Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA
Article
Phase transitions in small isotropic bicelles Erik F Kot, Sergey Alexandrovich Goncharuk, Alexander Sergeevich Arseniev, and Konstantin S Mineev Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03610 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
1
Phase transitions in small isotropic bicelles.
2
E.F. Kot1,2, S.A. Goncharuk1,3, A.S. Arseniev1,2, K.S. Mineev1,2*
3 4 5 6 7
1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences RAS, str. Miklukho-Maklaya 16/10, Moscow, 117997 Russian Federation 2 Moscow Institute of Physics and Technology, Institutsky per., 9, 141700, Dolgoprudnyi, Russian Federation 3 Lomonosov Moscow State University, Leninskiye Gory, 1, Moscow, 119991, Russian
8
Federation
9 10
Keywords: bicelles, isotropic bicelles, cholesterol, NMR spectroscopy, diffusion, 31P, phase transition.
11 12
ABSTRACT
13
Isotropic phospholipid bicelles are one of the most prospective membrane mimetics for the
14
structural studies of membrane proteins in solution. Recent works provided an almost full set of
15
data regarding the properties of isotropic bicelles, however, one major aspect of their behavior is
16
still under consideration: the possible mixing between the lipid and detergent in the bilayer area.
17
This problem may be resolved by studying the lipid phase transitions in bicelle particles. In the
18
present work, we investigate two effects - phase transitions of bilayer lipids and temperature-
19
induced growth of isotropic bicelles using the NMR spectroscopy. We propose an approach to
20
study the phase transitions in isotropic bicelles based on the properties of
21
bilayer-forming lipids. We show that phase transitions in small bicelles are "fractional", particles
22
with the liquid-crystalline and gel bilayers coexist in solution at certain temperatures. We study the
23
effects of lipid fatty chain type and demonstrate that the behavior of various lipids in bilayers is
24
reproduced in the isotropic bicelles. We show, that the temperature-induced growth of isotropic
25
bicelles is not related directly to the phase transition, but is the result of the reversible fusion of
26
bicelle particles. According to our data, rim detergents have also an impact on phase transitions:
27
detergents that resist the temperature-induced growth provide the narrowest and most expressed
28
transitions at higher temperatures. We demonstrate clearly that phase transitions take place even
29
in the smallest bicelles that are applicable for structural studies of membrane proteins by solution
30
NMR spectroscopy. This last finding, together with other data draws a thick line under the long-
31
lasting argument about the relevance of small isotropic bicelles. We show with certainty that the
32
small bicelles are able to reproduce the most fundamental property of lipid membranes - the
33
ability to undergo the phase transition. 1 ACS Paragon Plus Environment
31
P NMR spectra of
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
34
Introduction
35
Bicelles are membrane mimetics that are formed in the mixtures of membrane lipids and
36
specific detergents, such as the bile salt derivatives1 and short-chain phospholipids2–4. Bicelles are
37
known to form the discoidal particles5, with the lipids forming a patch of planar bilayer and
38
detergents associating into the "rim" of the disc. The size of particles is controlled by the ratio
39
between the lipid and rim-forming detergent (q) in quite a wide range, starting from 20 kDa. Large
40
bicelles (q>2-3 for various mixtures) were shown to orient spontaneously in the strong magnetic
41
field and are used in solid-state NMR spectroscopy to prepare the oriented bilayer samples6 and in
42
solution to create the anisotropic environment for the soluble proteins and measure the residual
43
dipolar couplings7. Due to the specified ability, such large particles are referred to as "anisotropic"
44
bicelles. In contrast, solutions with small particles which are not capable of spontaneous
45
orientation are called isotropic bicelles (IsoBs). IsoBs have several features that make them an
46
almost perfect membrane mimetic for structural studies of membrane proteins8–10. Unlike
47
detergent micelles, bicelles contain membrane lipids, that form the plane patch of membrane and
48
properly mimic some properties of the lipid bilayer11,12. The lipid composition of bicelles can be
49
varied to simulate the membranes of different cells or their microdomains13. Cholesterol,
50
sphingolipids, glycerolipids, lipids with unsaturated fatty chains or anionic headgroups can be
51
added to the isotropic bicelles14–18. IsoBs can be formed using the very mild rim-forming
52
surfactants, such as Facades19, that do not cause the unfolding of soluble globular domains of large
53
membrane proteins20. Unlike lipid-protein nanodiscs, IsoBs are cheaper and easier to prepare, size
54
of bicelles is lower, which makes them compatible with solution NMR spectroscopy. They allow the
55
exchange of matter between the particles in solution, which is convenient for the studies of
56
protein-protein interactions. IsoBs have found their application in X-ray crystallography21, however,
57
the widest use of this membrane mimetic was established in solution NMR studies of membrane
58
proteins8.
