Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 372: 451–464

DOI 10.1007/s00210-006-0039-4

REVIEW

Daniel Basting . Ines Lehner .

Mark Lorch . Clemens Glaubitz

Investigating transport proteins by solid state NMR

Received: 16 December 2005 / Accepted: 17 January 2006 / Published online: 28 February 2006
# Springer-Verlag 2006

Abstract Transporters form an interesting and complex revealed that 3–10% (3 % in Mycobacterium tuberculosis
class of membrane proteins. Many of them are potential and 10% in Salmonella typhimurium) of open reading
drug targets due to their role in translocation of ions, small frames are predicted to encode membrane transport
molecules and peptides across the membrane or due to their proteins (Cole et al. 1998; McClelland et al. 2001).
role in multidrug resistance. Hence elucidating their These transporters are vital for cell nutrition, environ-
structure and mechanism is of great importance and may mental sensing, ATP synthesis, protein/toxin secretion as
lead to a host of new drugs and methods to alter or inhibit well as influx and efflux of solutes. Furthermore, some of
their function. Solid state NMR is an emerging technique these transport proteins exhibit surprisingly broad sub-
for investigating transport proteins. Along with other strate specificity which allows them to play important
biochemical and biophysical techniques solid state NMR roles in multidrug resistance, cell volume regulation and
can provide data on drug binding, protein dynamics and peptide selection for translocation across membranes.
structure at the interface between structural biology and In order to fulfil their function, transport proteins must
functional analysis. Here, we review solid state NMR negotiate a cycle which includes steps associated with
applications to primary active and secondary transporters substrate recognition, binding, translocation and release.
involved in translocation of small molecules. We discuss This transport cycle may be coupled to ATP hydrolysis in
current experimental limitations and give an overall the case of ATP binding cassette (ABC) proteins or ion
perspective on how the technique may be used to address translocation in the case of secondary transporters.
some pertinent questions relevant to transporters. So far, 10 structures of transport proteins have been
determined (see Table 1) (Striebek 2005). This information
Keywords Drug resistance . EmrE . FucP . GalP . GusB . is clearly of great value, although this small number of
LacS . NupC . LmrA . Membrane . Multidrug . NMR . structures does not yet contain a representative from all
Protein . Transporter transporter families. Furthermore, we must remember that
each structure corresponds to a snapshot of the protein
during its transport cycle. Therefore, additional ap-
Introduction proaches are needed to elucidate the transport kinetics,
substrate-protein interactions or conformational changes
Transporters are important membrane proteins Membrane during substrate translocation.
proteins and specifically transporters provide the entry and
exit sites for proteins and solutes through biological lipid Solid state NMR for the study of membrane proteins Solid
membranes. Recently sequenced bacterial genomes have state NMR (ssNMR) has been widely used in membrane
biophysics, to study membrane bound peptides and
Daniel Basting and Ines Lehner have contributed equally to this increasingly for investigations of larger integral membrane
article. proteins. Areas of application cover protein structure and
dynamics, lipid-protein, substrate-protein, and substrate-
D. Basting . I. Lehner .
M. Lorch . C. Glaubitz (*) membrane interactions (Fig. 1). Static ssNMR applied to
Institute for Biophysical Chemistry, Centre for Biomolecular macroscopically ordered membrane samples has been used
Magnetic Resonance, J.W. Goethe Universität, to elucidate secondary structure and topological organisa-
Marie-Curie Str. 9, tion of membrane bound peptides (Ketchem et al. 1997).
60439 Frankfurt am Main, Germany
e-mail: glaubitz@em.uni-frankfurt.de However, for larger membrane proteins, that fall within the
Tel.: +49-69-79829927 scope of this review, static ssNMR techniques are of
Fax: +49-69-79829929 limited use. Instead, magic angle sample spinning (MAS)
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Table 1 Overview of publicly available transport protein structures
Protein Function Family Organism Resolution PDB

GlpT Sugar uptake Major facilitator superfamily (MFS) E. coli 3.30 Å 1PW4
LacY Lactose uptake Major facilitator superfamily (MFS) E. coli 3.50 Å 1PV6, 1PV7
AcrB Multidrug efflux Resistance-nodulation-cell division (RND) E. coli 3.50 Å 1IWG, 1OY6, 1OY8,
1OYD, 1OYE
EmrE Multidrug efflux Small multidrug resistance (SMR) E. coli 3.70 Å 1S7B, 2F2M
BtuCD Vitamin B12 ATP-binding cassette (ABC) E. coli 3.20 Å 1L7V
uptake
MsbA (closed) Lipid and drug ATP-binding cassette (ABC) E. coli 4.50 Å 1JSQ
export
MsbA (open) Lipid and drug ATP-binding cassette (ABC) Vibrio cholera 3.80 Å 1PF4
export
SecYEB Protein General secretory pathway (Sec) Methanococcus 3.50 Å 1RHZ, 1RH5
translocation jannaschii
AmtB Ammonium Ammonium transporter (Amt) E. coli 1.40 Å 1U77, 1U7G, 1U7C,
transport 1XQE, 1XQF
GltPH Glutamate Dicarboxylate / amino acid:cation Pyrococcus 3.50 Å 1XFH
transport symporter (DAACS) horikoshii
Data were compiled from a web-database maintained at the MPI for Biophysics, Frankfurt, Germany (Striebek 2005)

seems more promising as it imposes fewer restrictions The number of membrane proteins available for
with respect to sample preparations: The proteins can be biophysical studies has been dramatically increased by
studied in a number of states (discussed in detail below). the advent of microbial expression systems (e.g. Escher-
But most importantly, anisotropic NMR interactions such ichia coli, yeast and baculovirus) optimised for membrane
as chemical shift or dipolar couplings are averaged out by proteins, the availability of detergent screens and advanced
fast sample rotation (in practice 5–20 kHz) about the purification technologies, in addition to the sequencing of
magic angle (54.7° with respect to the magnetic field), a number of prokaryotic and eukaryotic genomes. For a
leading to the type of well resolved spectra shown in review, see (Wang et al. 2003). An overview of sample
Fig. 2. preparation and biophysical investigations possible at each
step is given in Fig. 3. Solid state NMR is increasingly
applied to isotope labelled membrane proteins in order to
derive data about protein structure and dynamics. The
possibility of a complete de novo structure determination
purely based on MAS NMR recoupling techniques was
first demonstrated for small insoluble peptides (Jaroniec et
al. 2004; Rienstra et al. 2002) and has been extended to
soluble proteins studied in the solid state (Castellani et al.
2002; Seidel et al. 2005; Zech et al. 2005) . However,
similar studies have not yet been successfully applied to
membrane proteins largely due to limitations in spectral
resolution. However, a number of studies have reported
promising MAS NMR spectra of membrane proteins
prepared as 2D crystals (Hiller et al. 2005), 3D crystals
(Lorch et al. 2005a) and also in proteoliposomes
(Andronesi et al. 2005). The latter is clearly the prepara-
tion of choice as it is closest to the protein’s native
environment. Lipid reconstituted samples also offer the
option of investigating the dynamics of the protein as it
binds ligands (Patching et al. 2004a), negotiates its
reaction cycle (Mason et al. 2005) or responds to changes
in the lipid environment (Yamaguchi et al. 2004).
Fig. 1 Solid state NMR can be used for investigating the interaction However, the currently very limited knowledge base
between substrate and protein (a), for studying changes in protein
conformation and dynamics upon substrate binding (b), for protein- does not yet allow us to derive general rules about how
lipid interactions (c) or for observing the binding of small molecules to prepare membrane proteins for solid state NMR and so
to lipid membranes (d) screens have to be performed for each individual protein.
 453

Fig. 2 Basic principles of solid state NMR. Large macromoleular proton decoupling and cross polarisation, and magic angle sample
complexes such as membrane proteins do not rotate fast enough to spinning at increasing rotation rates (from top to bottom) (b). MAS
average dipolar couplings or anisotropic chemicals shifts to zero, NMR combined with suitable pulse sequences for polarisation
which results in a complex NMR interaction network. Isotropic transfer, decoupling and recoupling of NMR interactions can then be
tumbling can be emulated by rotating the sample rapidly (1–20 kHz) used for example for resonance assignment and structural studies.
about the magic angle (MAS NMR) (a). The presence of all dipolar Shown here is a typical double–quantum–single–quantum correla-
couplings and anisotropic chemical shifts causes broad featureless tion spectrum of crystalline U–13C–glutamic acid (c)
spectra as shown here for 1–2–13C glycine, which improves by

