Communication
Cite This: Biochemistry 2017, 56, 5481-5484
Dodecyl--melibioside Detergent Micelles as a Medium for
Membrane Proteins
James M. Hutchison, Zhenwei Lu, Georey C. Li, Benjamin Travis, Ritesh Mittal,
Catherine L. Deatherage, and Charles R. Sanders*,
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37240, United States
Anatrace, 434 West Dussel Drive, Maumee, Ohio 43537, United States
*
S Supporting Information
ABSTRACT: There remains a need for new non-ionic
detergents that are suitable for use in biochemical and
biophysical studies of membrane proteins. Here we explore
the properties of n-dodecyl--melibioside (-DDMB)
micelles as a medium for membrane proteins. Melibiose
is D-galactose-(16)-D-glucose. Light scattering showed
the -DDMB micelle to be roughly 30 kDa smaller than
micelles formed by the commonly used n-dodecyl--
maltoside (-DDM). -DDMB stabilized diacylglycerol
kinase (DAGK) against thermal inactivation. Moreover,
activity assays conducted using aliquots of DAGK puried
into -DDMB yielded activities that were 40% higher than
those of DAGK puried into -DDM. -DDMB yielded
similar or better TROSY-HSQC NMR spectra for two
single-pass membrane proteins and the tetraspan mem-
brane protein peripheral myelin protein 22. -DDMB
appears be a useful addition to the toolbox of non-ionic
detergents available for membrane protein research.
S pectacular advances in membrane protein structural biology
over the past 20 years have been enabled, in part, by the
development of new and improved model membranes for
solubilizing and stabilizing these dicult-to-study biomole-
cules.19 Such media include nanodiscs, bicelles, new classes of
detergents, and lipidic cubic phases. Despite these advances,
there remains room for innovation. For example, there are few
examples of the successful use of non-ionic detergents in
nuclear magnetic resonance (NMR) studies of membrane
proteins. Given the widespread use of uncharged detergents
such as n-dodecyl--maltoside (-DDM) in crystallization/
diraction studies of many dierent membrane proteins, the
fact that -DDM, lauryl maltose neopentyl glycol (MNG), and
other uncharged detergents have thus far been of limited use
for NMR has been vexing. This paper was originally motivated
by the desire to nd an uncharged detergent that can be used to
solubilize membrane proteins for NMR studies. This led one of
the authors of this work to recall an alkyl glycoside rst
synthesized and subjected to preliminary characterization many
years ago, n-dodecyl--melibioside [-DDMB (Figure S1)].10
The disaccharide melibiose is distinct from maltose and most
other disaccharides in that its galactose and glucose units are
connected by an (16) glycosidic linkage, conferring a much
higher degree of conformational freedom between the two
sugar groups than is possible for non-16 glycosides. We
2017 American Chemical Society
pubs.acs.org/biochemistry
hypothesized that this enhanced conformational exibility
might attune its micelle properties and interactions with
membrane proteins in a way that would promote its utility in
both biochemical and biophysical studies, including solution
NMR spectroscopy. Preliminary testing of this hypothesis is
presented herein.
DDMB was synthesized and puried as described in the
Supporting Materials and Methods and Figure S2. Using an 8-
anilinonaphthalene-1-sulfonic acid (ANS) dye-based uores-
cence method,11 the critical micelle concentration of -DDMB
at 25 C was determined to be 0.3 mM. This is higher than that
of -DDM (0.2 mM12) and matches the value previously
determined for an unresolved mixture of and forms of
DDMB.13 Dynamic light scattering (DLS) was employed to
assess the size and aggregation number of -DDMB micelles
based on the assumption that they are spherical. Our DLS
measurements for micelles of pure -DDMB at 25 C and at
concentrations of <100 mM (Figure 1) indicate that the radius
of hydration (Rh) for -DDMB micelles is 2.9 nm and the
aggregation number is roughly 84, corresponding to an
aggregate molecular weight of 42 kDa. These results are similar
to those previously reported for an / anomeric mixture of
DDMB in the concentration range of 2040 mM.13 Multiangle
light scattering (MALS) was used to conrm the DLS result.
MALS determines the molecular weight of particles without
needing a shape factor or modeling. The MALS result of 46
kDa for the -DDMB micelle (Table S1) was in good
agreement with the value determined by DLS.
