1x10
12.5 nm
GB1
PglA
His6-Sortase A (S. aureus)
CaCl2
donor only: PglA(TAMRA)/PglC
FRET state: PglA(TAMRA)/PglC(Cy5-term.)
LPETGGG-YKKG
D
Fex 515nm (a.u.)
PglA
TAMRA
H-GGG-YKKG-OH
5x10
6
4x10
6
3x10
6
2x10
6
1x10
6
donor only: PglA(TAMRA)/PglC
FRET state: PglA(TAMRA)/PglC(Cy5-glob.)
6
0
0
550
600
Cy5 terminal
650
(nm)
700
750
550
600
650
(nm)
700
750
His6
Fig. 4. Analysis of cofacial localization of PgC and PglA in nanodiscs using FRET. (A) Preparation of fluorescent protein constructs. PglC Cys mutants were labeled
with Cy5 maleimide and PglA was labeled with TAMRA using a sortase-mediated transpeptidation with a TAMRA-containing peptide. (B) Graphic of the PglC/PglAND. Nanodisc dimensions and the R0 of the fluorophore pair are indicated. The locations of the fluorophores are based on structure prediction models (SI Text and
Fig. S3). (C) Corrected fluorescence emission spectra of PglC(Cy5-terminal)/PglA(TAMRA) nanodiscs (FRET state) and PglC(unlabeled)/PglA(TAMRA) nanodiscs (donor
only). (D) Corrected fluorescence emission spectra of PglC(Cy5-globular)/PglA(TAMRA) nanodiscs (FRET state) and donor-only nanodiscs. Data from nanodiscs
comprising the PglC species labeled in the globular segment reveal a higher FRET efficiency than nanodiscs containing terminally labeled PglC.
with respect to the PglA(TAMRA) donor using the unique
absorption signal of the fluorophore at 557 nm. PglC/PglA ensembles contained unlabeled PglC (∼50%), which led to a disproportional overestimation of unquenched donor for the two
nanodisc ensembles. To account for this high donor background,
the labeled fraction of reconstituted PglC in the nanodisc ensembles was quantified by UV spectroscopy to determine the
number of possible FRET states (see Methods and SI Text
for details).
To perform the FRET experiment, TAMRA was excited at
a low wavelength (515 nm) to minimize cross-excitation of the
Cy5 acceptor. The fluorescence emission showed defined signals
for the donor emission and excitation of the acceptor attributable to FRET (Fig. S5). The resulting spectra of the nanodisc
ensembles with different PglC mutants were corrected for the
number of non-FRET states in the donor fluorescence based on
UV quantification and for background contributions stemming
from direct excitation of the Cy5 fluorophore in the acceptor
fluorescence (SI Text), as determined by analysis of acceptor-only
samples of equal concentration (Fig. 4 C and D and Fig. S5).
The donor signal was quenched for donor/acceptor nanodisc
ensembles with both PglC mutants, revealing a higher FRET efficiency in the presence of the PglC(Cy5) acceptor labeled within
the globular protein segment (E = 0.57) compared with the terminally labeled PglC(Cy5) (E = 0.43). This trend was also observed in the intensity of the acceptor signals, revealing a stronger
signal for the PglC(Cy5-globular) species (Fig. 4 C and D). FRET
efficiencies were estimated based on donor signal decay upon
quenching, thereby avoiding the use of acceptor standards and
circumventing errors arising from endogenous acceptor quenching processes, such as those caused by different probe environments. Assuming the free rotation of the appended fluorophores,
the stronger energy transfer for PglC(Cy5-globular) relative to
PglC(Cy5-terminal) is good evidence for a model in which the
4 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1320852110
soluble globular domains of PglC and PglA obtain a cofacial arrangement in the nanodiscs, as illustrated in Fig. 4B.