The potential role of solid state NMR in the process of (neuropeptide) bound to its native G protein-coupled
drug discovery has been highlighted in a recent review receptor (GPCR) (Luca et al. 2003).
(Watts 2005). The structures of ligands and drugs can be
determined at their site of action by solid state NMR. This Solid state NMR studies on transporters So far, only a
aids in defining the ligand binding site. In addition, drug limited number of solid state NMR studies on transporters
partitioning, drug-lipid interactions and drug polymor- have been reported (Appleyard et al. 2000; Glaubitz et al.
phism can be assessed by solid state NMR. It has also 2000; Mason et al. 2004; Patching et al. 2004a,b, 2005;
recently been shown to be a method capable of de- Spooner et al. 1993, 1994a,b, 1998, 1999). Applications
termining the backbone structure of a peptide ligand cover in principle three areas: interactions of substrates
with the membrane, detection of substrates bound to
transporters, and methodological studies describing how to
prepare isotope labelled transporters.
The first solid state NMR applications to transporters
were presented by the laboratories of Watts and Henderson
who demonstrated the detection of substrate bound to the
sugar transporter GalP (Appleyard et al. 2000; Spooner et
al. 1993). This has triggered a number of further studies
aimed at obtaining structural information concerning the
position of the binding site (Spooner et al. 1998), as well
as the ligand/protein association constant (Patching et al.
2004a). Recently, it has also been shown that isotope
labelled transporters can be prepared at a quantity and
Fig. 3 Schematic diagram depicting transport protein preparation purity suitable for ssNMR (Mason et al. 2004).
and the biophysical techniques that can be utilised at each In the following, we will give an overview about
preparation stage published work on transporters and discuss perspectives
454

for further studies in the light of biochemical challenges. solution state NMR (Schwaiger et al. 1998). Unfortu-
This overview is not intended to serve as a technical nately, α-helical membrane proteins often have a low
introduction into solid state NMR. For this, the reader is spectral dispersion (Krueger-Koplin et al. 2004), and
referred to introductory texts such as Laws et al. (2002) or therefore uniformly labelled proteins yield very crowded
textbooks. NMR spectra with many overlapping peaks. To circum-
vent this problem, selective labelling of single amino acid
types can be achieved with a defined medium (synthetic
Sample preparation rich) containing all amino acids (Muchmore et al. 1989).
Selective labelling can be aided, and metabolic scrambling
Availability of transport proteins of NMR active nuclei avoided, with the use of auxotrophic
E. coli strains. However, auxotrophs are not available for
ssNMR imposes strict constraints on sample preparation all amino acids and usually support only low levels of
with respect to purity and homogeneity. The greatest protein overexpression. An alternative to auxotrophs
hurdles to overcome, however, are those of quantity and makes use of the T7 promoter and the action of the
concentration of the isotope labelled transport protein and/ antibiotic rifampicin (Arkin et al. 1996; Lee et al. 1995a).
or labelled ligand bound to the protein. Usually, the amount Rifampicin selectively binds to the E. coli RNA polymer-
of nuclear spins in the sample has to be in the order of μmol ase and blocks its transcription initiation, whilst the T7
for a decent signal-to-noise ratio. Considering that the RNA polymerase is not affected. This can be exploited by
active sample volume used for MAS NMR is typically in growing the E. coli in unlabelled media to the target cell
the order of 20–90 μl, the protein concentration must be in density, pelleting the cells and inducing in fresh isotope
the order of 3–20 mM. This means that 2–10 mg of a small labelled media (Almeida et al. 2001). Shortly after
12 kDa protein, like the small multidrug transporter EmrE, induction, rifampicin is added and thus it is assured that
needs to be inserted into such a small sample container. protein contaminants are not isotope labelled and E. coli
When studying proteoliposomes, the concentration prob- cell metabolism is reduced.
lem is compounded by the presence of lipids which take up
most of the rotor volume. Whilst functional assays can be L. lactis overexpression and labelling Overexpession and
performed on proteoliposomes with small protein/lipid mol labelling for NMR has been demonstrated for the ABC
ratio (1:1,000), ssNMR requires ratios of up to 1:100, but multidrug transporter LmrA (Mason et al. 2004). Ampli-
four-fifths of the sample volume is still taken up by lipids. fied expression in L. lactis offers a number of advantages.
Higher protein/lipid mol ratios are often prevented by the The cells grow rapidly to a high cell density and
risk of protein aggregation. overexpressed membrane proteins are found exclusively
Generally, the protein must be overexpressed in a host within the cytoplasmic membrane. Proteins can then be
system. This allows a range of uniform and amino acid solubilised directly from the membrane with mild
selective isotope labelling schemes to be applied, an detergents and purification is simplified by the small
essential step if the protein is to be studied directly. proteome size (Kunji et al. 2003).
Therefore, a suitable recombinant expression system must
be chosen. Overexpression of sufficient amounts of Cell Free Expression and labelling Cell free expression
transport proteins for solid state NMR has been successful systems have been produced from many eukaryotic and
in E. coli, Lactococcus lactis and a cell free expression prokaryotic cells including E. coli, rabbit reticulocyte and
system. wheat germ (Klammt et al. 2004; Madin et al. 2000;
Pelham and Jackson 1976). Crude cell extracts supply
Overexpression and labelling in E. coli For bacterial most of the necessary enzymes, ribosomes and cofactors
transporters, the classical E. coli expression is the system for expression. Currently, most published applications
of choice because of its low cost, the bacteria’s rapid utilise E. coli based coupled transcription/translation
doubling time, well understood genetics and the avail- expression systems. Early cell free expression systems
ability of a range of expression vectors and bacterial were performed in batch mode, but the yields were very
strains. Overexpression of bacterial transporters has been low due to extract deactivation and exhaustion of
repeatedly demonstrated (Auer et al. 2001; Curnow et al. substrates (Jewett and Swartz 2004). Development of
2004; Masi et al. 2003; Xie et al. 2004; Yerushalmi et al. continuous-flow reactors allowed dramatically prolonged
1995). A more detailed summary of expression levels of production times of up to 40 h but the yield only increased
various secondary membrane transporters is given in a marginally due to loss of translation components (Spirin et
review by Wang et al. (2003). al. 1988). More successful were approaches using semi-
E. coli is also the most used expression system for continuous cell free expression which yield up to 6 mg/ml
preparing isotope labelled proteins for NMR spectroscopy. for soluble proteins (Kigawa et al. 1999). Using E. coli
Complete labelling can be achieved by using minimal based coupled transcription/translation systems, integral
media which contains all necessary nutrients and an membrane proteins have been expressed and yields of
isotope enriched carbon and/or nitrogen source. These 1 mg/ml for GPCRs (Ishihara et al. 2005), 1.3 mg/ml for
well established standard procedures have been used, for MscL (Berrier et al. 2004) and 3 mg/ml for EmrE (Rotem
example, to prepare the E.coli transporter EmrE for et al. 2001) have been achieved . These proteins can be
 455