59
Recent works provided an almost full set of data regarding the behavior of IsoBs. Size and
60
concentration of detergents in monomeric form were determined for many types of bicelles as a
61
function of the lipid/detergent ratio and temperature5,12,22. This resulted in the ideal bicelle models
62
that fit the experimental data and describe the shape of bicelles, assuming the absence of
63
lipid/detergent mixing5,12,23. However, one major aspect of IsoBs behavior is still under
64
consideration: the possible mixing between the lipids and detergents in the planar area or rim of
65
the particles. According to the recent studies, small particles, applicable for the structural studies 2 ACS Paragon Plus Environment
Page 2 of 25
Page 3 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
66
by solution NMR spectroscopy, (radius less than 3.5 nm) and large IsoBs (greater than 3.5 nm)
67
behave similarly below the phase transition temperature of the bilayer-forming lipid and follow
68
the ideal bicelle models. However, the dramatic temperature-induced growth is observed for the
69
large bicelles12,22. Above some critical temperature, which is close to the phase transition
70
temperature of bilayer-forming lipid, large IsoBs begin to increase their radius. This growth is too
71
strong to be explained by the change of lipid packing properties that occurs during the phase
72
transition, and does not seem to stop at any particular temperature. This effect depends on the
73
type of membrane lipid and rim-forming agent. Thus, some researchers postulate that the small
74
IsoBs are characterized by the mixing between the lipid and detergent since they do not
75
demonstrate a temperature-induced growth and refer to the results of computer simulations22,24,
76
and others assume that the large IsoBs are subject to the mixing at high temperatures12. In our
77
previous work, we put forward a hypothesis that the state of lipids in small bicelles is the same as
78
observed in large bicelles below the phase transition temperature12. The indirect data, in
79
particular, the analysis of the protein-lipid contacts in the DMPC/CHAPS and DMPC/DHPC
80
mixtures, do not reveal any presence of the detergent in close proximity to the membrane protein
81
in small IsoBs regardless the temperature and size of the bicelles11,12. On the other hand, the
82
saturation transfer experiments with POPC/DHPC bicelles show that at least the transient contacts
83
are observed between the DHPC methyl groups and terminal residues of transmembrane helices in
84
such a mixture, while the lipid-protein contacts are prevailing for the buried part of the membrane
85
domains25.
86
One of the possible ways to investigate the lipid/detergent mixing and presence of lipid
87
bilayer in IsoBs is to study the lipid phase transitions and state of lipids in bicelles of various size
88
and compositions. Phase transitions in the isotropic DMPC/DHPC bicelles were already probed
89
using the infrared spectroscopy and the study revealed that such transitions do take place in
90
q=0.75 and q=1.0 mixtures, but seem to be absent in q=0.5 particles24. However, it is a single IsoB
91
system and the data obtained for q=0.5 bicelles do not allow to draw the definitive conclusion
92
about the absence or presence of the phase transition. Studies of phase transitions in various
93
systems with different temperature-dependent behavior are required. In the present work, we
94
investigate two effects in a variety of systems - the phase transitions and temperature-induced
95
growth of IsoBs using the 31P NMR spectroscopy and 1H NMR diffusion measurements.
96
Experimental Section 3 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
97
Page 4 of 25
Materials. Lipids and detergents (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-
98
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
99
phosphocholine
(DPPC),
(POPC),
1,2-dipalmitoyl-sn-glycero-3-
2-dihexanoyl-sn-glycero-3-phosphocholine
(DHPC),
3-[(3-
100
cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), Facade-EPC and Facade-EM),
101
sodium dodecyl sulfate (SDS) were the product of Avanti Polar Lipids Inc, USA. D2O was the
102
product of Cambridge Isotope Laboratory, USA.