Functional Reconstitution of PglC and PglA. Radioactivity tracer
studies were used to assess the activities of the enzymes in the
nanodisc assemblies. Specifically, undecaprenyl-[33P]phosphate
(Und-[33P]P) allowed for quantification of Und-P incorporation
and also provided an orthogonal radiolabel that was used in concert with [14C]- or [3H]-labeled UDP-sugars (UDP-[14C]diNAcBac
and UDP-[3H]GalNAc) for precise tracking of the Und-P–linked
species. To establish that Und-P incorporated into the nanodiscs,
PglC/PglA-NDs were prepared from PE/PG lipids (3:1) and 0.5 mol
percent Und-[33P]P. The assemblies were purified by Ni-NTA
chromatography, and the radioactivity present in the flow-through,
wash, and elution fractions was quantified by liquid scintillation
counting, which showed that ∼18% of the total radioactivity was associated with nanodiscs (Fig. 5A). Because only 20% of the nanodisc
ensembles included PglC, this level of Und-P incorporation was
consistent with a statistical distribution of Und-P.
To validate that PglC was functionally reconstituted into the
nanodiscs, PglC-NDs with Und-[33P]P were treated with soluble
UDP-[14C]diNAcBac. After incubation of the reaction for 1 h, an
organic-aqueous extraction of the reaction mixture was performed to assess incorporation of [14C], representing diNAcBac,
into Und-[33P]P-P-[14C]diNAcBac, which would extract into the
organic phase. The organic extract was analyzed by normal-phase
HPLC and elution fractions were collected and analyzed by
liquid scintillation counting. The 33P-signal corresponding to
Und-[33P]P eluted first, followed by [33P] and [14C] signals that
coeluted in a single fraction and corresponded to Und-[33P]P-P[14C]diNAcBac (Fig. 5B).
The PglC/PglA-NDs were prepared with Und-[33P]P and activity was assessed in a similar fashion, but using first unlabeled
UDP-diNAcBac and UDP-[3H]GalNAc (Fig. 5C), and then both
Hartley et al.
Discussion
We present an experimental platform that exploits membrane
bilayer nanodiscs for investigating the functional roles of linear
long-chain polyprenols in glycan assembly pathways. To establish
the potential of the approach, we have focused the current
studies on the first two membrane-committed steps in the C. jejuni
Pgl pathway carried out by the phosphoglycosyltransferase PglC
and the glycosyltransferase PglA, and have investigated coincorporation of the enzymes into nanodiscs using multiple biochemical and biophysical methods.
Protein Interactions at the Membrane Interface. We demonstrate
that PglC incorporates into nanodiscs as a functional monomer
Hartley et al.
% radioactivity
INAUGURAL ARTICLE
A
60
40
20
0
B
1
2
5
4
Fractions
3
6
Und-[33P]P
10
7
8
9
Und-[33P]P-P-[14C]Bac
pmoles
8
6
4
2
C
14
P
33
0
C
15
20
6
25
t (min)
30
Und-[33P]P-P-Bac-[3H]GalNAc
Und-[33P]P
BIOCHEMISTRY
pmoles
5
4
3
2
3
1
H
P
33
0
15
D
20
t (min)
25
30
Und-[33P]P-P-[14C]Bac-[3H]GalNAc
Und-[33P]P
5
pmoles
4
3
2
H
3
1
0
E
C
14
P
33
15
20
t (min)
25
30
Und-[33P]P
6
5
pmoles
UDP-[14C]diNAcBac and UDP-[3H]GalNAc (Fig. 5D). The [33P]signal corresponding to Und-[33P]P elutes first in both cases and
the disaccharide product is observed with coeluting [33P] and
[3H] or coeluting [33P], [14C], and [3H], consistent with the coupled
reactions of PglC and PglA. When UDP-diNAcBac is omitted
from the coupled reaction, no final product is observed (Fig.
5E), indicating that the sequential reactions of both enzymes
are required for production of the Und-P-P-disaccharide.