expressed either as precipitate or directly inserted into a 1998, 1994a). Nevertheless, in most cases proteins have to
hydrophobic environment when in the presence of be solubilised from membranes, purified and reconstituted
detergents and/or lipids (Fig. 4). However, yields in the into phospholipids. For one transporter, E. coli EmrE,
presence of detergents are reduced in a detergent depen- organic solvent based protein extraction has been described
dent fashion correlating with critical micellar concentra- (Yerushalmi et al. 1995), but in general, carefully selected
tion (CMC) (Berrier et al. 2004). Bacteriorhodopsin, for detergents have to be used for protein solubilisation.
example, has been expressed as aggregate in quantities Ideally, the detergent should fulfil several critical require-
sufficient for biophysical studies such as Fourier Trans- ments: it must be capable of solubilising the protein, the
form Infrared Spectroscopy (FTIR) and could be shown to protein must be stable, and finally it must be compatible
refold correctly into its native and active conformation with a reconstitution procedure to yield active protein.
using sodium dodecyl sulfate (SDS) and halobacterial Rarely does one detergent fulfil all requirements, and so the
lipids (Sonar et al. 1993). Meanwhile, small multidrug choice of detergent is often a compromise.
resistance (SMR) proteins, GPCRs and ion channels have Solubilisation is followed by one or more chromato-
been expressed successfully in the presence of detergents graphic purification steps, e.g. affinity, size exclusion, ionic
and lipids. Furthermore, they could be shown to be active or hydrophobic chromatography. Unfortunately, there is an
and were reconstituted successfully into artificial mem- inevitable trade-off between quantity, stability, activity and
branes (Berrier et al. 2004; Ishihara et al. 2005; Klammt et very pure protein preparations. The success of protein
al. 2004). In conclusion, in vitro expression is a viable purification is routinely monitored using SDS PAGE and
alternative to in vivo expression under some circum- western blot analysis. Protein homogeneity can also be
stances. The high yield of protein per mass of labelled evaluated using analytical size exclusion chromatography
amino acid and low detergent requirements make cell free and mass spectrometry. A very good review surveying
expression of some membrane proteins economically sample preparation and optimization was written by Wang
viable despite its inherent high reagent costs and its et al. (2003).
manifold of optimisation parameters (Guignard et al. High protein stability in detergent solution is necessary
2002; Jewett and Swartz 2004). Special consideration has for all applications. For example, detergents used during
to be given to prove the correct fold of the expressed protein crystallisation are screened for their ability to
protein. Direct manipulation of reaction conditions enables maintain the integrity of the protein for long periods of time
the use of cofactors and unnatural amino acids (Betton often judged by size exclusion chromatography. Long term
2003). And, of course, in vitro production of toxic proteins stability is also a prerequisite for solution state NMR,
is also possible. where the best detergent is usually judged by the quality of
the protein NMR spectrum. Using this strategy, a thorough
screen of 25 different detergents was performed on a
Solubilisation, purification, choice of detergents Staphylococcus aureus Smr (Krueger-Koplin et al. 2004)
where a number of promising candidates were found, such
Highly amplified expression levels were achieved for some as, for example, 1-palmitoyl-2-hydroxy-sn-glycero-3-
transporters (GalP, NucP, LacS, FucP) allowing substrate [phospho-RAC-(1-glycerol)] (LPPG). Despite the fact
binding studies by ssNMR of these proteins in their natural that the protein detergent complexes appeared to be larger
membranes (Patching et al. 2004a; Spooner et al. 1999, than 100 kDa, the rotational correlation time corresponded
to that of a 15–20 kDa protein tumbling isotropically in
solution. This has been interpreted as tumbling of the
protein inside the LPPG detergent micelle. Unfortunately,
the best detergents for high quality solution state NMR
spectra do not necessarily correspond to the detergents
found to be most suitable for maintaining a functional
folded protein or long term stability. For example, it has
been demonstrated using radioactive ligand binding assays
that detergent solubilised EmrE, (an Smr homologue) binds
the substrate tetraphenylphosphonium (TPP+) only in
dodecyl maltoside (DDM) amongst a dozen tested
detergents (Muth and Schuldiner 2000). The requirements
for ssNMR are similar to those above since the protein
should be stable and functional in detergent before the
protein is reconstituted into liposomes.

Reconstitution into lipid vesicles

Fig. 4 Schematic diagram of a semi–continuous E. coli based cell
free expression system with coupled transcription and translation Reconstitution of membrane proteins is commonly
(see text for details) (Klammt et al. 2004) achieved by one of three methods: mechanical means
456

such as freeze-thawing and sonication, dissolving protein different protein:lipid ratios (Chi-Rosso and Toole 1987).
and lipid in organic solvent with subsequent evaporation of Further characterisation of proteoliposomes can be
the solvent, or detergent mediated procedures. Although achieved by freeze fracture electron microscopy (Fig. 5)
reconstitution by codissolving of lipids and protein in to assess sample homogeneity and protein aggregation
organic solvents has been shown for EmrE (Yerushalmi et (Viitanen et al. 1986). Successful reconstitution also
al. 1995) and was used to prepare samples for solid state requires a functional test to verify the protein’s correct
NMR (Glaubitz et al. 2000), transport proteins have been fold within the liposome, for which transport, ligand
mainly reconstituted using detergent based methods which binding and ATPase assays in the case of ABC transporters
also facilitate a smooth transition to 2D crystallization. can be used. However, assays on proteoliposomes are not
Stability and activity of membrane proteins during and without their limitations and drawbacks. False negative
after the reconstitution process are mainly governed by the results will arise from drug binding sites being trapped on
detergent choice and homogeneous incorporation. In the inside of liposomes or within multilamellar complexes
general, detergent solubilised membrane proteins are and thus being inaccessible to the substrate.
mixed with lipids followed by a decrease of detergent
concentration which causes incorporation of the protein
into liposomes. Detergent removal can be achieved using Crystallisation
dialysis (Gorzelle et al. 1999), gel chromatography (Kiefer
et al. 1996), dilution (Curnow et al. 2004) or hydrophobic From the biochemical point of view, solid state NMR
absorption (Paternostre et al. 1988). The method of choice studies on proteoliposomes are clearly the approach of
depends mainly on detergent properties such as CMC and choice as this allows preparation of functional membrane
hydrophobicity, but also on the desired speed of reconsti- proteins. But it has been shown for two cases, the β-barrel
tution and completeness of detergent removal. Detergents OmpG and the α-helical diacylglycerol kinase (DGK), that
with a low CMC can only be removed efficiently by 2D (Hiller et al. 2005) or 3D crystals (Lorch et al. 2005a) of
hydrophobic adsorption, which is also the best method for membrane proteins can also be used for solid state NMR.
almost complete detergent removal (Allen et al. 1980; The advantage of crystals stems mainly from the higher
Holloway 1973), while dilution of detergents allows fastest protein concentration that can be achieved and from very
reconstitution. Rapid hydrophobic absorption of detergents well resolved ssNMR spectra which can be obtained under
by polystyrene beads seems to be the most promising favourable circumstances.
approach for SMR transport proteins (Curnow et al. 2004; These well resolved spectra have been attributed to high
Sikora and Turner 2005; Winstone et al. 2002), but has also ‘short-range’ order in crystalline samples compared to
been used for the ABC multidrug transporter LmrA. This proteoliposomes. Therefore, spectral improvements are
efflux pump has been reconstituted for solid state NMR by most likely due to restricted protein dynamics in crystals, as
mixing solubilised protein with preformed, detergent protein dynamics give rise to line-broadening due to
destabilised vesicles followed by detergent removal conformational exchange. The drawbacks of crystals are
through polystyrene beads (Mason et al. 2004). Phospho- the extensive screens needed for crystallisation conditions,
lipid adsorption onto the hydrophobic beads can be despite the fact that well diffracting crystals are not a
minimised by bead presaturation with lipids, and protein requirement.
adsorption has been shown to be negligible (Rigaud et al. 2D crystallisation is essentially a reconstitution process
1997). An excellent review on biobead based reconstitution optimised to induce local order and crystallinity at very
was written by Rigaud et al. (1998). high protein:lipid mol ratios of 1:15–30. During 2D
Successful reconstitution can be monitored using con- crystallisation slow, controlled, removal of detergent is
tinuous or discontinuous density gradient centrifugation to desired and usually a narrow range of near physiological
separate empty liposomes and proteoliposomes with

Fig. 5 Reconstitution of mem-

brane proteins. Protein incorpo-
ration into liposomes is
achieved by replacing detergents
with lipids which can be done
by a number of methods (see
text). Successful incorporation
is monitored by freeze fracture
electron microscopy as shown
here for the ABC multidrug
transport LmrA (16 k magnifi-
cation) (Mason et al. 2004). The
image was kindly provided by
Dr. W. Haase, MPI Biophysik
 457

crystallisation conditions is screened. This is a major

advantage compared to 3D crystallisation.