-DDM14 has previously been shown to form micelles in the
range of 6976 kDa,15 signicantly larger than the 42 kDa
micelles formed by -DDMB (see also the Rh comparisons in
panels A and B of Figure 1). -DDM micelles also exhibited a
linear temperature-dependent increase in size at higher
concentrations (i.e., 100 mM) not seen for -DDMB micelles
at 100 mM (Figure 1B). The Rh of -DDMB micelles was also
independent of both pH and ionic strength (panels C and D,
respectively, of Figure 1). The constancy of the properties of -
DDMB micelles across a wide range of relevant pHs,
temperatures, concentrations, and ionic strengths is useful
because -DDMB micelles can be employed under a wide range
Received: August 19, 2017
Revised: October 3, 2017
Published: October 5, 2017
5481 DOI: 10.1021/acs.biochem.7b00810
Biochemistry 2017, 56, 54815484
Biochemistry Communication

Figure 2. Thermal stability of DAGK in -DDMB and other

detergents. DAGK samples (0.2 mg/mL) were incubated at 70 C,
with aliquots being removed at time points and assayed in the standard
-DM/cardiolipin mixed miceller DAGK activity assay.17 The T1/2* of
Figure 1. Use of DLS to characterize the mean radius of hydration MNG represents the DAGK population that is sensitive to thermal
(Rh) of -DDMB and -DDM micelles. (A) The 10 mM DDMB inactivation in MNG/DAGK mixed micelles.
(black) or 10 mM DDM (red) detergent was dissolved in water and
subjected to a 1 C/min temperature ramp. (B) Example of a
temperature ramp used in panel A. (C) -DDMB micelle size as a suppress irreversible thermal inactivation of DAGK and much
function of pH. The buer was either 25 mM imidazole buered at pH better than DPC or -DM.16
7.5 or 6.5, or 25 mM sodium acetate buered at pH 5.5 or 4.5. (D) - We next tested the degree to which -DDMB can serve as a
DDMB micelles as a function of salt concentration. medium that supports the native functionality of a membrane
protein. We again employed DAGK, which was puried into
lipid-free -DDMB, MNG, -DDM, and -DM. Aliquots from
of conditions without concern about major changes in micellar these stock solutions were then transferred into DAGK assay
properties. mixtures prepared using several dierent types of micelles or
To test whether -DDMB micelles are smaller than -DDM detergent/cardiolipin mixed micelles, including -DM/cardio-
micelles in the presence of a membrane protein, we used size lipin mixed micelles (the traditional standard assay con-
exclusion chromatography (SEC)MALS to examine the sizes dition17). As shown in Figure 3, DAGK has only low activity
of micelles containing Escherichia coli diacylglycerol kinase (1 unit/mg) when assays are performed in lipid-free -
(DAGK), a 42 kDa homotrimeric mostly helical protein with DDMB micelles, as is also the case for lipid-free -DM, MNG,
three transmembrane segments per subunit. The -DDMB and -DDM. However, the presence of cardiolipin (CL) in the
DAGK complex is roughly 30 kDa smaller than the -DDM
DAGK complex (Table S2), indicating that the smaller micelle
size of -DDMB extends to its complex with DAGK.
We next tested the ability of -DDMB to maintain the
stability of a membrane protein. DAGK was puried into -
DDMB and into each of four commonly used detergents [n-
dodecylphosphocholine (DPC), n-decyl--maltoside (-DM),
MNG, and -DDM] and incubated at 70 C. Aliquots were
removed as a function of time and subjected to the standard
DAGK activity assay (in DM/cardiolipin mixed micelles) to
determine t1/2 values for irreversible inactivation of the enzyme,
a measure of thermostability.16 As shown in Figure 2, DAGK is
much more stable in -DDM and -DDMB (T1/2 values of 8.5
0.5 and 9.9 1.7 h, respectively) than in DPC or -DM,
where T1/2 in each case was <4 h. In the case of MNG, there
seem to be two populations of DAGK/MNG mixed micelles:
an 40% population in which DAGK is sensitive to thermal
inactivation (T1/2 = 5.6 h) and another 60% population that
appears to be resistant to thermal inactivation over a period of
Figure 3. DAGK activity assays started with detergent aliquots of
many hours. This heterogeneity may reect the fact that the DAGK diluted various micelle and mixed micelle assay mixtures.