Because all of the substrates in the assays were discretely and
quantifiably labeled with radioisotopes, the percent conversion of
the nanodisc-associated Und-[33P]P could be determined. After 1
h we observed that ∼50% of the Und-[33P]P had been converted
to corresponding Und-[33P]P-P-linked product (Fig. 5 B–D). This
finding is consistent with a model in which the Und-P is statistically distributed in the nanodisc with the phosphate oriented
equally toward both faces of the membrane bilayer. Because it is
well established that polyprenyl-phosphates do not undergo passive membrane flipping (41, 42), this finding would correlate with
all of the available Und-P being processed through the active
sites of the enzymes on the same face of the disc.
To monitor the time course of the coupled PglC/PglA reaction
and production of the product, a similar method to that described
above using unlabeled Und-P, UDP-diNAcBac, and UDP-[3H]
GalNAc, was applied and aliquots of the reaction mixture were
quenched at multiple time points. The data show a rapid and efficient production of the labeled Und-P-P-diNAcBac-[3H]GalNAc
product (Fig. 6A). To assess for activity because of endogenous
Und-P in the native E. coli lipid extract, the nanodiscs were prepared without added Und-P, and indeed a low level of background activity was observed (Fig. 6A). When this experiment
was repeated with nanodiscs prepared with a lipid mixture composed
of synthetic PE (67.0%) and PG (23.2%), as well as cardiolipin
(9.8%)—which is comparable to the predominant E. coli membrane
lipid components—background activity was not observed. Therefore, this observation is consistent with the commercial E. coli
lipid extract containing a low level of endogenous Und-P.
To demonstrate the importance of colocalization in nanodiscs
for enzyme activity, the enzymatic reaction rates were determined
in nanodiscs and in detergent-disrupted nanodiscs. Two identical
nanodisc samples were diluted with either buffer or detergent and
then assayed for activity. The reaction rate of detergent-disrupted
nanodiscs was ∼12.5-times slower than the reaction rate of the
undisrupted sample (Fig. 6B).
Finally, evidence for a static colocalization of the PglC and
PglA enzymatic reactions at the nanodisc surface was obtained
by evaluating the reaction rates at different PglC/PglA-ND dilutions. Assuming that nanodiscs are stable macromolecular complexes containing cofacially oriented PglC, PglA, and Und-P, then
the observed activity rate should not depend on the concentration
of PglC, PglA, or Und-P, but rather the absolute amount of these
components. To test this theory, an equimolar amount of nanodiscs was assayed at three different dilutions and the concentrations of the soluble UDP-sugars were kept constant. The initial
reaction rates reflected in the slopes of the reaction turnover are
the same for all dilutions (Fig. 6C). This finding is consistent with
the formation of nanodiscs containing stable cofacially oriented
PglC and PglA in tandem with available Und-P substrate.
4
3
2
1
0
H
3
P
33
15
20
t (min)
25
30
Fig. 5. Functional PglC/PglA-NDs demonstrated with triple orthogonal
radiolabeling. (A) PglC/PglA-NDs were prepared with Und-[33P]P and purified by Ni-NTA chromatography. The flow-through (fraction 1), washes
(fractions 2–5) and elutions (fractions 6–9) were analyzed by liquid scintillation counting and it was determined that ∼18% of the Und-[33P]P
coeluted with nanodiscs. (B–E) Functional characterization of PglC/PglAND radiolabeled products by HPLC. (B) PglC-NDs produce Und-[33 P]P-P[14 C]diNAcBac. (C ) PglC/PglA-NDs produce Und-[33P]P-P-diNAcBac-[3 H]
GalNAc. (D) PglC/PglA-NDs produce triple-labeled Und-[ 33P]P-P-[14C]
diNAcBac-[ 3H]GalNAc. (E ) Negative control for the coupled reaction of
PglC/PglA containing Und-[ 33P]P and UDP-[ 3H]GalNAc and lacking UDPdiNAcBac. No double-labeled product is observed.