Solid state NMR on transporters

Sugar transporters

Sugar transporter GalP

The first solid state NMR studies on a transporter were

carried out on GalP, a member of the major facilitator
superfamily (MFS). GalP is a galactose-H+ symport protein
located in the inner membrane of E. coli. It is closely
related to sugar transport systems in higher organisms,
including Glut1 in humans (Baldwin and Henderson 1989).
GalP has a molecular weight of 50 kDa, is predicted to
consist of 6 transmembrane helices and is thought to
function as a homodimer. The protein can be overexpressed
in E.coli where it is targeted to the inner membrane. These
GalP-containing membranes can be extracted and worked
on directly, removing the need for any further purification
steps and allowing the protein to be studied in its native
membrane. Such samples have been used for ssNMR based
ligand binding studies.
The approach presented by Spooner et al. (1994a) relies
on the effect of molecular dynamics on cross polarisation
(CP) (Schaefer et al. 1977). Cross polarisation is usually Fig. 6 13C–CPMAS spectra from E. coli membranes containing
used to transfer polarisation via dipolar couplings from GalP (a) and with bound D–[1–13C]glucose (b). Expanded regions
protons to less sensitive nuclei such as 13C in order to show an increase of signal upon addition of 0.25 μmol (c) and
1 μmol of D–[1–13C]glucose (d). Adapted, with the authors’
enhance their signal intensity. Spooner et al. (1994a) have permission, from Spooner et al. (1994a)
used CP as a ‘dynamic filter’ since dipolar couplings are
reduced with molecular motions causing decay in signal
intensity. A GalP substrate such as D-[1-13C]glucose will microsecond to millisecond range and was found to be very
therefore be visible under CP conditions when bound to similar for bound substrate, protein and lipid resonances.
GalP in a fluid membrane, while no cross polarisation will This has been interpreted by the authors as an indication
take place for its free state. Typical 13C-CPMAS spectra of that slow collective motions influence all membrane
GalP containing membranes with and without D-[1-13C] components equally.
glucose are shown in Fig. 6. Two resonances are detected The presented relaxation and CP data show that the
because glucose is present in two anomeric forms. The lifetime of the transporter-substrate complex corresponds
chemical shifts of glucose are very similar to those detected to the ms timescale of the NMR experiment, i.e. slow
in solution, but the peak intensities (Fig. 6d) reveal a exchange takes place. These findings are consistent with a
greater spectral contribution from the β-form of glucose previously suggested ‘mobile-barrier’ mechanism, in
indicating its preferred binding to GalP. It was also possible which the substrate-transporter complex is alternatively
to show that 13C-labelled D-glucose and D-galactose were exposed to both sides of the membrane by conformational
displaced from GalP with singly labelled [7-OCO13CH3] fluctuations.
forskolin, a known inhibitor of sugar transport in GalP The general application of cross-polarisation as a
(Appleyard et al. 2000). No alternative binding site for dynamic filter to detect substrates bound to transporters
forskolin was found. is limited by the presence of large background signals
The timescales of the molecular dynamics of bound D- arising from the 13C natural abundance in lipids and
[1-13C]glucose have been determined from NMR relax- proteins. However, the use of multiply 13C -labelled
ation measurements (Spooner et al. 1994a). The proton substrates with strongly dipolar coupled nuclei in close
spin-lattice relaxation in the laboratory frame T1Z is proximity offers a solution: the application of double-
influenced by high frequency motions (10−9 s) and was quantum filtering NMR experiments will only select those
found to be much faster for bound substrate compared to its coupled nuclei while suppressing the 13C natural abun-
crystalline state. Therefore, the substrate in its binding site dance background (Lee et al. 1995b). This approach has
undergoes fast motions on the nanosecond timescale. The been attempted on doubly labelled glucose and forskolin.
relaxation T1ρ in the rotating frame of the spin-locking field Unfortunately, it was found that in fluid membranes at 2°C
used for CP is sensitive to low frequency motions in the no substrate detection was possible, but freezing samples at
458

−35°C allowed an efficient double-quantum filtering and been used for MAS NMR substrate binding studies on the
hence substrate detection, most likely due to restrictions of fully active mutant LacS (K373C) (Spooner et al. 1999).
molecular motion. At this temperature, both bound and Cross polarisation experiments on D-[1-13C]galactose
non-bound substrates start to become immobilised which revealed that both galactose anomers are bound by LacS,
makes dynamic filtering difficult (Appleyard et al. 2000). though there is no significant chemical shift change
The problem of strong 13C natural abundance back- compared to galactose in solution. The cross polarisation
ground has also been addressed by preparing 13C-depleted signal follows classical saturation binding behaviour with
membranes containing GalP. E. coli, expressing GalP, were respect to the substrate concentration. Consequently, the
grown on 13C-depleted glucose (Patching et al. 2004b). authors were able to determine a Kd of 4 mM for D-[1-13C]
The interfering 13C background signals were significantly galactose directly from solid state NMR data.
reduced to 13C≤0.07% (compared to natural 13C abundance After attaching a nitroxide maleimide spin label to C373,
of 1.1%). Based on this very low background, the located in the interhelix loop 10–11, the 13C CPMAS
simultaneous detection of both sugar substrate and inhib- signals of bound D-[1-13C]galactose disappeared almost
itor by cross polarisation was possible. completely. Based on this paramagnetic signal quenching,
the authors estimated the sugar binding site to be within
15 Å of the nitroxide spin label at residue C373 and
Fucose transporter FucP concluded that the interhelix loop 10–11 is in close
proximity to the substrate binding site.
The L-fucose-H+ symport protein FucP from E. coli has a
monomeric size of 47 kDa and is predicted to form 12
transmembrane segments (TMS). High expression levels, Nucleoside transporter NupC and glucuronide
FucP constitutes 20% total inner membrane protein, transporter GusB
enabled ssNMR studies directly on native membranes
(Spooner et al. 1998). Further improvements on the quantitative characterisation
FucP is known to transport D-arabinose, L-galactose and of substrate binding by MAS NMR were presented by
L-fucose, but not their respective stereoisomers (Muiry et Henderson, Middleton and colleagues on the bacterial
al. 1993). It binds its ligands weakly making competitive transporters GusB and NupC (Patching et al. 2004a). GusB
binding assays impractical. However, detection of substrate is a 50 kDa glucuronide-H+ symporter from E. coli. It
binding by solid state NMR has been done following the transports glucuronides, which are often conjugated to
approach discussed for GalP. It was possible to show by hormones, xenobiotics and drugs excreted from the human
cross polarisation that both substrate anomers of D-[1-13C] body (Liang et al. 2005). The 43 kDa nucleoside
arabinose and L-[13C6]galactose are bound equally well. transporter NupC from E. coli is a member of the
To measure the substrate exchange time, the authors concentrative nucleoside transporter (CNT) family. It
went a step further by suggesting a dephased delayed cross shares structural motifs with the three human members of
polarisation (DDCP) experiment: All magnetisation of this family (hCNT1-3) and thus can serve as a experimental
protons associated with the membrane is dephased, i.e. model for the clinically relevant mammalian proteins
removed, and only magnetisation of protons free in (Patching et al. 2005). For both proteins, a 20–50-fold
solution, such as in unbound substrate, is retained. During amplification of their expression levels has been shown
a variable mixing time, this free substrate can bind to FucP (Patching et al. 2004a). Hence, the concentration of the
which is detected by cross polarisation. The substrate protein in the native membrane is high enough for substrate
exchange time has been estimated by analysing the cross binding studies to be carried out. It had been demonstrated
polarisation signal intensity as a function of these mixing for LacS, that a Kd can be obtained from signal intensities
times. It was found that a very slow exchange between free of bound substrate, filtered by cross polarisation, as a
and bound substrates takes place (>10−1 sec). function of substrate concentration (Spooner et al. 1999).
This normally requires substrate titration to the sample into
the MAS rotor or the preparation of many different
Lactose Transporter LacS samples. Patching et al. (2004a) suggest an alternative.
They show that binding constants and dissociation rate
The 69 kDa lactose transport protein LacS from Strepto- constants koff can be determined directly from a single
coccus thermophilus belongs to the glycoside-pentoside- sample. This was achieved by following the cross
hexuronide (GPH):cation symporter family. It is predicted polarisation signal intensity as a function of the time
to fold into 12 TMS and appears to form a functional period during which cross polarisation takes place (contact
dimer with one sugar translocation channel per monomer time). The method is based on a statistical analysis of
(Veenhoff et al. 2001). Members of the GPH family are substrate binding kinetics during 1H-13C cross polarisation.
characterised by sequence conservation in helices II, IV If substrate binds, cross polarisation is enabled and
and in the interhelical loop between helices 10 and 11 magnetisation builds up during the residence time of the
(Poolman et al. 1996). Overexpression in Streptococcus substrate in the binding site, after which the substrate
thermophilus results in 25% of the total protein content in returns to its free state. The longer the contact time, the
native membranes being LacS. These membranes have higher is the probability that a binding event will occur
 459