properties of MNG are close to the borderline between being DAGK was puried into -DDMB, -DM, -DDM, or MNG micelles
detergent-like and being lipid-like, such that MNG may be able (stock) and then assayed using reaction mixtures at 30 C. The
to form more than one type of kinetically stable complex with standard assay condition is labeled DM1CL. 1CL refers to the
some membrane proteins. In any case, these results indicate fact that the mole fraction of CL in the mixture is the same (1) as in
that -DDMB is similar to -DDM in terms of its ability to the standard assay procedure.17

5482 DOI: 10.1021/acs.biochem.7b00810

Biochemistry 2017, 56, 54815484
Biochemistry Communication

assay mixtures as a lipid activator dramatically enhances DAGK

activity in -DDM, MNG, and -DDMB. The activities
observed in -DDMB/CL and -DDM/CL micelles are not
as high as for the corresponding reactions performed in -DM/
CL or MNG/CL micelles, suggesting subtle dierences in
DAGK structure and/or dynamics that result in suboptimal
catalytic properties in -DDMB/CL and -DDM/CL mixed
micelles relative to -DM/CL and MNG/CL conditions. When
the same DAGK stock is used, MNG with cardiolipin gives an
activity higher than that of DM with cardiolipin. A surprising
observation is that DAGK prepared in -DDMB and then
aliquoted (250-fold diluted) into either -DM/CL, -DDM/
CL, or -DDMB/CL mixed micelle assay mixtures yielded
activities higher than those of the corresponding assays initiated
using small aliquots of DAGK prepared in -DM, -DDM, or
MNG micelles and then transferred to these same detergent/
CL mixtures. Indeed, the observed specic activity of 166 15
units/mg for DAGK puried into -DDMB and then aliquoted
into -DM/CL mixed micelles is much higher than the highest
previously reported 30 C mixed micellar specic activity for
DAGK, which is 120 units/mg.18,19 This suggests that DAGK
prepared in -DM (and, most likely, many other detergent
types) micelles is susceptible to irreversible misfolding, a
problem that is avoided when the enzyme is prepared in -
DDMB. Indeed, DAGK has long been known to be prone to
misfolding in detergent micelles.20,21 These results show that -
DDMB not only provides a medium that supports the thermal
stability of DAGK but also seems to enhance its eciency of
folding during and following purication (see Supporting
Materials and Methods). On the other hand, -DDMB is
typical of most detergents22,23 in that it cannot itself satisfy the
requirement of DAGK for the presence of a lipid cofactor for
maximal catalytic activity.24,25 Figure 4. NMR spectra of single-span membrane proteins in -DDMB
We next examined -DDMB micelles as a medium for NMR and other detergents: (A) 400 M C99 in 9% -DDMB (black) and
studies of membrane proteins. TROSY-HSQC spectra were 400 M C99 in 9% LMPG (red), (B) 400 M C99 in 9% -DDMB
collected for three dierent membrane proteins: (i) human (black) and 300 M C99 in 9% -DDM (red), (C) 230 M Notch-
Notch1 transmembrane domain (Notch-TMD) residues TMD in 5% -DDMB (black) and 300 M Notch-TMD in 1% LMPC
17211771 (a single-span protein), (ii) the 99-residue C99 (red), (D) 230 M Notch-TMD in 5% -DDMB (black) and 300 M
domain of the human amyloid precursor protein, the immediate Notch-TMD in 4.5% -DDM (red), (E) 350 M PMP-22 in 20% -
DDMB (black) and 700 M PMP-22 in 20% TDPC (red), and (F)
precursor of the amyloid- polypeptide (also with a single
350 M PMP-22 in 20% -DDMB (black) and 400 M PMP-22 in
transmembrane segment), and (iii) 160-residue human 20% -DDM (red).
peripheral myelin protein 22 (a mostly helical protein with
four transmembrane segments). Each of these proteins was Table S2). The 15N line widths of C99 in a -DDM micelle
puried into -DDMB micelles, into -DDM micelles, and into were 12% larger than the line widths in the -DDMB micelle.