103
Sample preparation. Here and below q* denotes the effective lipid/detergent ratio, taking
104
into account the presence of the detergent in the monomeric form12. To prepare the bicelles, ~10
105
mg of lipids in a dry powder were dissolved by the necessary amount of 10% stock solution of
106
CHAPS, DHPC, Facade-EPC or Facade-EM and then the total volume of the sample was adjusted to
107
500 ul by the addition of 30 mM NaPi buffer, pH 7.0, containing 10% of D2O. The resulting mixture
108
was frozen and then heated to 40-50 °C in the ultrasonic bath for 3-4 times. To vary the q ratio,
109
the solution was either diluted by the stock solution of detergent or added to the necessary
110
amount of lipid in dry powder and put into the ultrasonic bath for 5-6 minutes at 40 °C. For the
111
dilution experiments, 10% w/w solution of bicelles at necessary q was prepared. To vary the
112
concentration of bicelles, necessary portions of the initial solution were diluted by the 3.0-5.0
113
mM solution of CHAPS, depending on the bilayer lipid and q* of bicelles.
114
To prepare the lipid-protein nanodiscs (LPNs) samples, necessary amounts of MSP1D1 were
115
added to the mixture of DMPC and sodium cholate (1:1) dissolved in the ND-buffer (20 mM Tris pH
116
8.0, 100 mM NaCl) and incubated for 1h. Total lipid/MSP ratio was adjusted to 65. To assemble
117
LPNs, the Biobeads SM-2 (Biorad, USA) (1 g per 70 mg of detergent) were added and the
118
suspension was shaken at room temperature for 12-16 h26. The resulting solution was filtered (to
119
remove Biobeads), centrifuged at 14000g for 20 min, then the supernatant was purified by the
120
size-exclusion chromatography and concentrated to 500 ul.
121
31
P NMR experiments.
31
P NMR spectra were recorded on the Bruker Avance 600 MHz
122
spectrometer equipped with the broadband double resonance probe. A single pulse experiment
123
was employed using the π/6 pulse of 4 us and relaxation delay of 3 s, acquisition time of 2.7 s with
124
broadband WALTZ16 proton decoupling at the field strength of 1.42 kHz. A pre-acquisition delay of
125
10 min was used before each experiment to ensure the equilibrium state of the system. Spectra
126
were referenced indirectly with respect to the proton signal of trimethylsilylpropanoic acid (TSP) at
127
0.0 ppm using the gyromagnetic ratio. To determine the chemical shifts and to measure the
128
fractions of gel/liquid-crystalline bicelles, the 31P spectra were deconvoluted using the Lorentzian 4 ACS Paragon Plus Environment
Page 5 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
129
lineshapes and non-linear regression in Wolfram Mathematica software. The temperature was
130
varied in either direction to exclude the hysteresis and control the equilibrium state of the sample.
131
31
P diffusion experiments were performed using the double stimulated echo experiment27,
132
designed to suppress the effects of convection, with diffusion delay equal to 500 ms and encoding
133
gradient equal to 5 ms, with the gradient pulse strength being varied linearly from 1 to 52 G/cm in
134
32 iterations. The pulse sequence included the broadband WALTZ16 proton decoupling at the field
135
strength of 1.42 kHz both during the relaxation delay and acquisition. Summary duration of the
136
diffusion experiment was equal to 8 hours. The data analysis included the simultaneous
137
deconvolution of two DPPC peaks with Lorentzian lineshapes and serial processing of the whole
138
dataset with the common peak positions and line widths in Wolfram Mathematica software, to
139
increase the reliability of approximation. Six repetitions were made at each temperature/q* and
140
paired Student test was used to measure the reliability of the changes (p-values).
141
1
H NMR experiments. All diffusion 1H NMR spectra were recorded on the Bruker Avance
142
700 spectrometer with the working frequency of protons equal to 700 MHz, equipped with the
143
room-temperature triple resonance TXI probe. Diffusion was measured using the double
144
stimulated echo pulse sequence with the efficient suppression of convection effects and of the
145
intense solvent signal28. Diffusion experiments were recorded with 32 scans and 32 increments of
146
the encoding gradient pulse (gradient pulse strength was varied linearly from 12 to 52 G/cm).