during that period. It is then possible to simulate the effect but without NupC, and then applied to membranes with
of many random binding events with different substrate- NupC. Using this filtering trick in conjunction with the
transporter lifetimes, substrate binding constants and method described above for the LacS measurements, a
varying contact times on the CP signal intensity. As a value for Kd of 2.6 mM was obtained.
result, experimental CP build-up curves can be analysed to
obtain values for Kd and koff. The authors were able to
demonstrate and verify this approach for the binding of Multidrug Efflux Pumps
[1-13C]-β-glucuronide for which Kd and koff values of
4.17 mM and 698 s−1 have been obtained (Fig. 7). It was Antibiotic resistance of pathogenic bacteria is a major
even possible determine binding constants for unlabelled worldwide problem. Bacteria rapidly acquire resistance to
ligands by carrying out competition binding experiments. new antibiotics and their widespread use over a number of
A slightly different situation was observed for the years means they no longer provide effective control
binding of [1′-13C]uridine to NupC: using cross polarisa- against many infectious diseases. Several resistance
tion, a signal from bound ligand was identified in native E. mechanisms, namely, drug inactivation, target alteration,
coli membrane with and without induced expression of prevention of drug influx and active drug extrusion are
NupC. The chemical shift did not differ significantly in recognised and have been found to act synergistically. One
either case, but signal intensity was higher in the presence of these mechanisms, active drug extrusion, can be
of protein, indicating that nonspecific binding to the assigned to membrane-bound primary or secondary efflux
membrane or other membrane proteins takes place. Any pumps. The primary efflux pumps, otherwise known as
quantitative analysis would have to take this signal ABC transporters, utilise energy-derived from ATP hy-
contribution into account by either difference spectroscopy drolysis for active transport of substrates. Among the
or suitable filtering techniques. The method used by secondary transporters, four families, SMR (small multi-
Patching et al. (2004a) relies on cross polarisation with drug resistance), multidrug and toxic compound extrusion
polarisation inversion (CPPI) normally applied as a (MATE), resistance nodulation cell division (RND) and
spectral editing technique to simplify NMR spectra (Wu major facilitator superfamily (MFS), have currently been
and Zilm 1993). After cross polarisation, magnetisation is described (Putman et al. 2000).
transferred back from 13C to 1H for a short period of time,
tp. Depending on the length of this time period and the
motional characteristic of the observed molecular group, SMR protein EmrE
the carbon signal will disappear. If specifically and
nonspecifically bound substrates differ in rates and Proteins of the SMR family are small multidrug efflux
amplitudes of their anisotropic motion, signal from non- pumps of 10–12 kDa with a predicted four helix trans-
specifically bound populations can be eliminated by membrane topology and have been found in archeae and
appropriate choice of tp. The experiment was first bacteria (De Rossi et al. 1998; Grinius et al. 1992; Ninio
calibrated on native membranes containing [1′-13C]uridine, and Schuldiner 2003). In one case, SMR proteins have

Fig. 7 The cross polarisation

build–up of [1–13C]–β–glucu-
ronide bound to GusB can be
analysed using a statistical ap-
proach in terms of KD and koff.
Adapted, with the authors’ per-
mission, from Patching et al.
(2004a)
460

been detected in up to 31% of clinical isolates of methicillin

resistant S. aureus (Noguchi et al. 2005).
SMR proteins contain approximately 110 amino acids
and are characterised by five amino acid sequence motifs,
highlighted in the topology diagram (Fig. 8a) of EmrE, the
most studied SMR protein.
It has been suggested that EmrE transports a diverse
array of aromatic and positively charged substrates in a
proton/drug antiport fashion (Paulsen et al. 1996). Direct
structural information is only available for EmrE. Solution
state NMR of EmrE in the highly artificial environment of a
chloroform:methanol:water mixture achieved a secondary
structure determination, but no tertiary structure could be
proposed (Schwaiger et al. 1998). Low resolution tertiary
structures both with and without bound substrate were
achieved more recently by electron microscopy of 2D
crystals (Tate et al. 2001; Ubarretxena-Belandia et al.
2003). The electron density reveals that three helices in
each monomer are crossing the membrane and are nearly
perpendicular to the membrane plane whereas the remain-
ing fourth helix is highly tilted (Tate et al. 2001;
Ubarretxena-Belandia et al. 2003). A 3.8 Å x-ray crystal-
lographic structure of EmrE suggests a highly unusual
dimer of conformational heterodimers as the functional
unit. Monomers of the conformational heterodimer are
roughly inverted with respect to each other. Helices 1–3
form a six helix bundle and helix 4 of one monomer is
positioned nearly parallel to the membrane surface, while
the remaining helix 4 of the second monomer protrudes
from the membrane (Ma and Chang 2004). A new 3.7 Å x-
ray structure of EmrE in complex with tetraphenylphos-
phonium (TPP+) confirms the opposite direction of both
subunits, but agrees with the transmembrane arrangement
of all helixes detected by electron microscopy (Pornillos et
al. 2005).
Despite these data, the oligomeric state of SMR proteins
remains highly controversial. Biochemical and structural
data supporting dimers, trimers or tetramers have been
reported and are summarised in Table 2.
Solid state NMR studies of EmrE followed a similar
approach to those described for GalP. 31P-cross-polarisa- Fig. 8 Topology of the SMR transporter EmrE with highly
tion dynamics was used to differentiate between bound and conserved residues (black) and signature sequence motifs (grey)
unbound TPP+, which has been shown to bind to EmrE (a). A 31P CP MAS spectrum of EmrE in complex with TPP+ in
DMPC bilayers reveals two resonances (b). The chemical shift of
(Muth and Schuldiner 2000). In contrast to GalP, expres- peak 1 is similar to that of free TPP+ and is attributed to nonspecific
sion levels of EmrE are not sufficient to perform binding interactions with the protein while resonance 2 is shifted by 4 ppm
studies in the native membrane. Therefore, experiments which is assigned to specifically bound substrate (Glaubitz et al.
have to be performed on purified EmrE in proteoliposomes. 2000). While cross polarisation filters and enhances only signals
from immobile components such as bound ligands, direct polarisa-
TPP+ is known to diffuse into the membrane (Ahmed and tion (by a simple 90° pulse followed by proton decoupling) excites
Krishnamoorthy 1990), but once there remains very all 31P nuclei equally. Here, the spectral contribution from unbound
mobile. Consequently, the TPP+ signal is not enhanced TPP+ is much higher compared to the bound fraction
by cross polarisation in either DMPC (Glaubitz et al. 2000)
or E. coli liposomes. A signal from TPP+ can, however, be data clearly show that the TPP+-EmrE complex is stable on
seen when the sample is polarised directly (i.e. without the the ms timescale, but further experiments are needed to
motion filters imparted by cross polarisation). When EmrE understand the mechanism of substrate binding and
is included in the liposomes two signals appear (Fig. 8c). translocation in the light of contradicting biochemical
Based on the chemical shifts, the cross polarisation data (see Table 2). Solid state NMR with the help of
dynamics and signal enhancement upon adding more suitable labelling schemes could help to observe changes in
substrate, the resonances have been interpreted as arising local structure and dynamics upon substrate binding. Of
from nonspecific and specific bindings sites. The ssNMR special interest are highly conserved key residues (Fig. 8a),
 461
Table 2 Summary of experimental evidence for the oligomeric homologues of mammalian transporters. Most notably, in
states of EmrE (see text for details) some cases very high expression levels have been reported
Oligomeric Experimental methodology Reference which made substrate binding studies on native membranes
state possible. It has been shown that bound substrate can be
selectively filtered by cross polarisation allowing binding
Dimer EPR spectroscopy (Koteiche et al. 2003) constants and dissociation rates to be determined. For most
EM structure (Tate et al. 2001) and cases, except for EmrE, weak substrate binding with Kd
(Tate et al. 2003) values in the mM range was found without significant
Cysteine crosslinking (Soskine et al. 2002) changes in substrate chemical shifts. The substrate-
whole cell growth assay (Jack et al. 2000) transporter complex lifetimes were estimated to be in the
analytical ultracentrifugation (Butler et al. 2004) range of milliseconds. One central difficulty is certainly
and size exclusion that isotope labelled substrate is needed and that the use of
chromatrography cross polarisation as a dynamic filter requires the mem-
Trimer In vivo and in vitro negative (Yerushalmi et al. brane to be in its fluid phase. Furthermore, as in the case of
13
dominance 1996) C or 31P labels, natural abundance signals of lipids or
Radioactive ligand binding (Muth and Schuldiner other membrane components could obscure the substrate
2000) signal. One solution is offered by double-quantum filtering
Tetramer Functional complementation of (Elbaz et al. 2004) which has the disadvantage that it normally requires frozen
in vitro produced protein samples. This eliminates the dynamic filter effect provided
X-ray crystallography structure (Ma and Chang 2004) by CP measurements and hence makes it impossible to
distinguish between bound and unbound species. Never-
theless, these approaches hold significant potential for drug
which could be isotope labelled using the methods screening especially in cases where native membrane can
discussed above. In combination with double-quantum be used, but also for purified and reconstituted samples.
filtering, only those resonances arising from labelled The above mentioned examples show, so far, that the
residues could be selected (Lorch et al. 2005b). main focus has been clearly on drug-protein interaction
studies, but solid state NMR on other membrane proteins
has yielded unique information about conformations and
ABC Transporter LmrA dynamics. Therefore, first experiments on purified, recon-
stituted and labelled transporters have begun. This work is
The L. lactis multidrug efflux pump LmrA is a member of in its early stages mainly due to the difficulty of developing
the ABC transporter family. It forms a homodimer of two suitable methods to overexpress, isotope label and purify
64 kDa subunits each containing 6 transmembrane helices sufficient amounts of membrane transporters for NMR and
and one nucleotide binding domain. It has been shown to to asses their activity in a reliable fashion. Furthermore,
be a functional homologue of the human P-glycoprotein transporters, especially multidrug efflux pumps, may have
(van Veen et al. 1998). Substrates of both proteins to be highly flexible to accommodate a number of different
accumulate within the membrane in the interface region substrates during the transport cycle. This may cause
as shown by 1H-MAS NMR (Siarheyeva et al. , submitted). further difficulties when it comes to sample preparation,
Overexpression of LmrA in L.lactis allows sufficient protein stability and NMR spectroscopy. In principle, solid
amounts of protein to be obtained for NMR as is the case state NMR can be applied at various stages of sample
for all other transporters discussed above. Solid state NMR preparation, but membrane proteins are ideally studied in a
studies on such complex membrane proteins have been reconstituted form.
limited due to stability, concentration and reconstitution In the authors’ opinion, transporters belong to the most
problems. Recently, it has been shown that it is possible to exciting membrane proteins as they have to undergo a
overcome these restrictions by extensive reconstitution complex translocation cycle consisting of substrate bind-
screens and residues selective labelling schemes (Mason et ing, translocation and substrate release in an energy-
al. 2004). Using this approach, protein dynamic studies dependent fashion. Considering the progress in solid state
have been reported using residue selective 2H-NMR (Lorch NMR methodology and advancements in membrane
et al. 2005b). protein sample preparation as reported here, the authors
expect solid state NMR to make important contributions to
the understanding of the mechanism of energy dependent
Conclusions and perspectives substrate translocation across membranes in the near
future.
The number of solid state NMR studies on transporter
proteins reported so far is still very small and limited to Acknowledgements Material for Fig. 5 was kindly provided by Dr.
bacterial systems, but both ABC and secondary systems are W. Haase, MPI Biophysik, for Fig. 6 by Prof. Anthony Watts, Oxford
covered. Primary and secondary transporters are involved and for Fig. 7 by Dr. David Middleton, Manchester. The authors
in diverse functions such as multidrug efflux, sugar and acknowledge the support by the Frankfurt Special Research Centre
(SFB 628) ‘Functional Membrane Proteomics’.
nucleoside transport. Some bacterial transporters are
462