(Figs. 2 and 5, and Fig. S1), which is unique in showing the
oligomeric state of a phosphoglycosyltransferase being probed
in a lipid bilayer. Furthermore, we show that PglC and PglA
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8
through the application of triple isotopic-labeling approaches
using the uniquely labeled substrates Und-[33P]P, UDP-[14C]
diNAcBac, and UDP-[3H]GalNAc (Fig. 5D). Importantly, the
ability to carry out quantitative analysis of activities, for example
in the disc disruption and disc dilution assays (Fig. 6 B and C),
reveals the impact of enzyme colocalization in a 2D membrane.
The results from such studies are crucial for deepening our understanding about how membrane-bound enzymes in multistep
pathways function in vivo.
Although the current studies have focused on PglC and PglA,
the native Pgl pathway comprises five glycan assembly enzymes
(PglC, PglA, PglJ, PglH, and PglI, Fig. 1A), and intriguingly only
PglC and PglI are predicted to be integral membrane proteins by
sequence analysis (12). Based on this established foundation of
methods, the nanodisc platform should be readily applicable for investigating the specific roles of PglC and PglI in recruiting the entire
cluster of enzymes to the membrane bilayer interface where they
function. Additionally, the opportunities for orthogonal isotopic
labeling of substrates and intermediates will enable studies to examine whether substrates are transferred directly between sequential enzymes in the pathway. Because substrate processivity in glycan
assembly pathways is poorly understood, this approach promises to
provide the experimental system for addressing this key issue.
PglC/PglA-Nanodiscs
control w/o Und-P (E. coli lipids)
control w/o Und-P (POPE/POPG/CA)
(pmoles)
6
4
2
0
B
0
8
20
t (min)
40
60
intact PglC/PglA-Nanodiscs
disintegrated Nanodiscs
6
physically and functionally interact in the presence of a lipid
bilayer. Nonrandom, colocalization of PglC and PglA provides
clear evidence that these enzymes undergo a specific protein–
protein interaction that is dependent on the lipid bilayer interface and does not occur in the presence of detergent (Fig. 6B
and Fig. S6). In addition, the sequential activities of the two
enzymes in the nanodisc platform are clearly demonstrated
FRET Analysis of Protein Interactions in Nanodiscs. In contrast to
vesicular membrane model systems such as liposomes, both faces
of the nanodisc are accessible for interactions with substrates and
enzymes. Therefore, we developed orthogonal labeling strategies
to prepare fluorescently modified variants of PglC and PglA to
enable a FRET analysis to assess the topological relationship
between the globular domains of the enzymes in the nanodiscs.
The studies clearly reveal a preference for a cofacial relationship
(pmoles)
Fig. 6. Time course of activity in PglC/PglA-NDs as measured by radioactivity. (A)
Comparison of activity in PglC/PglA-NDs prepared with lipid extracts (E. coli) or
synthetic lipid compositions with and without Und-P. (B) Reaction efficiency
of PglC/PglA-NDs compared with a detergent-disrupted PglC/PglA-NDs. (C)
Initial reaction rates of PglC/PglA-NDs in sequentially diluted reactions.
Structure/Function Relationships at the Lipid Bilayer. It is well
documented that nanodiscs can be assembled from a variety of
phospholipids, which may reflect native or nonnative compositions. In these studies we demonstrated that disc preparations
made with native E. coli lipids result in a measureable background activity in the absence of added Und-P, presumably because the native E. coli lipid extract included a small amount of
endogenous Und-P (Fig. 6A). This background activity is completely abolished with discs assembled with synthetic phospholipid components. Importantly, addition of dosed quantities
of Und-P restores robust activity that can be readily quantified by
using isotopically labeled substrates. These studies therefore
show that the nanodisc platform will allow tremendous versatility
in quantifying the specific effects of lipid bilayer composition on the
functional efficiency of the sequential membrane enzyme activities.