147
Summary length of the encoding gradient pulse was 4.0 ms, duration of the diffusion delay was
148
300-400 ms. The gradients were calibrated using the diffusion coefficient of residual water in 100%
149
D2O at 25 °C. The at least 30-minute interval was used after the change of the temperature to
150
ensure the equilibrium state of the system. To minimize the effects of signal overlap and magnetic
151
field inhomogeneity, the integral of the most intense and narrow peak, corresponding to N-(CH3)n
152
moieties, was used to determine the diffusion coefficients of DMPC and CHAPS. This approach
153
appeared to be the most error-resistant in comparison to averaging of several peaks of the
154
compounds. Additionally, use of the most narrow peak allows the more adequate averaging of the
155
diffusion coefficients between the particles of various size.
156
Data analysis. To analyze the diffusion coefficients and convert them to the radii of particles,
157
we used the formalism, described in details in our previous work12. The analysis included the
158
corrections for the diffusion obstruction due to the high concentration of particles and due to the
159
discoidal shape of bicelles29,30. Experimental errors were determined using the Monte-Carlo
160
analysis. 5 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 25
161
Results and Discussion
162
Detection of phase transitions in IsoBs. 31P NMR is actively used to study the state of lipids
163
in membrane mimetics, however, mostly for the systems with large particles. One of the most
164
common applications of
165
field31,32. This process is temperature-dependent, and, at some point, the isotropic chemical shift
166
of phosphorous (close to 0 ppm) becomes anisotropic (shifted by 10-20 ppm), which indicates the
167
orientation of particles.
168
monomeric detergent in IsoBs5,24. Thus, we expected to observe the changes in 31P chemical shifts
169
or line widths upon the lipid phase transition or as a manifestation of mixing between the lipid and
170
detergents in either the rim or planar area of IsoBs. We first tested the DPPC/CHAPS system: size
171
of these bicelles was determined for the wide range of q*12 and phase transition of DPPC occurs
172
at a convenient temperature of 41 ºC, allowing the detection of NMR spectra 30-40º below and
173
above the critical temperature.
31
P is monitoring of the orientation of large bicelles in strong magnetic
31
P spectra were also utilized to measure the concentration of the
174
First experiments were performed at q*=1.5, under these conditions particles with the
175
radius of 5.1 nm (at 35 ºC) are formed and expressed temperature-induced growth of bicelles is
176
observed12. At high temperatures, the single and narrow peak is observed in the 1D
177
spectra of IsoBs solution at -0.8 ppm (Figure 1, left panel). Surprisingly, below the phase transition,
178
this single peak becomes split into the narrow peak and wide peak, which is shifted upfield. The
179
populations of two peaks depend on the ambient temperature: intensity of the wide peak
180
increases and of the narrow peak - decreases at lower temperatures until only the single wide
181
peak is left in the NMR spectra at 10 ºC. The temperature coefficients of 31P chemical shift for the
182
narrow peak are the same above and below the critical temperature and are equal to 2.5 ppb/K.
183
Temperature coefficient for the wide peak is much higher and is equal to 8.5 ppb/K (Figure 2A).
184
Thus, we can make two conclusions. First of all,
185
transitions in IsoBs (at least for DPPC): it is manifested in NMR spectra by the presence of wide,
186
upfield shifted and broad peak with the relatively high temperature coefficient of chemical shift.
187
Second, the phase transition in isotropic bicelles is "fractional". At some temperatures gel and
188
liquid-crystalline lipids coexist in solution. This may be interpreted in two ways - either some lipids
189
are in liquid or gel phases within the same bicelle, or one fraction of bicelles is in the gel phase
190
and the other fraction is liquid-crystalline. Two situations could be distinguished clearly by
191
measuring the diffusion coefficients for broad and narrow peaks in NMR spectra.