References Glaubitz C, Groger A, Gottschalk K, Spooner P, Watts A, Schuldiner

S, Kessler H (2000) P-31-CP-MAS NMR studies on TPP+
bound to the ion-coupled multidrug transport protein EmrE.
Ahmed I, Krishnamoorthy G (1990) Enhancement of transmem- FEBS Lett 480:127–131
brane proton conductivity of protonophores by membrane- Gorzelle BM, Nagy JK, Oxenoid K, Lonzer WL, Cafiso DS,
permeant cations. Biochim Biophys Acta 1024:298–306 Sanders CR (1999) Reconstitutive refolding of diacylglycerol
Allen TM, Romans AY, Kercret H, Segrest JP (1980) Detergent kinase, an integral membrane protein. Biochemistry
removal during membrane reconstitution. Biochim Biophys 38:16373–16382
Acta 601:328–342 Grinius L, Dreguniene G, Goldberg EB, Liao CH, Projan SJ (1992)
Almeida FC, Amorim GC, Moreau VH, Sousa VO, Creazola AT, A staphylococcal multidrug resistance gene product is a
Americo TA, Pais AP, Leite A, Netto LE, Giordano RJ, Valente member of a new protein family. Plasmid 27:119–129
AP (2001) Selectively labeling the heterologous protein in Guignard L, Ozawa K, Pursglove SE, Otting G, Dixon NE (2002)
Escherichia coli for NMR studies: a strategy to speed up NMR NMR analysis of in vitro-synthesized proteins without
spectroscopy. J Magn Reson 148:142–146 purification: a high-throughput approach. FEBS Lett
Andronesi OC, Becker S, Seidel K, Heise H, Young HS, Baldus M 524:159–162
(2005) Determination of membrane protein structure and Hiller M, Krabben L, Vinothkumar KR, Castellani F, van Rossum
dynamics by magic-angle-spinning solid-state NMR spectros- BJ, Kuhlbrandt W, Oschkinat H (2005) Solid-state magic-angle
copy. J Am Chem Soc 127:12965–12974 spinning NMR of outer-membrane protein G from Escherichia
Appleyard AN, Herbert RB, Henderson PJ, Watts A, Spooner PJ coli. Chembiochem 6:1679–1684
(2000) Selective NMR observation of inhibitor and sugar Holloway PW (1973) A simple procedure for removal of Triton X-
binding to the galactose-H(+) symport protein GalP, of 100 from protein samples. Anal Biochem 53:304–308
Escherichia coli. Biochim Biophys Acta 1509:55–64 Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T, Shirouzu M,
Arkin IT, Russ WP, Lebendiker M, Schuldiner S (1996) Determin- Yokoyama S (2005) Expression of G protein coupled receptors
ing the secondary structure and orientation of EmrE, a multi- in a cell-free translational system using detergents and
drug transporter, indicates a transmenbrane four-helix bundle. thioredoxin-fusion vectors. Protein Expr Purif 41:27–37
Biochemistry 35:7233–7238 Jack DL, Storms ML, Tchieu JH, Paulsen IT, Saier MHJ (2000) A
Auer M, Kim MJ, Lemieux MJ, Villa A, Song J, Li XD, Wang DN broad-specificity multidrug efflux pump requiring a pair of
(2001) High-yield expression and functional analysis of homologous SMR-Typ proteins. J Bacteriol 182:2311–2313
Escherichia coli glycerol-3-phosphate transporter. Biochemis- Jaroniec CP, MacPhee CE, Bajaj VS, McMahon MT, Dobson CM,
try 40:6628–6635 Griffin RG (2004) High-resolution molecular structure of a
Baldwin SA, Henderson PJ (1989) Homologies between sugar peptide in an amyloid fibril determined by magic angle
transporters from eukaryotes and prokaryotes. Annu Rev spinning NMR spectroscopy. PNAS 101:711–716
Physiol 51:459–471 Jewett MC, Swartz JR (2004) Mimicking the Escherichia coli
Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A (2004) cytoplasmic environment activates long-lived and efficient cell-
Cell-free synthesis of a functional ion channel in the absence of free protein synthesis. Biotechnol Bioeng 86:19–26
a membrane and in the presence of detergent. Biochemistry Ketchem RR, Roux B, Cross TA (1997) High-resolution polypep-
43:12585–12591 tide structure in a lamellar phase lipid environment from solid
Betton JM (2003) Rapid translation system (RTS): a promising state NMR derived orientational constraints. Structure
alternative for recombinant protein production. Curr Protein 5:1655–1669
Pept Sci 4:73–80 Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD,
Butler PJG, Ubarretxena-Belandia I, Warne T, Tate CG (2004) The Breer H (1996) Expression of an olfactory receptor in
Escherichia coli multidrug transporter EmrE is a dimer in the Escherichia coli: purification, reconstitution, and ligand bind-
detergent-solubilised State. J Mol Biol 340:797–808 ing. Biochemistry 35:16077–16084
Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T,
Oschkinat H (2002) Structure of a protein determined by solid- Yokoyama S (1999) Cell-free production and stable-isotope
state magic-angle-spinning NMR spectroscopy. Nature labeling of milligram quantities of proteins. FEBS Lett
420:98–102 442:15–19
Chi-Rosso G, Toole BP (1987) Hyaluronate-binding protein of Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, Ruterjans H,
simian virus 40-transformed 3T3 cells: membrane distribution Glaubitz C, Bernhard F (2004) High level cell-free expression
and reconstitution into lipid vesicles. J Cell Biochem and specific labeling of integral membrane proteins. Eur J
33:173–183 Biochem 271:568–580
Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Koteiche HA, Reeves MD, Mchaourab HS (2003) Structure of the
Gordon SV, Eiglmeier K, Gas S, Barry CE, Tekaia F, Badcock substrate binding pocket of the multidrug transporter EmrE:
K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, site-derected spin labeling of transmembrane segment 1.
Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornby Biochemistry 42:6099–6105
T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver Krueger-Koplin RD, Sorgen PL, Krueger-Koplin ST, Rivera-Torres
K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, IO, Cahill SM, Hicks DB, Grinius L, Krulwich TA, Girvin ME
Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor (2004) An evaluation of detergents for NMR structural studies
K, Whitehead S, Barrell BG (1998) Deciphering the biology of of membrane proteins. J Biomol NMR 28:43–57
Mycobacterium tuberculosis from the complete genome se- Kunji ERS, Slotboom DJ, Poolman B (2003) Lactococcus lactis as
quence. Nature 393:537–544 host for overproduction of functional membrane proteins.
