Microscale NMR screening of new detergents for membrane protein structural biology

Article abstract of DOI:10.1021/ja077863d

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly ²H,¹⁵N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ ¹⁵N,¹H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.

Full text of DOI:10.1021/ja077863d

Published on Web 05/14/2008
Microscale NMR Screening of New Detergents for Membrane
Protein Structural Biology
Qinghai Zhang,*,† Reto Horst,† Michael Geralt,† Xingquan Ma,† Wen-Xu Hong,†
M. G. Finn,‡,§ Raymond C. Stevens,†,‡ and Kurt Wu¨thrich*,†,‡,§
Department of Molecular Biology, Department of Chemistry, Skaggs Institute for Chemical
Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received October 16, 2007; E-mail: qinghai@scripps.edu; wuthrich@scripps.edu
Abstract: The rate limiting step in biophysical characterization of membrane proteins is often the availability
of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear
magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins
for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a
series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane
proteins, using the previously well characterized ꢀ-barrel E. coli outer membrane protein OmpX as a test
case. Fold screening was thus achieved with microgram amounts of uniformly 2H,15N-labeld OmpX and
affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient
selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for
membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that
yield 2D [15N,1H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.
However, its use for the study of IMPs is still limited,9–12 for
three major reasons: (1) Reconstitution of IMPs with amphiphilic
Introduction
Integral membrane proteins (IMPs) are embedded in lipid
bilayers, performing a wide variety of essential functions in
cellular biochemistry and cell-cell communication. IMPs are
encoded by about one-third of a typical mammalian genome
and comprise more than half of all current human drug
targets.1–3 The structural characterization of IMPs has been an
important challenge for decades, which has only recently been
addressed with increasing success, primarily by X-ray crystal-
lography. The use of detergents to extract and purify IMPs from
their natural membrane environments is crucial for X-ray
crystallography, which further requires the additional challenging
step of making high-quality crystals containing the IMP,
detergent, and possibly other additives.4,5 In large part due to
the difficulties of these operations, only a relatively small
number of IMP structures have been solved to date.6
molecules gives rise to a substantial increase in molecular mass
of the NMR target. (2) IMPs tend to be unstable in non-native
environments. (3) Sample preparation is demanding due to low
yields in membrane protein production and the requirement of
stable-isotope labeling. While significant progress has been made
in using solution NMR for larger particles.13 the difficulties of
sample preparation must so far be addressed on a case-by-case
basis.
Integral membrane proteins have a pronounced tendency to
aggregate or fold incorrectly in the detergents used to extract
them from their native membrane lipids; this is a critical problem
for their structural, biochemical, and biophysical characteriza-
tion. Reconstitution of IMPs into detergent micelles by refolding
is beneficial, because it allows the investigator to take advantage
of more efficient synthesis of isotope-labeled proteins in an
aggregated form or possibly also by cell-free methods.12,14–20
Solution NMR spectroscopy has found broad application as
a complementary technique to X-ray crystallography in the
structural characterization of biological macromolecules.7,8
(7) Wu¨thrich, K. Angew. Chem., Int. Ed. 2003, 42, 3340–63.
(8) Ferentz, A. E.; Wagner, G. Q. ReV. Biophys. 2000, 33, 29–65.
(9) Sanders, C. R.; So¨nnichsen, F. Magn. Reson. Chem. 2006, 44, S24–
40.
† Department of Molecular Biology
‡ Department of Chemistry.
§ Skaggs Institute for Chemical Biology.
(10) Sanders, C. R.; Oxenoid, K. Biochim. Biophys. Acta 2000, 1508, 129–
45.
(1) Hopkins, A. L.; Groom, C. R. Nat. ReV. Drug DiscoVery 2002, 1,
727–30.
(11) Arora, A.; Tamm, L. K. Curr. Opin. Struct. Biol. 2001, 11, 540–7.
(12) Ferna´ndez, C.; Wu¨thrich, K. FEBS Lett. 2003, 555, 144–50.
(13) The size limitations of solution NMR, exemplified in many early
studies of small membrane-associated peptides or membrane protein
fragments, have been remarkably improved by higher field instru-
mentation and new techniques such as uniform and selective isotope
labeling and heteronuclear multidimensional NMR spectroscopy. For
example, transverse relaxation-optimized spectroscopy (TROSY)
[Wu¨thrich, K.; Wider, G. Encycl. Nucl. Magn. Reson 2002, 9, 468–
477. ] has pushed the size limit beyond 100 kDa also in applications
to the study of IMPs.
(2) Lundstrom, K. Curr. Protein Pept. Sci. 2006, 7, 465–70.