31
31
P NMR
P NMR can be used to monitor the lipid phase
6 ACS Paragon Plus Environment
Page 7 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
192
Langmuir
P NMR diffusion experiments. We recorded the 31P DOSY spectra of q*=1.5 DPPC/CHAPS
31
193
bicelles at 25 ºC and treated separately the signals in NMR spectra (Figure S1). The experiment
194
revealed the substantially different diffusion coefficients for the two peaks, which supports that
195
the broad and narrow signals in
196
solution: liquid and gel-phase bicelles. Bicelles in the fluid phase had radii equal to 4.00±0.08 nm,
197
while the diffusion of gel-phase bicelles can be described with the radius of 5.82±0.11 nm (Figure
198
3). This is counterintuitive because the melting of bicelles should result in the increase of radius
199
according to the ideal bicelle model12, due to the increased area per lipid headgroup, observed in
200
liquid bilayers in comparison to the gel bilayers33. However, we observe the opposite, the size of
201
fluid-phase bicelles is decreased substantially. This can be considered as an evidence in favor of the
202
mixing between the lipid and detergent in liquid-crystalline bicelles. If liquid-crystalline and gel
203
phase bicelles have different morphology and structure, this could result in the presence of two
204
subsets of particles with various extent of mixing between the lipid and detergent. In addition,
205
such a mixing could explain the reduced phase transition temperature of DPPC in bicelles,
206
compared to the bilayer membrane. However, we suppose that it is unlikely for several reasons.
207
When the whole pool of particles is measured by 1H diffusion, the radius of liquid-crystalline
208
bicelles above the phase transition is greater than the radius of bicelles in the gel phase. At 40 ºC
209
radius of DPPC/CHAPS q*=1.5 bicelles equals 6.9±0.2 nm. This is greater than the size of IsoBs at
210
35 ºC, the starting point of phase transition (5.1±0.1 nm), and is greater than the radius of gel
211
phase bicelles at 25 ºC . Thus, it is not a general property of bicelles in the liquid-crystalline phase
212
to be smaller than gel-phase particles, the increased size of gel bicelles is observed only during the
213
"fractional" phase transition. Therefore, this effect cannot be explained by the different
214
morphology and lipid/detergent mixing in two kinds of particles. Additionally, the migration of
215
detergents to the bilayer area of bicelles should not decrease, but rather increase the size of
216
particles, because such mixing is equivalent to the growth of the effective q ratio.
217
The reduced size of
31
P spectra correspond to the different types of particles in
bicelles with liquid-crystalline lipids, observed below the critical
218
temperature, may be explained, assuming that smaller bicelles have the lower propensity to
219
undergo phase transition than the bigger particles. Bicelles are not a continuous media, they
220
represent a set of individual particles, which follow some kind of size distribution. In turn, rim
221
detergents are rather mobile and may disturb the packing of lipids in IsoBs, which are in the direct
222
contact with the rim. Smaller bicelles contain the greater amounts of detergents per lipid
223
molecule, which further disturbs the lipid packing. Using the simple ideal CHAPS-based bicelle 7 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
224
model12 one could estimate that 4.0 nm bicelles contain 72 lipid molecules, with 22 (31%) lipids
225
being in the direct contact with the rim surfactants. In turn, 5.5 nm bicelles contain 178 lipids, with
226
36 (20%) interacting to the rim, and this implies the less distorted lipid bilayer and higher phase
227
transition temperature. Thus, the phase transition inside the one particular bicelle is a stochastic
228
event, which has the probability that may depend on the size of the particle. Therefore, there
229
should be a stochastic distribution of bicelles between the fluid and gel phases within the same
230
sample.
231
In addition, there may be a difference in the free energy of CHAPS in the rim of bicelles in
232
gel and liquid-crystalline phases. If the presence of CHAPS in the rim of gel-phase bicelles was less
233
favorable than in the rim of the liquid-crystalline IsoBs, this would redistribute the detergent
234
between the two subsets of particles. Gel bicelles would contain less detergent per lipid and
235
become larger, while liquid-crystalline particles would be formed with the lower q* and become
236
smaller. This assumption is in agreement with the concentration of monomeric detergent in our
237
systems. In bicelles that do not undergo the phase transition (DMPC/CHAPS with q=1.0) the
238
concentration of monomeric CHAPS in solution is almost constant (3.5 mM) and does not depend
239
on the temperature12. In DPPC/CHAPS q*=1.35 bicelles the concentration of monomeric CHAPS
240
starts to increase below the critical temperature from initial 3.0 mM, until it reaches its maximum
241
of 7-8 mM at 15 C, which is equal to the CMC of CHAPS in the absence of lipids (Figure S2). Thus,
242
smaller bicelles would remain in the fluid phase at the given temperature. When temperature is
243
decreased, these particles may become frozen, which results in the observed changes in the
244
populations of bicelles with gel and liquid-crystalline lipids.