The current studies take the versatility of nanodiscs one step
further by showing the opportunity to investigate glycan assembly
enzymes that require a membrane-bound substrate, in this case
a polyprenyl-phosphate derivative. To our knowledge, this demonstration of enzymes acting on coincorporated membranebound substrates in nanodiscs is unique. Previously, the activity of
a series of diverse polyprenyl-phosphates were previous investigated using PglC as a detergent micelle preparation (16).
The studies revealed that although unsaturation of the α-isoprene
and a particular combination of E and Z isoprene units were key
features determining enzyme activity, the length of the polyprenyl
chain had significantly less impact on protein activity. However, in
a detergent-based assay, the effects of polyprenol length on membrane partitioning and dynamics would have been taken out of
consideration. With the nanodisc approach, we are now in a
unique position to quantitatively assess structure/function relationships of native and nonnative polyprenyl-linked substrates
and phospholipids directly in a membrane bilayer, which will enable us to make measurements that are simply inaccessible through
any other in vitro or in vivo system. Furthermore, we will be able to
assess the impact of capturing the substrates and enzymes in
a 2D bilayer, which is likely to have profound implications for
substrate availability and propensity of the turnover (42–44).
4
2
0
C
0
1.0
2
t (min)
4
6
40
3-fold dilution
2-fold dilution
undiluted
30
negative control (w/o UDP-diNAcBac)
0.6
20
0.4
10
0.2
0.0
conversion (%)
(pmoles)
0.8
0
2
4
t (min)
6
6 of 8 | www.pnas.org/cgi/doi/10.1073/pnas.1320852110
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Hartley et al.
Conclusion
The application of nanodiscs to the well-characterized C. jejuni
pgl pathway provides an exciting experimental platform for
addressing the evolutionary purpose of linear polyprenols in
glycan biosynthetic pathways. The present study presents key
steps in establishing the platform and demonstrates that the
system delivers robust quantitative measurements of activity that
will be valuable for investigating the specific roles of polyprenyllinked substrates in the integrated function of glycan assembly
enzymes typified by components of the bacterial pgl pathway.
The study highlights the interaction of PglC and PglA, which are
responsible for the first two membrane-committed steps in the
pgl pathway. Coincorporation of these enzymes into nanodiscs is
characterized by multiple biophysical methods that support the
cospatial and cofacial localization of the proteins. In tandem
with PglC and PglA, the undecaprenyl-phosphate substrate is
also incorporated into nanodiscs and the functional reconstitution
of a PglC–PglA complex is verified through biochemical assays.
This work represents a proof-of-concept demonstrating that
nanodiscs can be used for the precise manipulation and study
of polyprenol-dependent pathways. The study is also unique in
providing a set of evidence supporting the hypothesis that enzymes dependent on polyprenyl-phosphates associate into biosynthetic macromolecular complexes.
Methods
Protein Expression, Purification, and Site-Specific Modification of PglC and PglA
Variants. Standard techniques of molecular biology and biochemistry were
used to clone, express, and purify His6-GB1-TEV-PglC, the accompanying
single Cys mutants (C7 and S145C), His8-TEV-PglA, and PglA-LPETGG-His6.
PglC single Cys mutants were modified using Cy5 maleimide and PglA was
modified using a sortase-mediated ligation (40) with GGGYK(TAMRA)KG.
Detailed protocols are included in SI Text. See Tables S1 and S2 for primers
used in preparation of PglC and PglA DNA constructs.
Materials. All phospholipids, radioactive compounds, amino acid derivatives
and fluorescent labeling agents were purchased from commercial vendors.
Undecaprenol was extracted from the leaves of the staghorn sumac (Rhus
typhina) and phosphorylated using chemical or enzymatic methods (SI Text).
Und-[33P]P was prepared by using Streptococcus mutans kinase and [γ33P]
ATP (46). UDP-diNAcBac was prepared using a chemoenzymatic approach
Hartley et al.