(3) Wallin, E.; von Heijne, G. Protein Sci. 1998, 7, 1029–38.
(4) Membrane protein purification and crystallization, a practical guide,
2nd ed.; Hunte, C., von Jagow, G., Scha¨gger, H., Eds.; Academic Press:
San Deigo, CA, 2003.
(5) Methods and Results in Crystallization of Membrane Proteins; Iwata,
S., Ed.; International University Line: La Jolla, CA, 2003.
(6) See H. Michel’s and S. White’s summaries at http://www.mpibp-
frankfurt.mpg.de/michel/public/memprotstruct.html
blanco.biomol.uci.edu/Membrane_Proteins_xtal.html.
and
http://
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2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 7357–7363 7357

A R T I C L E S
Zhang et al.
A number of IMPs, mostly ꢀ-barrel proteins, have been
successfully refolded into detergents, lipids, or detergent-lipid
mixtures, and the available comparisons have shown the refolded
structures to exhibit nearly identical properties to the native
proteins.15,21–26 In general, however, our knowledge of how to
correctly fold a large pool of IMPs is still very limited, lagging
far behind that for soluble proteins.27 It is therefore of great
value to further explore appropriate folding conditions for IMP
targets of biochemical and structural interest. Synthetic chem-
istry has a role to play in this endeavor, since the properties of
small-molecule amphiphiles contribute in many ways to the
isolation, stabilization, and characterization of IMPs.
In solution NMR studies, detergent micelles, as the smallest
membrane-replacing agents, are the prime choice for IMP
reconstitution.9–11,15–17,28 The number of detergents suitable for
NMR analysis of IMPs is very limited, and only a few have
been characterized for potential general use, including ꢀ-D-
octylpyranoglucoside (OG), n-dodecylphosphocholine (DPC),
1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and single-
chain lysophopholipid analogues (e.g., LPPG, 1-palmitoyl-2-
hydroxy-sn-glycero-3-[phosphor-RAC-(1-glycerol); LMPC,
1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine).9,29–31
Fold screening of soluble proteins has been performed using
microcoil1D1HNMRspectroscopy.32,33Membraneprotein-detergent
mixed micelles are more challenging targets. The detergents
used to reconstitute and solubilize the proteins introduce a
background that interferes with a straightforward interpretation
of the protein signals in 1D 1H NMR spectra, and the molecular
weight of the protein-detergent micelles is significantly larger
than that for the proteins alone, typically 50-100 kDa even for
small membrane proteins.12,16,21 As a result, it is necessary to
use isotope-labeled IMP-detergent mixed micelles for screening
by NMR.
Selection of an appropriate detergent for NMR is a significant
challenge. Experience from IMP crystallization so far provides
little help: as might be expected given the different requirements
of intermolecular association in the two situations, a detergent
that is successful for crystallizing a specific IMP is often
unsuitable for its NMR characterization. In a previous study,30
a selection of commercially available detergents were fold-
screened with 2D [15N,1H]-HSQC experiments, using a con-
ventional 5 mm NMR probe, and the single-chain lipid-like
LPPG was singled out as a good detergent for selected R-helical
membrane proteins. However, with conventional NMR probe
technology, milligram amounts of protein are generally required
for each experiment, which makes serial detergent screening
impractical. We report here the screening of both commercial
and newly synthesized detergents for successful NMR sample
preparation of a well-characterized IMP with the use of an NMR
microprobe, which requires only microgram quantities of protein
for each measurement.
Methods
Detergent Synthesis and Purification. The synthesis of phos-
phocholine detergents followed the standard procedures.34 Details
of the synthesis and characterization of new detergents may be
found in the Supporting Information. Briefly, phosphocholines with
branched alkyl chains were synthesized directly from the respective
commercially available secondary alcohols. The ester-linked de-
tergents were made by coupling of the hydroxyl group of 1-hy-
droxylethylphosphocholine (for the preparation of single chain
detergents) or 138-Fos (for dialkyl chain detergents) with individual
carboxylic acids. The amide-linked detergents were prepared by
coupling the amine group of 1-aminoethylphosphocholine (for
single-chain detergents) or 168-Fos (for dialkyl chain detergents)
with the respective carboxylic acid chloride. The coupling procedure
is described in the Supporting Information. Each detergent used
for refolding and NMR experiments was prepared in 100 mg
batches, purified by reversed-phase HPLC (after silica-gel column
chromatography) to >99% purity, and its structure was confirmed
by NMR and by mass spectrometry (Supporting Information).
Determination of Critical Micelle Concentration (CMC).