245
This may be tested by measuring the size of bicelles in the gel and fluid phases for the
246
mixture prepared with smaller q*. At q*=1.5 the fraction of liquid-crystalline lipids is too low at 20
247
ºC to perform the accurate measurements. For this reason, we prepared the q*=1.35 DPPC/CHAPS
248
bicelles and recorded the 31P diffusion spectra at 20 and 25 ºC. The radii of both the gel phase and
249
liquid-crystalline bicelles decreased to 5.53±0.19 nm (p=0.02) and 3.85±0.04 nm (p=0.009) at 25
250
ºC in comparison to the q*=1.5 mixture (Figure 3). Upon the cooling of the system to 20 ºC, the
251
radius of gel phase bicelles had increased to 5.98±0.47 nm, however, the low accuracy of data does
252
not allow a definitive conclusion (p=0.063), whereas the size of liquid-crystalline bicelles had
253
clearly decreased to 3.53±0.13 nm (p=0.0012). Thus, these data are in agreement with the above-
254
stated hypothesis.
8 ACS Paragon Plus Environment
Page 8 of 25
Page 9 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
255
We need to point out here that the coexistence of IsoBs with gel and liquid-crystalline lipids
256
has the important practical consequences. First, IsoBs should not be applied for the structural
257
studies below the critical temperature of the bilayer lipid. One would always prefer to have the
258
homogeneous samples, while IsoBs below the critical temperature would provide highly
259
polydisperse solutions, with highly varied lipid packing properties. In addition, IsoBs cannot be
260
utilized to investigate the effects of lipid phase transition on the properties of membrane proteins,
261
for the same reasons.
262
To summarize the paragraph, we show that the phase transition of bilayer lipids is fractional
263
in IsoBs - small liquid-crystalline particles and large gel bicelles coexist in solution within the broad
264
range of temperatures. Such a behavior is likely to be caused by the different propensity of small
265
and large bicelles to undergo the phase transitions of bilayer lipids.
266
Effect of q*. The effect of bicelle size (q* ratio) on the parameters of lipid phase transitions
267
can be investigated in more details to understand the properties of the smallest particles. For this
268
purpose, we prepared several DPPC/CHAPS mixtures with q* varied from 0.6 to 1.5 and recorded
269
the 31P spectra at temperatures ranging from 5 to 70 ºC. As was revealed by this experiment, the
270
fractional phase transitions do occur even in very small bicelles, e.g. at q=0.6, when the radius of
271
particles is equal to 2.7 nm (this is equivalent to q*=0.3 DMPC/DHPC particles, which are widely
272
applied for solution NMR spectroscopy of membrane proteins34). The chemical shift dependencies
273
on the temperature of both gel and fluid phase bicelles are almost identical at all tested q* (Figure
274
2A). This indicates that no substantial difference is observed between the lipid packing parameters
275
in small and large IsoBs. However, some properties of phase transition are different in smaller
276
particles. In particular, the fraction of gel-phase bicelles is reduced at low q* (Figure 2B). If we
277
define the effective critical temperature (Te) as a point with 50% of bicelles in the gel state, then Te
278
is reduced from 32.5 ºC at q*=1.5 to 17 ºC at q*=0.6. This is in agreement with our hypothesis,
279
suggested above, that small bicelles are less likely to be "frozen" than the larger ones. The
280
presence of phase transition even in smallest particles seems to in part resolve the long-lasting
281
argument about the relevance of small IsoBs5,24. As we show here, small bicelles are able to
282
reproduce one of the major properties of the lipid bilayer membranes - gel to the liquid-crystalline
283
phase transition.