the micelle type previously observed to yield the most favorable A more demanding test case for NMR is provided by human
NMR spectra for each protein following extensive screen- PMP22, a tetraspan membrane protein. Despite more than a
ing.2628 A TROSY-HSQC spectrum was collected for each decade of eort to optimize NMR conditions for this disease-
sample. Figure 4 shows that -DDMB yields spectra for C99 linked protein, the best NMR spectra acquired to date have
and Notch-TMD that are comparable in quality with those of been in tetradecylphosphocholine (TDPC) micelles and are
the detergents previously shown to yield the best spectra for highly unsatisfactory, characterized by the absence of many
these proteins [lyso-myristoylphosphatidylglycerol (LMPG) for peaks and considerable heterogeneity of line widths among the
C99 and lyso-myristoylphosphatidylcholine (LMPC) for peaks that can be observed.28,29 As shown in Figure 4E, PMP22
Notch-TMD]. Figure 4 also shows that -DDMB also yields presents an improved spectrum when puried in -DDMB
better spectra for C99 and Notch-TMD than -DDM does. In relative to that of TDPC. The levels of quality of the spectra
both cases, certain peaks observed when the proteins are in - from -DDM and -DDMB are more similar (Figure 4F);
DDMB are too broad to detect in -DDM. For example, there however, the -DDMB spectrum exhibits more peaks in the
are a pair of black peaks near 111 ppm in the 15N dimension for crowded region between 8.5 and 7.5 H ppm.
Notch-TMD in -DDMB for which there are no corresponding In conclusion, detergents remain a nearly ubiquitous tool in
red peaks from -DDM. The average 15N line widths for Notch biochemical and biophysical studies of membrane proteins,
in the -DDM micelle are 32% larger than the line widths for usually being employed during purication and reconstitution,
Notch in -DDMB, most likely reecting the fact that -DDM as well as often being a component of the nal model
micelles are signicantly larger than -DDMB micelles (see membrane system in which the membrane protein is
5483 DOI: 10.1021/acs.biochem.7b00810
Biochemistry 2017, 56, 54815484
Biochemistry
characterized. Here, studies of -DDMB show that it forms
micelles smaller than the commonly used -DDM micelles,
even though both detergents have n-dodecyl tails and
disaccharide headgroups. -DDMB is just as good as -DDM
at maintaining the thermal stability of a complex membrane
enzyme, DAGK. Moreover, -DDMB is better than any
detergent yet tested as a medium in which to purify DAGK
in a way that avoids misfolding. Finally, for three dierent
membrane proteins, -DDMB was seen to yield NMR spectra
of a quality similar to or even higher than that previously
observed for the best of the previously tested detergents. This is
an important development for non-ionic detergents, which have
rarely been used as membrane-mimetic media in previous
NMR studies. We hope these observations will encourage both
further characterization of -DDMB and exploration of
applications for which it may be uniquely well suited.
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.7b00810.
Materials and methods, Figures S1 and S2, extended
gure captions, Tables S1 and S2, and supporting
references (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: chuck.sanders@vanderbilt.edu.
ORCID
Charles R. Sanders: 0000-0003-2046-2862
Funding
J.M.H. was supported by National Institutes of Health (NIH)
Grant T32 CA00958229. The NMR instrumentation used in
this work was supported by NIH Grant S10 RR026677 and
National Science Foundation Grant DBI-0922862. This work
was supported by NIH Grants RO1 GM106672, RO1
AG056147, and RO1 NS095089.
Notes
The authors declare the following competing nancial
interest(s): Anatrace (the employer of authors B.T. and
R.M.) is a producer and vendor of -DDMB and the other
detergents employed in this work.
REFERENCES
(1) Seddon, A. M., Curnow, P., and Booth, P. J. (2004) Biochim.
Biophys. Acta, Biomembr. 1666, 105117.
(2) Bayburt, T. H., and Sligar, S. G. (2010) FEBS Lett. 584, 1721
1727.
(3) Dorr, J. M., Scheidelaar, S., Koorengevel, M. C., Dominguez, J. J.,
Schafer, M., van Walree, C. A., and Killian, J. A. (2016) Eur. Biophys. J.
45, 321.
(4) Warschawski, D. E., Arnold, A. A., Beaugrand, M., Gravel, A.,
Chartrand, E., and Marcotte, I. (2011) Biochim. Biophys. Acta,
Biomembr. 1808, 19571974.