CMC values of detergents were determined by monitoring the
fluorescence of the ammonium salt of 8-anilino-1-naphtalenesulfonic
acid (ANS), which becomes highly fluourescent (λex ) 405 nm;
λem ) 465 nm) when incorporated into the hydrophobic micellar
environment.35 Solutions containing 10 µM ANS and a range of
concentrations for each detergent were examined on a DXT880
multiplate spectrofluorimeter (Beckman Coulter). The CMC is
defined as the breakpoint in the plot of fluorescence intensity vs
concentration.
(14) Etezady-Esfarjani, T.; Hiller, S.; Villalba, C.; Wu¨thrich, K. J. Biomol.
NMR 2007, 39, 229–238.
(15) Arora, A.; Abildgaard, F.; Bushweller, J. H.; Tamm, L. K. Nat. Struct.
Biol. 2001, 8, 334–8.
(16) Ferna´ndez, C.; Adeishvili, K.; Wu¨thrich, K. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 2358–63.
(17) Hwang, P. M.; Choy, W. Y.; Lo, E. I.; Chen, L.; Forman-Kay, J. D.;
Raetz, C. R.; Prive´, G. G.; Bishop, R. E.; Kay, L. E. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 13560–5.
(18) Roosild, T. P.; Greenwald, J.; Vega, M.; Castronovo, S.; Riek, R.;
Choe, S. Science 2005, 307, 1317–21.
(19) Ferna´ndez, C.; Hilty, C.; Bonjour, S.; Adeishvili, K.; Pervushin, K.;
Wu¨thrich, K. FEBS Lett. 2001, 504, 173–8.
(20) Koglin, A.; Klammt, C.; Trbovic, N.; Schwarz, D.; Schneider, B.;
Scha¨fer, B.; Lo¨hr, F.; Bernhard, F.; Do¨tsch, V. Magn. Reson. Chem.
2006, 44, S17–23.
(21) Ferna´ndez, C.; Hilty, C.; Wider, G.; Gu¨ntert, P.; Wu¨thrich, K. J. Mol.
Biol. 2004, 336, 1211–1221.
(22) Pautsch, A.; Schulz, G. E. Nat. Struct. Biol. 1998, 5, 1013–7.
(23) Pautsch, A.; Schulz, G. E. J. Mol. Biol. 2000, 298, 273–82.
(24) Vogt, J.; Schulz, G. E. Structure 1999, 7, 1301–9.
(25) Huang, K. S.; Bayley, H.; Liao, M. J.; London, E.; Khorana, H. G.
J. Biol. Chem. 1981, 256, 3802–9.
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44.
(27) Booth, P. J.; Curnow, P. Curr. Opin. Struct. Biol. 2006, 16, 480–8.
(28) Bicelles (a mixture of detergents and lipids) and amphipols (am-
phiphilic polymer detergents) have recently been used in the solution
NMR study of native integral membrane proteins: (a) Poget, S. F.;
Cahill, S. M.; Girvin, M. E. J. Am. Chem. Soc. 2007, 129, 2432–3.
(b) Zoonens, M.; Catoire, L. J.; Giusti, F.; Popot, J. L. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 8893–8.
Expression and Purification of OmpX in D2O Medium. OmpX
was precloned in the pET 3b plasmid and transformed into E. coli
BL-21 (DE3) pLysS (Stratagene) competent cells for expression.
One colony was used to inoculate a culture flask containing 20
mL of LB broth with the necessary antibiotics and shaken at 37 °C
overnight. The cell culture was adapted to D2O by inoculating 4
mL of standard M9 minimal medium containing 33% v/v D2O with
40 µL of this LB culture and shaken at 37 °C overnight. 100 µL of
the 33% D2O culture were then used to inoculate 10 mL of M9
(29) Vinogradova, O.; So¨nnichsen, F.; Sanders, C. R. J. Biomol. NMR 1998,
11, 381–6.
(30) Krueger-Koplin, R. D.; Sorgen, P. L.; Krueger-Koplin, S. T.; Rivera-
Torres, I. O.; Cahill, S. M.; Hicks, D. B.; Grinius, L.; Krulwich, T. A.;
Girvin, M. E. J. Biomol. NMR 2004, 28, 43–57.
(31) Brown, L. R.; Bo¨sch, C.; Wu¨thrich, K. Biochim. Biophys. Acta 1981,
642, 296–312.
(32) Peti, W.; Norcross, J.; Eldridge, G.; O’Neil-Johnson, M. J. Am. Chem.
Soc. 2004, 126, 5873–8.
(34) Tegoulia, V. A.; Rao, W. S.; Kalambur, A. T.; Rabolt, J. R.; Cooper,
S. L. Langmuir 2001, 17, 4396–404.
(35) De Vendittis, E.; Palumbo, G.; Parlato, G.; Bocchini, V. Anal. Biochem.
1981, 115, 278–86.