284
Effect of lipid chain length and saturation. So far, we used the 31P NMR to study only the
285
single bicelle system, namely, DPPC/CHAPS. However, the applicability of the approach and general
286
character of the conclusions needs to be tested with the other kinds of lipids and rim-forming 9 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 25
287
surfactants. For that reason, we investigated the effect of lipid fatty acids on the 31P spectra and
288
phase transitions and prepared the DMPC/CHAPS and POPC/CHAPS mixtures. POPC/CHAPS q*=1.2
289
bicelles behaved as was expected: only the single and narrow peak was observed in the 31P NMR
290
spectra with the temperature coefficient of 3-3.5 ppb/K, and chemical shifts close to the observed
291
for the liquid-crystalline DPPC/CHAPS bicelles (Figure 4, S3). In other words, no phase transition is
292
observed for the POPC/CHAPS IsoBs, which is expected, because the phase transition temperature
293
of POPC bilayers is below 0 ºC. DMPC behaved similarly to DPPC in CHAPS-based bicelles: two 31P
294
signals, broad and narrow, appeared in NMR spectra below 20 ºC at q*=1.5 (critical temperature of
295
DMPC bilayers equals 24 ºC). The broad peak is shifted upfield and is characterized by the big
296
temperature coefficient, 8 ppb/K. Like for DPPC, the efficient phase transition temperature is
297
reduced for lower q* (17 ºC at q*=1.5 and 8 ºC at q*=1). Thus, we can conclude here that the
298
behavior of
299
clearly reflects the phase transitions. Moreover, the relationships between the critical
300
temperatures of the phase transition for various lipids in bilayers are reproduced in the IsoBs.
31
P signals in NMR spectra is similar for all phosphatidylcholine lipids in bicelles and
301
Effect of the rim-forming agent. Effect of the rim-forming agents on lipid phase transition in
302
isotropic bicelles is also very important. In our previous works, we characterized the bicelles
303
formed by CHAPS, CHAPSO, DHPC, bile salt, Facade-EM, and Facade-EPC in mixtures with the
304
DMPC phospholipid12,20. The listed surfactants allowed obtaining the bicelles with various
305
magnitudes of the temperature-induced growth and various ability to maintain the native fold of
306
membrane protein water-soluble domains20. Here we selected the most prospective detergents:
307
DHPC, Facade-EM and Facade-EPC and applied the
308
behavior and lipid/detergent mixing in IsoBs. In all three systems, the fractional phase transition
309
was observed, with parameters of 31P spectra similar to those of DMPC/CHAPS (Figure 5 A,B). The
310
efficient critical temperature was in all cases depending on q* in a manner, described previously -
311
the Te value was reduced at low lipid/detergent ratios (Figure S4). Like it was in the case of
312
DPPC/CHAPS, for all tested detergents, phase transitions were observed even in the mixtures with
313
the relatively small particles, applicable for solution NMR studies: q*=0.45 DMPC/DHPC (Figure S5,
314
3.0 nm), q*=1.0 DMPC/CHAPS (3.1 nm), q*=1.1 DMPC/Facade-EPC (2.9 nm). Thus, we can state
315
that lipid phase behavior is similar in bicelles, formed at various q* and by various surfactants.
31
P NMR spectroscopy to estimate the phase
316
However, there was the major difference between all rim-forming detergents observed with
317
respect to the number of lipids in the fluid and gel phases at various temperatures. If we take
318
bicelles of similar hydrodynamic radii (3.4 nm) at 10 ºC, we find that in the DMPC/Facade-EPC 10 ACS Paragon Plus Environment
Page 11 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
319
bicelles the fraction of lipid in the gel state is maximal (95%), in the DMPC/DHPC mixture the
320
amount of "frozen" bicelles equals 69%, while the DMPC/CHAPS and DMPC/Facade-EM provide
321
only 45-48% of bicelles in the gel phase (Figure 5C). It is noteworthy that the detergents that
322
provide the least expressed temperature-induced growth of IsoBs (Facade-EPC and DHPC) retain
323
the most expressed phase transition of bilayer lipids. This completely disproves our previous
324
hypotheses that the temperature-induced growth of bicelles directly reflects the lipid phase
325
transition and that Facade-EPC is mixed with the lipid in both the bilayer and rim areas of
326
bicelles12,20.
327
Another important observation refers to the properties of the bicelle rim. DHPC and Facade-
328
EPC contain the phosphatidylcholine moieties and are revealed in 31P NMR spectra. According to
329
the NMR data, the behavior of rim signals in NMR spectra is completely different to the behavior
330
of lipids. No phase transition is observed for the rim of IsoBs. Single and narrow peaks of DHPC and
331
Facade-EPC are present in
332
behavior of liquid-crystalline DMPC phosphate groups. In other words, while the lipids are in the
333
gel phase, the rim is always "liquid" (Figure 5B). This definitely implies the absence of the
334
detectable mixing between the detergents and lipids in the bilayer area of bicelles, at least in the
335
gel state. Such clear segregation and distinct temperature-dependent behavior of the detergent
336
and lipid signals also implies the absence of lipid molecules in the rim of the bicelles. Rim and
337
planar areas definitely have the different lipid packing properties, therefore a separate peak
338
should be observed for such rim-placed lipids, which is absent from the NMR spectra.