(5) Raschle, T., Hiller, S., Etzkorn, M., and Wagner, G. (2010) Curr.
Opin. Struct. Biol. 20, 471479.
(6) Caffrey, M., and Cherezov, V. (2009) Nat. Protoc. 4, 706731.
(7) Chae, P. S., Rasmussen, S. G. F., Rana, R. R., Gotfryd, K.,
Chandra, R., Goren, M. A., Kruse, A. C., Nurva, S., Loland, C. J.,
Pierre, Y., Drew, D., Popot, J. L., Picot, D., Fox, B. G., Guan, L.,
Gether, U., Byrne, B., Kobilka, B., and Gellman, S. H. (2010) Nat.
Methods 7, 10031008.
5484
Communication
(8) Durr, U. H. N., Gildenberg, M., and Ramamoorthy, A. (2012)
Chem. Rev. 112, 60546074.
(9) Garavito, R. M., and Ferguson-Miller, S. (2001) J. Biol. Chem.
276, 3240332406.
(10) Sanders, C. R., and Prestegard, J. H. (1990) Biophys. J. 57, 33a.
(11) Miguel, M. D., Eidelman, O., Ollivon, M., and Walter, A. (1989)
Biochemistry 28, 89218928.
(12) VanAken, T., Foxall-VanAken, S., Castleman, S., and Ferguson-
Miller, S. (1986) Methods Enzymol. 125, 2735.
(13) Kegel, L. L., Szabo, L. Z., Polt, R., and Pemberton, J. E. (2016)
Green Chem. 18, 44464460.
(14) Rosevear, P., VanAken, T., Baxter, J., and Ferguson-Miller, S.
(1980) Biochemistry 19, 41084115.
(15) Oliver, R. C., Lipfert, J., Fox, D. A., Lo, R. H., Doniach, S., and
Columbus, L. (2013) PLoS One 8, e62488.
(16) Zhou, Y. F., Lau, F. W., Nauli, S., Yang, D., and Bowie, J. U.
(2001) Protein Sci. 10, 378383.
(17) Czerski, L., and Sanders, C. R. (2000) Anal. Biochem. 284, 327
333.
(18) Koehler, J., Sulistijo, E. S., Sakakura, M., Kim, F. J., Ellis, C. D.,
and Sanders, C. R. (2010) Biochemistry 49, 70897099.
(19) Van Horn, W. D., Kim, H. J., Ellis, C. D., Hadziselimovic, A.,
Sulistijo, E. S., Karra, M. D., Tian, C. L., Sonnichsen, F. D., and
Sanders, C. R. (2009) Science 324, 17261729.
(20) Van Horn, W. D., Kim, H. J., Ellis, C. D., Hadziselimovic, A.,
Sulistijo, E. S., Karra, M. D., Tian, C., Sonnichsen, F. D., and Sanders,
C. R. (2009) Science 324, 17261729.
(21) Gorzelle, B. M., Nagy, J. K., Oxenoid, K., Lonzer, W. L., Cafiso,
D. S., and Sanders, C. R. (1999) Biochemistry 38, 1637316382.
(22) Li, Q. X., Mittal, R., Huang, L. J., Travis, B., and Sanders, C. R.
(2009) Biochemistry 48, 1160611608.
(23) Vinogradova, O., Sonnichsen, F., and Sanders, C. R., II (1998) J.
Biomol. NMR 11, 381386.
(24) Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239
6247.
(25) Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 5062
5069.
(26) Beel, A. J., Mobley, C. K., Kim, H. J., Tian, F., Hadziselimovic,
A., Jap, B., Prestegard, J. H., and Sanders, C. R. (2008) Biochemistry 47,
94289446.
(27) Deatherage, C. L., Lu, Z. W., Kim, J. H., and Sanders, C. R.
(2015) Biochemistry 54, 35653568.
(28) Sakakura, M., Hadziselimovic, A., Wang, Z., Schey, K. L., and
Sanders, C. R. (2011) Structure 19, 11601169.
(29) Mobley, C. K., Myers, J. K., Hadziselimovic, A., Ellis, C. D., and
Sanders, C. R. (2007) Biochemistry 46, 1118511195.
DOI: 10.1021/acs.biochem.7b00810
Biochemistry 2017, 56, 54815484