(33) Peti, W.; Page, R.; Moy, K.; O’Neil-Johnson, M.; Wilson, I. A.;
Stevens, R. C.; Wu¨thrich, K. J. Struct. Funct. Genom. 2005, 6, 259–
67.
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Microscale NMR for Membrane Protein Structural Biology
A R T I C L E S
minimal medium with 66% v/v D2O and similarly shaken overnight.
A uniformly 2H,15N-labeled OmpX sample was prepared by
inoculating 1 L of standard M9 minimal medium containing 99%
D2O (Spectra Gases) and 1 g/L of [15N]-ammonium chloride (CIL)
with the resulting 10 mL of D2O-adapted culture. This culture was
shaken at 37 °C until the cell density reached an OD600 of
approximately 1.0, and protein expression was then induced with
1 mM isopropyl-R-D-thiogalactopyranoside. Upon reaching the
stationary growth phase after approximately 4 h, the cells were
harvested at 5000 g for 10 min. TE buffer (20 mM Tris-HCl at pH
) 8.0, 5 mM EDTA) was used to resuspend the cell pellet in a
volume corresponding to approximately 10 times the wet cell mass
in grams and sonicated for 25 min using a Misonix 3000 sonicator.
The solution was pelleted at 4300 g for 1 h, and the supernatant
was discarded. The remaining insoluble pellet was resuspended in
the same volume of TE buffer plus 2% (v/v) Triton X-100 at room
temperature, pelleted at 4300 g for 30 min, and the supernatant
was discarded. Triton was then removed by repeating the same
procedure with TE buffer. OmpX was recovered from the inclusion
bodies by resuspending the cell debris in TE buffer plus 6 M urea
for 2 h at room temperature. Remaining cellular debris were then
pelleted at 48 000 g for 20 min at 4 °C, and the unfolded OmpX in
the supernatant was recovered for ion exchange purification.
¨
Purification steps were performed using an AKTA purifier
(Amersham/GE) with a 5 mL HiTrap Q HP anion exchange column
(Amersham/GE). OmpX was bound to the column using buffer A
(20 mM Tris-HCl at pH ) 8.5, 6 M urea) and eluted with a linear
NaCl gradient of 0-1 M at a rate of 33.3 mM per minute. All
impure fractions were exchanged into buffer A and repurified, using
the previously mentioned steps. The pooled pure OmpX fractions
were concentrated to 1 mL using Vivaspin 2 (Sartorius) concentra-
tors with a 3 kDa molecular weight cutoff. The concentrated sample
was desalted with buffer A using a 5 mL HiTrap desalting column
(Amersham/GE).
Reconstitution of OmpX into Detergent Micelles for SDS
Gel Electrophoresis. Unfolded OmpX (11.9 mg/mL, 20 mM Tris
at pH 8.5, 6 M urea) was diluted 30-fold with 1% (w/v) detergent
solution at 25 °C (1% detergent concentration is well above the
CMC for each of the commercial or newly synthesized detergents
used). The detergents were predissolved in either of two buffers,
50 mM citrate with 1 mM EDTA for the pH range 3.4-6.0 or 50
mM Tris-HCl with 1 mM EDTA for the pH range 6.0-9.8. The
solution was incubated at 25 °C for 3 h before running the SDS
gel electrophoresis (using 15% polyacrylamide gels and without
boiling the samples). The gels were stained with Coomassie blue,
and the fractions of refolded protein were estimated with
densitometry.
Reconstitution of OmpX into Detergent Micelles for NMR
Spectroscopy. A previously established protocol16 for OmpX
reconstitution in DHPC (Avanti Polar Lipids) detergent micelles
was modified for microscale experiments with a variety of widely
different detergents (Figure 1). At 4 °C, 100 µL of unfolded OmpX
at a concentration of 10 mg/mL were added to 600 µL of refolding
buffer (20 mM Tris-HCl at pH ) 8.5, 5 mM EDTA, 600 mM L-Arg,
and 2% (w/v) detergent) over a period of 4 h with vigorous stirring.
The resulting OmpX solution was vigorously stirred at 4 °C for
∼16 h. The detergent-refolded OmpX was then collected and
exchanged into NMR buffer (20 mM sodium phosphate at pH )
6.8, 100 mM NaCl, 0.3% NaN3, 10% D2O, detergent) by way of
concentration and dilution using Vivaspin 500 concentrators (10 000
molecular weight cutoff), alternatively using NMR buffer with and
without 2% (w/v) of detergent. The final sample was concentrated
to 50 µL.