Curnow P, Lorch M, Charalambous K, Booth PJ (2004) The Biochim Biophys Acta-Biomembr 1610:97–108
reconstitution and activity of the small multidrug transporter Laws DD, Bitter HML, Jerschow A (2002) Solid-state NMR
EmrE is modulated by non-bilayer lipid composition. J Mol spectroscopic methods in chemistry. Angew Chem Int Ed
Biol 343:213–222 41:3096–3129
De Rossi E, Branzoni M, Cantoni R, Milano A, Riccardi G, Ciferri Lee KM, Androphy EJ, Baleja JD (1995a) A novel method for
O (1998) mmr, a Mycobacterium tuberculosis gene conferring selective isotope labeling of bacterially expressed proteins.
resistance to small cationic dyes and inhibitors. J Bacteriol J Biomol NMR 5:93–96
180:6068–6071 Lee YK, Kurur ND, Hemle M, Johannessen OG, Nielsen NC, Levitt
Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S (2004) In vitro MH (1995b) Efficient dipolar recoupling in the NMR of
synthesis of fully functional EmrE, a multidrug transporter, and rotating solids. A sevenfold symmetric radiofrequency pulse
study of its oligomeric state. Proc Natl Acad Sci USA sequence. Chem Phys Lett 242:304–309
101:1519–1524
 463
Liang WJ, Wilson KJ, Xie H, Knol J, Suzuki S, Rutherford NG, Patching SG, Herbert RB, O’Reilly J, Brough AR, Henderson PJ
Henderson PJ, Jefferson RA (2005) The gusBC genes of (2004b) Low 13C-background for NMR-based studies of
Escherichia coli encode a glucuronide transport system. ligand binding using 13C-depleted glucose as carbon source
J Bacteriol 187:2377–2385 for microbial growth: 13C-labeled glucose and 13C-forskolin
Lorch M, Fahem S, Kaiser C, Weber I, Mason AJ, Bowie JU, binding to the galactose-H+ symport protein GalP in Escher-
Glaubitz C (2005a) How to prepare membrane proteins for ichia coli. J Am Chem Soc 126:86–87
solid-state NMR: a case study on the alpha-helical integral Paternostre M-T, Roux M, Rigaud J-L (1988) Mechanisms of
membrane protein diacylglycerol kinase from E. coli. Chem- membrane protein insertion into liposomes during reconstitu-
biochem 6:1693–1700 tion procedures involving the use of detergents. 1. Solubiliza-
Lorch M, Lehner I, Siarheyeva A, Basting D, Pfleger N, Manolikas tion of large unilamellar liposomes (Prepared by Reverse-Phase
T, Glaubitz C (2005b) NMR and fluorescence spectroscopy Evaporation) by Triton X-100, Octyl Glucoside, and Sodium
approaches to secondary and primary active multidrug efflux Cholate. Biochemistry 27:2668–2677
pumps. Biochem Soc Trans 33:873–877 Paulsen IT, Skurray RA, Tam R, Saier MHJ, Turner R, Weiner JH,
Luca S, White JF, Sohal AK, Filippov DV, van Boom JH, Goldberg EB, Grinius LL (1996) The SMR family: a novel
Grisshammer R, Baldus M (2003) The conformation of family of multidrug efflux proteins involved with the efflux of
neurotensin bound to its G protein-coupled receptor. Proc lipophilic drugs. Mol Microbiol 19:1167–1175
Natl Acad Sci USA 100:10706–10711 Pelham HR, Jackson RJ (1976). An efficient mRNA-dependent
Ma C, Chang G (2004) Structure of the multidrug resistance efflux translation system from reticulocyte lysates. Eur J Biochem
transporter EmrE from Escherichia coli. PNAS 101:2852–2857 67:247–256
Madin K, Sawasaki T, Ogasawara T, Endo Y (2000) A highly Poolman B, Knol J, van der Does C, Henderson PJ, Liang WJ,
efficient and robust cell-free protein synthesis system prepared Leblanc G, Pourcher T, Mus-Veteau I (1996) Cation and sugar
from wheat embryos: plants apparently contain a suicide system selectivity determinants in a novel family of transport proteins.
directed at ribosomes. Proc Natl Acad Sci USA 97:559–564 Mol Microbiol 19:911–922
Masi M, Pages JM, Pradel E (2003) Overexpression and purification Pornillos O, Chen YJ, Chen AP, Chang G (2005) X-ray structure of
of the three components of the Enterobacter aerogenes AcrA- the EmrE multidrug transporter in complex with a substrate.
AcrB-TolC multidrug efflux pump. J Chromatogr B Analyt Science 310:1950–1953
Technol Biomed Life Sci 786:197–205 Putman M, van Veen HW, Degener JE, Konings,WN (2000)
Mason AJ, Siarheyeva A, Haase W, Lorch M, van Veen H, Glaubitz Antibiotic resistance: era of the multidrug pump. Mol
C (2004) Amino acid type selective isotope labelling of the Microbiol 36:772–773
multidrug ABC transporter LmrA for solid-state NMR studies. Rienstra CM, Tucker-Kellogg L, Jaroniec CP, Hohwy M, Reif B,
FEBS Lett 568:117–121 McMahon MT, Tidor B, Lozano-Pérez T, Griffin RG (2002) De
Mason AJ, Turner GJ, Glaubitz C (2005) Conformational hetero- novo determination of peptide struchture with solid-state
geneity of transmembrane residues after the Schiff base magic-angle spinning NMR spectroscopy. PNAS
reprotonation of bacteriorhodopsin: 15N CPMAS NMR of 99:10260–10265
D85N/T170C membranes. Febs J 272:2152–2164 Rigaud JL, Mosser G, Lacapere JJ, Olofsson A, Levy D, Ranck JL
McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, (1997) Bio-Beads: an efficient strategy for two-dimensional
Courtney L, Porwollik S, Ali J, Dante M, Du FY, Hou SF, crystallization of membrane proteins. J Struct Biol
Layman D, Leonard S, Nguyen C, Scott K, Holmes A, Grewal 118:226–235
N, Mulvaney E, Ryan E, Sun H, Florea L, Miller W, Stoneking Rigaud J-L, Levy D, Mosser G, Lambert O (1998) Detergent
T, Nhan M, Waterston R, Wilson RK (2001) Complete genome removal by non-polar poystyrene beads. Eur Biophys J
sequence of Salmonella enterica serovar typhimurium LT2. 27:305–319
Nature 413:852–856 Rotem D, Sal-man N, Schuldiner S (2001) In vitro monomer
Muchmore DC, McIntosh LP, Russell CB, Anderson DE, Dahlquist swapping in EmrE, a multidrug transporter from Escherichia
FW (1989) Expression and nitrogen-15 labeling of proteins for coli, reveals that the Oligomer is the functional unit. J Biol
proton and nitrogen-15 nuclear magnetic resonance. Methods Chem 276:48243–48249
Enzymol 177:44–73 Schaefer J, Stejskal EO, Buchdahl R (1977) Magic-angle C-13 Nmr
Muiry JA, Gunn TC, McDonald TP, Bradley SA, Tate CG, analysis of motion in solid glassy polymers. Macromolecules
Henderson PJ (1993) Proton-linked L-rhamnose transport, 10:384–405