INAUGURAL ARTICLE
exploiting PglF, PglE, and PglD of the pgl pathway (47). For the preparation
of UDP-[14C]diNAcBac, [14C]acetyl-CoA was applied in the PglD-catalyzed step.
Nanodisc Preparation and Characterization. Nanodisc assembly methods were
based on established protocols (27, 29), and involved incubation of the
protein components (MSP and PglC in the presence and absence of PglA)
with membrane lipids containing Und-P at defined ratios. Self-assembly of
nanodiscs was initiated by detergent removal with hydrophobic adsorbants
(e.g., biobeads). Nanodisc assemblies are purified using the unique His6-tag
handle on PglC by using Ni-NTA affinity chromatography. Nanodisc assemblies were characterized using SDS/PAGE, SEC, and DLS. Specific quantification of proteins was carried out using Coomassie blue staining followed by
gel densitometry. Detailed protocols are described in SI Text.
FRET Analysis. Nanodiscs for FRET analysis were prepared by creating nanodisc
ensembles containing the following reconstituted variants of PglC and PglA: (i)
PglA(TAMRA) and unlabeled PglC; (ii) PglA(TAMRA) and PglC(Cy5-terminal);
(iii) PglA(TAMRA) and PglC(Cy5-globular); (iv) unlabeled PglA and PglC(Cy5terminal); (v) unlabeled PglA and PglC(Cy5-globular). The concentrations of the
PglA(TAMRA) containing nanodisc ensembles i–iii were adjusted to identical
concentration on basis of the donor TAMRA UV-absorption signal. Acceptor-only
samples iv and v were adjusted in concentration to match samples ii and iii,
respectively, applying the Cy5-absorption signals. FRET data were acquired by
excitation at 515 nm to minimize direct acceptor excitation. Because of the
different labeling strategies, the PglA construct was quantitatively labeled,
whereas labeling of PglC was incomplete. To minimize errors resulting from
different degrees of labeling between the two species, the fractions of labeled versus nonlabeled PglC were stringently quantified after nanodisc assembly
to account for overestimation of the donor fluorescence because of the population
of non-FRET PglC/PglA-NDs with unlabeled PglC. These non-FRET states were
represented by a fraction of 45% in case of the PglC-globular species and 56% in
case of PglC-terminal. To account for this, the “donor-only” emission spectrum (i)
was scaled according to the amount of the non-FRET states in each sample. The two
resulting donor-only curves were used to correct the spectra of the donor/acceptor
FRET samples (ii and iii). Subsequently, acceptor signal contributions stemming
from direct excitation of the acceptor were subtracted using data from acceptor
only samples (iv and v; full analysis is presented in Fig. S5 and SI Text).
Activity Analysis. Enzyme function in the nanodisc assemblies was assessed
using isotopically labeled substrates (Und-[33P]P, UDP-[14C]diNAcBac, and
UDP-[3H]GalNAc). For all concentrations and specific activities, see SI Text.
For product analysis by HPLC, four nanodisc preparations containing PglC
alone or PglC and PglA were prepared with 0.5 mol percent Und-[33P]P. PglCNDs were incubated with UDP-[14C]diNAcBac. PglC/PglA-NDs were incubated
with unlabeled UDP-diNAcBac and UDP-[3H]GalNAc, or UDP-[14C]diNAcBac
and UDP-[3H]GalNAc. As a control reaction, PglC/PglA-NDs were incubated
with UDP-[3H]GalNAc in the absence of UDP-diNAcBac.
After 1 h, the reactions were quenched in chloroform:methanol (2:1, 1 mL)
and the organic layer was washed with chloroform:methanol: 0.1 M aqueous
potassium chloride (3:48:47, 3× 400 μL). The organic layer was dried and
resuspended in chloroform:methanol (4:1, 100 μL) for analysis using NPHPLC. Fractions of 1 mL were collected and each fraction was dried under an
N2 stream. Radioactivity was determined by liquid scintillation counting.