Figure 1. Flowchart for microscale OmpX reconstitution in detergent
micelles. Solid lines indicate the route to reconstituted OmpX for structural
studies, and dashed lines indicate the salvage pathways for precipitated
OmpX. The buffers are described in the Methods section. Asterisks indicate
procedural steps that were decisively improved with the use of the new
detergents (see Table 3).
recorded as described previously.16,36 Details of the parameter
settings are given in the legend of Figure 4. Data processing and
analysis were carried out using TOPSPIN 1.3 (Bruker) and
XEASY,37 respectively.
Results and Discussion
OmpX is a 148-residue, 16.5 kD outer membrane protein with
an eight-stranded ꢀ-barrel structure and structure-grade NMR
spectra available for comparison.16,21 In general, the transmem-
brane domains of ꢀ-barrel proteins are less hydrophobic than
those of helical IMPs, making the former highly soluble in
chaotropic reagents as a starting point for refolding studies.
Twenty-three commercially available detergents (Table 1)
were selected for OmpX reconstitution, which included the most
popular representatives of the sugar (glucoside and maltoside),
zwitterionic (Fos-choline series and lauryldimethylamine-N-
oxide), cholate (sodium cholate, CHAPS, and CHAPSO), and
amphipol (PMAL series: amphiphilic polymer detergents)
classes; selected structures are shown in the Supporting Infor-
mation (Figure S1).
OmpX is resistant to denaturation by SDS (as are many
ꢀ-barrel membrane proteins38–43), yet this detergent inhibits
(36) Pervushin, K.; Riek, R.; Wider, G.; Wu¨thrich, K. Proc. Natl. Acad.
Sci. U.S.A. 1997, 94, 12366–71.
(37) Bartels, C.; Xia, T.; Billeter, M.; Gu¨ntert, P.; Wu¨thrich, K. J. Biomol.
NMR 1995, 6, 1–10.
(38) Kleinschmidt, J. H. Chem. Phys. Lipids. 2006, 141, 30–47.
(39) Tamm, L. K.; Hong, H.; Liang, B. Biochim. Biophys. Acta 2004, 1666,
250–63.
(40) Ohnishi, S.; Kameyama, K. Biochim. Biophys. Acta 2001, 1515, 159–
66.
NMR Spectroscopy. All NMR experiments were recorded at
25 °C on a Bruker DRX-700 spectrometer (BrukerBiospin, Billerica,
MA) equipped with a 1 mm TXI microprobe. The 1 mm NMR
capillaries were filled with 7 µL of the solution containing the mixed
OmpX-detergent micelles, using a 10 µL Gilson Syringe (Gilson
Co., Reno, NV). 2D [15N,1H]-TROSY correlation experiments were
(41) Conlan, S.; Bayley, H. Biochemistry 2003, 42, 9453–65.
(42) Pocanschi, C. L.; Apell, H. J.; Puntervoll, P.; Hogh, B.; Jensen, H. B.;
Welte, W.; Kleinschmidt, J. H. J. Mol. Biol. 2006, 355, 548–61.
(43) Surrey, T.; Ja¨hnig, F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7457–
61.
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A R T I C L E S
Zhang et al.
Table 1. List of Commercial Detergents Used (Anatrace, Inc.) and Estimated OmpX Refolding Yields ((5% error) by SDS-PAGE Assay
detergent
yield (%)
detergent
yield (%)
detergent
yield (%)
n-decyl-ꢀ-D-maltopyranoside
n-undecyl-ꢀ-D-maltopyranoside
n-dodecyl-ꢀ-D-maltopyranoside
n-tridecyl-ꢀ-D-maltopyranoside
CYMAL-6
CYMAL-7
C-HEGA-11
MEGA-8
40
55
55
60
55
60
35
<5
Triton X-100
Fos-choline-10
Fos-choline-11
Fos-choline-12
Fos-choline-13
Fos-choline-14
n-octyl-ꢀ-D-gluopyranoside
n-nonyl-ꢀ-D-gluopyranoside
20
>90
>90
>90
>90
>90
<5
sodium cholate
CHAPS
CHAPSO
PMAL-C10
PMAL-C12
PMAL-C16
lauryldimethylamine-N-oxide
<5
<5
<5
<5
<5
10
80
10
detergents, and amphipols were least efficient, with <5%
refolding in most of the cases. The larger maltose headgroup
provided some improvement in refolding yield (40-60%), and
lauryldimethylamine-N-oxide (LDAO) also gave a good yield
(80%).
A majority of known favorable NMR detergents also fall into
the phosphocholine class.9,29–31 In order to improve upon the
phosphocholine theme, we introduced two types of modifications
to make the detergents more closely resemble natural phospho-
lipids (Figure 3). First, a spacer group to mimic the glycerol
motif in lipids was inserted between the charged, potentially
denaturing phosphocholine headgroup and the alkyl chain
(Figure 3a). Second, short branches were added to mimic the
dialkyl chain structure of lipids while maintaining their solubility
(Figure 3b). Lysophospholipids and DHPC represent the former
and latter patterns, respectively, and are generally regarded as
mild phospho-detergents.