and its comparison with L-fucose transport in Enterobacteri- Schwaiger M, Lebendiker M, Yerushalmi H, Coles M, Gröger A,
aceae. Biochem J 290:833–842 Schwarz C, Schuldiner S, Kessler H (1998) NMR investigation
Muth TR, Schuldiner S (2000) A membrane-embedded glutamate is of the multidrug transporter EmrE, an integral membrane
required for ligand binding to the multidrug transporter EmrE. protein. Eur Biophys J 254:610–619
Embo J 19:234–240 Seidel K, Etzkorn M, Heise H, Becker S, Baldus M (2005) High-
Ninio S, Schuldiner S (2003) Characterization of an archaeal resolution solid-state NMR studies on uniformly [13C,15N]-
multidrug transporter with a unique amino acid composition. labeled ubiquitin. Chembiochem 6:1638–1647
J Biol Chem 278:12000–12005 Sikora CW, Turner RJ (2005) Investigation of ligand binding to the
Noguchi N, Suwa J, Narui K, Sasatsu M, Ito T, Hiramatsu K, Song multidrug resistance protein EmrE by isothermal titration
JH (2005) Susceptibilities to antiseptic agents and distribution calorimetry. Biophys J 88:475–482
of antiseptic-resistance genes qacA/B and smr of methicillin- Sonar S, Patel N, Fischer W, Rothschild KJ (1993) Cell-free
resistant Staphylococcus aureus isolated in Asia during 1998 synthesis, functional refolding, and spectroscopic characteriza-
and 1999. J Med Microbiol 54:557–565 tion of bacteriorhodopsin, an integral membrane protein.
Patching SG, Baldwin SA, Baldwin AD, Young JD, Gallagher MP, Biochemistry 32:13777–13781
Henderson PJ, Herbert RB (2005) The nucleoside transport Soskine M, Steiner-Mordoch S, Schuldiner S (2002) Crosslinking of
proteins, NupC and NupG, from Escherichia coli: specific membrane-embedded cysteines reveals contact points in the
structural motifs necessary for the binding of ligands. Org EmrE oligomer. PNAS 99:12043–12048
Biomol Chem 3:462–470 Spirin AS, Baranov VI, Ryabova LA, Ovodov SY, Alakhov YB
Patching SG, Brough AR, Herbert RB, Rajakarier JA, Henderson (1988) A continuous cell-free translation system capable of
PJ, Middleton DA (2004a) Substrate affinities for membrane producing polypeptides in high yield. Science 242:1162–1164
transport proteins determined by 13C cross-polarization magic- Spooner PJR, Watts A, Henderson PJF (1993) Magic-angle spinning
angle spinning nuclear magnetic resonance spectroscopy. J Am Nmr-studies on the galactose-H+ symport protein of Escher-
Chem Soc 126:3072–3080 ichia-Coli. Biophys J 64:A51–A51
464
Spooner PJR, Rutherford NG, Watts A, Henderson PJF (1994a) Nmr Watts A (2005) Solid-state NMR in drug design and discovery for
observation of substrate in the binding-site of an active sugar-H membrane-embedded targets. Nature Reviews Drug Discovery
+ symport protein in native membranes. Proc Natl Acad Sci 4:555–568
USA 91:3877–3881 Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD
Spooner PJR, Watts A, Henderson PJF (1994b) Functional- (2003) Practical aspects of overexpressing bacterial secondary
characteristics of the sugar-H+ symport proteins from magic- membrane transporters for structural studies. Biochim Biophys
angle-spinning Nmr. Biophys J 66:A38–A38 Acta 1610:23–36
Spooner PJR, O’Reilly WJ, Homans SW, Rutherford NG, Winstone TL, Duncalf KA, Turner R (2002) Optimization of
Henderson PJF, Watts A (1998) Weak substrate binding to expression and the purification by organic extraction of the
transport proteins studied by NMR. Biophys J 75:2794–2800 integral membrane protein EmrE. Protein Expr Purif
Spooner PJ, Veenhoff LM, Watts A, Poolman B (1999) Structural 26:111–121
information on a membrane transport protein from nuclear Wu XL, Zilm KW (1993) Complete spectral editing in CPMAS
magnetic resonance spectroscopy using sequence-selective NMR. J Magn Reson Ser A 102:205–213
nitroxide labeling. Biochemistry 38:9634–9639 Xie H, Patching SG, Gallagher MP, Litherland GJ, Brough AR,
Striebek M (2005) Membrane proteins of known structure. MPI fuer Venter H, Yao SYM, Ng AML, Young JD, Herbert RB,
Biophysik, http://www.mpibp-frankfurt.mpg.de/michel/public/ Henderson PJF, Baldwin SA (2004) Purification and properties
memprotstruct.html of the Escherichia coli nucleoside transporter NupG, a
Tate CG, Kunji ER, Lebendiker M, Schuldiner S (2001) The paradigm for a major facilitator transporter sub-family. Mol
projection structure of EmrE, a proton-linked multidrug Membr Biol 21:323–336
transporter from Escherichia coli, at 7 A resolution. Embo J Yamaguchi S, Tuzi S, Bowie JU, Saito H (2004) Secondary structure
20:77–81 and backbone dynamics of Escherichia coli diacylglycerol
Tate CG, Ubarretxena-Belandia I, Baldwin JM (2003) Conforma- kinase, as revealed by site-directed solid-state C-13 NMR.
tional changes in the multidrug transporter EmrE associated Biochimica Et Biophysica Acta-Proteins and Proteomics
with substrate binding. J Mol Biol 332:229–242 1698:97–105
Ubarretxena-Belandia I, Baldwin JM, Schuldiner S, Tate CG (2003) Yerushalmi H, Lebendiker M, Schuldiner S (1995) EmrE, an E. coli
Three-dimensional structure of the bacterial multidrug trans- 12kDa Multidrug Transporter, exchanges toxic cations and H+
porter EmrE shows it is an asymmetric homodimer. Embo J and is soluble in organic solvents. J Biol Chem 270:6856–6863
22:6175–6181 Yerushalmi H, Lebendiker M, Schuldiner S (1996) Negative
van Veen HW, Callaghan R, Soceneantu L, Sardini A, Konings WN, dominance studies demonstrate the oligomeric structure of
Higgins CF (1998) A bacterial antibiotic-resistance gene that EmrE, a multidrug antiporter from Escherichia coli. J Biol
complements the human multidrug-resistance P-glycoprotein Chem 271:31044–31048
gene. Nature 391:291–295 Zech SG, Wand AJ, McDermott AE (2005) Protein structure
Veenhoff LM, Heuberger EH, Poolman B (2001) The lactose determination by high-resolution solid-state NMR spectrosco-
transport protein is a cooperative dimer with two sugar py: application to microcrystalline ubiquitin. J Am Chem Soc
translocation pathways. Embo J 20:3056–3062 127:8618–8626
Viitanen P, Newman MJ, Foster DL, Wilson TH, Kaback HR (1986)
Purification, reconstitution, and characterization of the lac
permease of Escherichia coli. Methods Enzymol 125:429–452