Activity analysis for assessing the effects of lipid composition and detergent-based nanodisc disruption was carried out using PglC/PglA-NDs
prepared with unlabeled Und-P. Reactions were initiated by the addition of
UDP-diNAcBac and UDP-[3H]GalNAc, and aliquots were quenched at various
time points and then extracted to isolate organic soluble radiolabeled
products (Und-P-P-diNAcBac-[3H]GalNAc). For nanodisc disruption, we used
Triton X-100 as a nondenaturing detergent at a concentration of 0.175%
(vol/vol), which is sufficient to completely disrupt the nanodiscs as verified by
Ni-NTA chromatographic analysis (Fig. S6 and SI Text). Additionally, measurements of the coupled PglC/PglA reaction show that activity is maximal at
0.175% (vol/vol) Triton X-100 (Fig. S6).
For the dilution assays, Und-[33P]P was used to enable accurate quantification
of the disc-loaded substrate and the rates of enzyme-catalyzed conversions
were measured by quantifying [3H]GalNAc incorporation into the Und-[33P]
P-P-glycan products. Nanodiscs were assayed at 0.05 μM Und-[33P]P, 0.025 μM
Und-[33P]P, or 0.017 μM Und-[33P]P, in volumes of 50 μL, 100 μL, and 150 μL,
such that all three reactions contained equimolar amounts of the disc preparation at different dilutions. The concentrations of UDP-diNAcBac and UDP[3H]GalNAc remained constant. Aliquots corresponding to 15% of the total
starting volume were quenched and extracted, as described above.
PNAS Early Edition | 7 of 8
BIOCHEMISTRY
of the globular domains; FRET efficiency is higher when PglC is
labeled on the globular domain compared with N-terminal labeling, which places the label on the distal side of the membrane
(Fig. 4 C and D). At the current time, further quantitative analysis
of the FRET efficiencies would be premature, because structural
information is not yet available on either PglC or PglA. The finding
that the globular domains are cofacially oriented is corroborated
by activity analyses and the functional reconstitution of the PglC/
PglA-ND. In particular, in experiments using precisely quantified
Und-[33P]P, all of the available of Und-P is converted to final
product (Fig. 5 B–D). If the major population of discs did not
include PglC and PglA oriented with cofacial active sites, the
extent of conversion would be lower because assemblies with the
two enzymes oriented on opposite faces would not be able to process two sequential reactions to afford Und-PP-diNAcBac-GalNAc.
Although the FRET analyses in the present studies were
carried out with an ensemble of discs including only two protein
species, the opportunities for fluorescent labeling of other enzymes in the glycan assembly phase of the pathway (PglJ, PglH,
and PglI) using similar orthogonal labeling approaches suggests
that the nanodisc platform can be extended to the entire pathway. In this context, it should be noted that although the analysis
of nanodisc species as an ensemble was relatively complicated
because of incomplete labeling of one of the two enzymes, singlemolecule approaches would avoid this complication and render
analysis of more than two differentially labeled species feasible.
Single-molecule approaches have already been applied to nanodisc systems and, therefore, all of the components are in place for
taking the current inquiries on the pgl pathway and the roles of
polyprenols in general to the next level of complexity (45).
ACKNOWLEDGMENTS. The authors thank Prof. Stephen Sligar and Dr. Yelena
Grinkova for valuable technical advice and for providing samples of membrane scaffold protein for our initial nanodisc studies. Also, the authors
are grateful to Dr. Karen Allen, Dr. Angelyn Larkin, and Vinita Lukose
for their valuable feedback regarding the manuscript. This work was
supported by National Institutes of Health Grant GM039334 (to B.I.); an
American Chemical Society award of a Fellowship in Medicinal Chemistry
(to M.D.H.) and a Leopoldina Fellowship Program LPDS 2009-38 (to P.E.S.).
The Massachussetts Institute of Technology Biophysical Instrumentation Facility is also acknowledged.
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