We synthesized 42 new phosphocholine detergents (Figures
3 and S2) to test these design principles. The polar spacer groups
inserted between the zwitterionic phosphocholine and the alkyl
chain include secondary hydroxyl and amine groups (138-Fos
and 168-Fos), ethylene oxide (185-Fos), ester (115-Fos, 141-
Fos, and 142-Fos), amide (179-Fos, 173-Fos, and 169-Fos), and
others (see Figure S2). For dialkyl chain detergents, the short
chain was conveniently appended through an ester or amide
bond or preinstalled on a secondary alcohol (branched deter-
gents). Partially fluorinated detergents (e.g., 169-Fos) were also
synthesized for their reportedly milder action.45
Similar refolding experimentations to Figure 2b yielded the
data summarized in Table 2. Quite a number of the newly
synthesized phosphocholine detergents could refold OmpX as
efficiently as commercial ones. We have synthesized and
evaluated other classes of new detergents, and the present study
now corroborates that the phosphocholine series is among the
top performers among all our new detergent structures for
refolding OmpX. From the results shown in Table 2, it is
apparent that detergents bearing dialkyl chains (branched
through either an ester or amide linkage) are much less effective
in refolding OmpX than analogues with single straight alkyl
chains. Almost all the best candidates, with >90% refolding,
are either single alkyl chain detergents or those with a single
methyl group on the branch (30-Fos, 31-Fos, 34-Fos, and 38-
Fos).
To extend the electrophoresis assay of Figure 2b, we used
uniform 2H- and 15N-labeling of the protein OmpX and TROSY-
type 2D [15N,1H]-correlation NMR spectroscopy to assess the
fold of OmpX in mixed micelles with different detergents. To
accomplish this goal, a screening protocol using microcoil NMR
equipment was first tested with OmpX-DHPC mixed micelles
(OmpX/DHPC). OmpX/DHPC has previously been studied in
Figure 2. (a) SDS-gel electrophoresis of OmpX samples at pH 8.5. Lane
1: unfolded OmpX in 6 M urea. Lane 2: OmpX refolded in DDM. Lane 3:
OmpX refolded in TPC. (b) pH effect on OmpX refolding. F is the fraction
of the OmpX that was refolded. The protein (0.4 mg/mL), urea (0.2 M),
and detergent (1 wt %/vol) were incubated at room temperature for 3 h,
followed by quick analysis by SDS-PAGE. The fraction of folded OmpX
was determined by densitometric analysis of the Coomassie stained gels
((5% error). The relatively high concentration ensured that each detergent
was present above CMC; the long incubation time ensured completion of
the refolding process before the start of each measurement.44
refolding of the protein once it is denatured. Thus, folded and
unfolded OmpX can be easily distinguished by SDS gel
electrophoresis, migrating at 18 and 16 kD, respectively, on the
standard Laemmli gels (15% polyacrylamide, Figure 2a).
Interestingly, the folded and unfolded OmpX migrated in a
reversed order (12 and 19 kD, respectively) on Bis-Tris gels (4
- 12% gradient polyacrylamide). This observation of the
inverted running sequence of the folded and unfolded forms of
ꢀ-barrel membrane proteins on different SDS gel matrices has
not been recorded before to the best of our knowledge. The
readout by SDS-PAGE was used to determine the OmpX folding
efficiency in the presence of two well-known detergents,
n-dodecyl-ꢀ-D-maltopyranoside (DDM) and Fos-choline-13
(TPC), as a function of pH (Figure 2b). OmpX refolding was
thus found to be more efficient in TPC than in DDM in general
and was increasingly favored at higher pH, with >90% folded
protein achieved at pH 8.0 in TPC. The addition of folding
additives, such as L-arginine and trimethylamine oxide, had no
observable effect (data not shown).
Standardized folding conditions (pH 8.5 and 1% detergent
concentration) were then used to screen the 23 aforementioned
commercial detergents. As summarized in Table 1, the Fos-
choline series (hydrophobic chain length 10-14 carbons) were
the only candidates to support almost complete refolding of
OmpX. In contrast, detergents with only glucose as the polar
head (including octyl- and nonylglucoside), cholate-based
(44) Kleinschmidt, J. H.; Wiener, M. C.; Tamm, L. K. Protein Sci. 1999,
8, 2065–71.
(45) Chaudier, Y.; Zito, F.; Barthe´le´my, P.; Stroebel, D.; Ame´duri, B.;
Popot, J. L.; Pucci, B. Bioorg. Med. Chem. Lett. 2002, 12, 1587–90.
9
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Microscale NMR for Membrane Protein Structural Biology
A R T I C L E S
Figure 3. Representative examples of designed phosphocholine structures. (a) Single chain detergents. (b) Dialkyl-chain phospholipid (DOPC) and lipid-
like detergents. The branched alkyl-chain-containing detergents (34-Fos and 193-Fos) were assigned to both classes. Detergents examined by NMR are
shown in blue and red, with the latter exhibiting the best performance.
great detail, so that nearly complete polypeptide backbone
assignments and a solution structure are available.16,21 The 2D
[15N,1H]-TROSY correlation spectrum of OmpX/DHPC mea-
sured with the 1 mm TXI microprobe shows a wide dispersion
of cross peaks with roughly uniform intensities and line shapes
(Figure 4a). Comparison with the previously determined chemi-
cal shift list (BMRB accession code 4936) showed that all cross
peaks were visible, with the sole exception of the four peaks
belonging to the polypeptide segment E32-S35. This shows
that our screening protocol, which required only about 10 µL
of protein solution (active volume in the NMR microcoil ) 7
µL), provided high-quality [15N,1H]-correlation maps that can
be used as diagnostic fingerprints to assess the conformation of
OmpX in mixed micelles with detergents.
micelles with five newly synthesized detergents. The structures
are representative of distinct classes (spacer groups) that fully
refolded the protein (138-Fos, CMC 0.1%; 179-Fos, CMC
0.062%; 115-Fos, CMC 0.025%; 34-Fos, CMC 0.036%; 185-
Fos, CMC 0.01%). A widely used commercial phosphocholine
detergent (TPC, CMC 0.027%) was also used for comparison.
OmpX was readily reconstituted into these detergent micelles
at high concentrations (100 µL of 10 mg/mL unfolded OmpX
was refolded into 600 µL of detergent solution by slow addition);
no protein precipitation was observed, and SDS gel electro-
phoresis experiments confirmed complete solubilization.
The 2D [15N,1H]-TROSY correlation spectra of mixed
OmpX/138-Fos and OmpX/179-Fos micelles (Figure 4b and
c) are both closely similar to the spectrum of OmpX/DHPC,
with respect to the number of cross-peaks, their chemical shift
dispersion, and the peak intensity distribution, indicating that
Using the electrophoresis results to guide the selection of
candidates, we examined [2H,15N]-labeled OmpX in mixed
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A R T I C L E S
Zhang et al.
Table 2. List of Newly Synthesized Phosphocholine Detergents
and Estimated OmpX Refolding Yields ((5% Error) by SDS-PAGE
Assay
Table 3. Microscale OmpX Reconstitution with Different
Detergents
OmpX precipitation
in refolding buffera
OmpX precipitation
in NMR bufferb
NMR
qualityc
detergent
detergenta
yield (%)
detergenta
yield (%)
detergenta
yield (%)
DHPC
138-Fos
179-Fos
115-Fos
TPC
∼30%
0%
∼40%
0%
++
++
+
30-Fos
31-Fos
34-Fos
35-Fos
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
>90
168-Fos
167-Fos
165-Fos
140-Fos
172-Fos
117-Fos
37-Fos
190-Fos
177-Fos
192-Fos
141-Fos
137-Fos
176-Fos
33-Fos
>90
84
71
70
69
67
63
60
60
58
57
56
55
55
171-Fos
142-Fos
193-Fos
173-Fos
170-Fos
175-Fos
178-Fos
194-Fos
174-Fos
195-Fos
169-Fos
144-Fos
145-Fos
41-Fos
55
52
50
49
46
45
44
44
43
30
28
28
25
<5
0%
0%
0%
0%
0%
0%
0%
0%
-
-
38-Fos
34-Fos
185-Fos
-
115-Fos
116-Fos
180-Fos
179-Fos
185-Fos
228-Fos
229-Fos
182-Fos
138-Fos
0%
0%
-
a Recoverable OmpX loss due to precipitation during refolding.
b Recoverable OmpX loss due to buffer exchange (Figure 1). c 2D
[
15N,1H]-TROSY spectra (Figure 4) rated “++” for spectra suitable for
a structure determination, “+” for spectra with detergent artifacts, and
“-” for spectra showing that OmpX was solubilized, but not
reconstituted.
a Detergent structures can be found in Figures 3 and S2.
138-Fos or in 179-Fos can efficiently be achieved, whereas with
DHPC several recovery cycles were needed in order to obtain
an NMR sample (Figure 1).
The 2D [15N,1H]-TROSY correlation spectra of the mixed
micelles with OmpX/115-Fos, OmpX/TPC, OmpX/34-Fos, and
OmpX/185-Fos (Figure 4d-g) all show a cluster of broad lines
in the center of the spectrum and variable numbers of additional,
resolved cross peaks of unequal shapes and intensities, which
indicates that these protein samples are not homogeneously
folded and probably form nonspecific soluble aggregates with
these detergents.
In conclusion, the combined use of preliminary screening of
a library of candidate detergents for OmpX refolding using SDS
gel electrophoresis and further characterization of the OmpX
conformation in mixed micelles with selected detergents by 2D
[15N,1H]-TROSY correlation NMR spectroscopy resulted in the
identification of two new detergents, 138-Fos and 179-Fos, that
afford reconstitution of OmpX in its native form more efficiently
than DHPC. These two single-chain phosphocholine detergents
are the structural mimics of the lysophospholipids that have been
shown to retain the folded conformation of several R-helical
membrane proteins in solution NMR studies.30 The use of
microcoil NMR technology was key to this success, reducing
the cost of the protein as well as the novel amphiphiles, which
are often initially made in small quantities.47–53
An important result is the observation that dialkyl chain lipid-
like detergents are not as efficient as straight-chain detergents
in refolding OmpX, which is consistent with the known
difficulty of refolding ꢀ-barrel proteins into lipids with long
double-alkyl chains.42,43 Presumably, detergents with two
hydrophobic chains self-assemble into larger structures with
smaller surface curvature than the single-chain analogues, and
the smaller micelles formed by single-chain detergents tend to
exhibit more rapid exchange with the detergent monomers.
Figure 4. 2D [15N,1H]-TROSY correlation NMR spectra of uniformly
[2H,15N]-labeled OmpX reconstituted in mixed micelles with different
detergents: (a) DHPC; (b) 138-Fos; (c) 179-Fos, where the vertical band
of peaks near 8.0 ppm represents t1-noise from the signal of the amide
proton of 179-Fos; (d) 115-Fos; (e) TPC; (f) 34-Fos; (g) 185-Fos. The
spectra were collected with the following parameters: data size 50 (t1) ×
1024 (t2) complex points; t1max ) 25 ms; t2max ) 86 ms; 300 scans per t1
increment, overall measurement time ) 9 h per experiment. Before Fourier
transformation, the data matrices were multiplied with an exponential
window function in the acquisition dimension and with a 75°-shifted sine
bell window46 in the indirect dimension.
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OmpX was homogeneously reconstituted and adopted a similar
conformation in the new detergents as in DHPC. A decisive
advance, which results in significant savings of detergent and
working time, is that there was no precipitation during the
microscale reconstitution in 138-Fos or in 179-Fos, whereas
more than 50% of the protein precipitated during each cycle of
reconstitution in DHPC (Table 3). Therefore, reconstitution in
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7362 J. AM. CHEM. SOC. VOL. 130, NO. 23, 2008

Microscale NMR for Membrane Protein Structural Biology
A R T I C L E S
These properties might contribute to the high refolding efficiency
for OmpX.54,55
similar results; toward this end, we will continue our amphiphile
synthesis and characterization efforts with new classes of
detergents and with other IMPs.
Minor structural changes of the detergent chemical structures
can have dramatic effects on their ability to complex with
membrane proteins in solution. We and others have observed
the same type of sensitivity to detergent modification in
crystallization studies of IMPs.4 Thus, among the six phospho-
choline detergents studied here by NMR spectroscopy, 115-
Fos and 179-Fos differ only in the substitution of an ester bond
by an amide linkage, but only 179-Fos was found to properly
reconstitute OmpX. The two new molecules found to support
full folding of OmpX under NMR conditions, 138-Fos and 179-
Fos, are superior to the previously used DHPC and to lyso-
phospholipids in terms of detergent stability, since the ester
bonds in DHPC and in lysophospholipids are prone to hydrolysis
at elevated temperature and basic pH.56–58 It is likely that
different proteins will require different detergents to achieve
Acknowledgment. We thank Prof. G. Chang for providing some
of the commercial detergents and Prof. J. Kelly and P. Dawson for
helpful discussions. This work was supported by the Joint Center
for Innovative Membrane Protein Technologies (NIH Roadmap
Grant GM073197) and the Joint Center for Structural Genomics
(NIH Grant U54 GM074898). K.W. is the Cecil H. and Ida M.
Green Professor of Structural Biology at The Scripps Research
Institute.
Supporting Information Available: Structures of the com-
mercial and newly synthesized detergents; synthetic procedures
and characterization data for representative detergents. Complete
ref 51. This material is available free of charge via the Internet
at http://pubs.acs.org.
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