339
31
P spectra, their temperature-dependent behavior is similar to the
31
P NMR spectra of lipid-protein nanodiscs and large IsoBs. The belt protein of lipid-protein
340
nanodiscs (LPNs) may be also considered as the rim of bicelle-like particle35. Taking into account
341
that the phase transition was found to take place in DMPC LPNs36, it would be interesting to
342
compare the properties of
343
There was no sign of fractional phase transition found - only the single peak was observed in NMR
344
spectra at all temperatures in the range 5-60 ºC. However, the temperature coefficients of
345
chemical shifts clearly reflect the presence of phase transition. While above the Tm the signal
346
follows the behavior of DMPC in liquid-crystalline DMPC/CHAPS or DMPC/Facade-EPC bicelles,
347
below the critical point the slope of the temperature dependence of chemical shift is increased
348
and the properties of 31P spectra are similar to those of the "frozen" bicelles (Figure 6B). Moreover,
349
the temperature dependence of 31P chemical shifts in LPNs is almost identical to the dependence
350
observed for the q*=3 DMPC/Facade-EPC bicelles that have the same radius of ca. 4.9 nm20,36,37,
31
P NMR spectra of these particles to IsoBs at various temperatures.
11 ACS Paragon Plus Environment
31
P
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
351
and is to a high extent similar to the dependencies recorded for the q*=3 DMPC/Facade-EM and
352
q=1 DMPC/DHPC mixtures. Moreover,
353
phase transition temperature corresponded to the position of the broad peak in NMR spectra of
354
small IsoBs. Thus, we can conclude, that from the viewpoint of headgroups, the lipid packing
355
properties are similar in the LPNs and large IsoBs. Additionally, we state that the phase transition
356
of lipids may be observed via the 31P NMR spectroscopy even for such large particles with the radii
357
close to or exceeding 5 nm. Considering LPNs as a reference bilayer-containing membrane
358
mimetic we can state that behavior of lipids in LPNs is also the same as in small IsoBs, according to
359
the chemical shifts of signals in 31P NMR spectra.
31
P NMR spectra of very large IsoBs and LPNs below the
360
Parameters of phase transitions and temperature-induced growth depend on the
361
concentration of IsoBs. Apart from the temperature, lipid contents, and detergent, the overall lipid
362
concentration is also known to be important for the phase behavior of both anisotropic and
363
isotropic bicelles38. To investigate the influence of this parameter on the phase behavior of IsoBs,
364
we diluted the initial sample of DPPC/CHAPS q*=1.5 bicelles 10-fold (to 0.3 % w/w from initial 4%)
365
with the solution containing 3 mM of CHAPS, corresponding to the concentration of free CHAPS in
366
such a mixture12. This procedure did not affect the average size of bicelles at 35 ºC, according to
367
the 1H diffusion measurements and did not alter substantially the efficient phase transition
368
temperature of lipids (Figure 7A,B). On the other hand, the dilution resulted in a much narrower
369
phase transition: the fraction of bicelles in the fluid phase is decreased to zero already at 27 ºC at
370
0.3% instead of 10 ºC for 4% solution. Dilution as well affected the chemical shifts of 31P signals in
371
NMR spectra. The chemical shift decay with the decrease of temperature becomes much steeper
372
for the gel lipids at high dilution and appears to reach a plateau at -1.15 ppm and 20 ºC. Altogether
373
this may be interpreted by assuming that the parameters of phase transitions depend on the
374
particle collision rate in solution, which explains the majority of effects, described above. Collisions
375
may cause the melting of bicelles, which explains the presence of the fraction of bicelles in the
376
liquid-crystalline state below the critical temperature. Additionally, collisions do not allow the
377
complete freezing of lipids in the gel bicelles, which explains the less steep chemical shift
378
dependence on temperature at high concentrations and the absence of plateau at low
379
temperatures. This finding may also explain the previously reported extremely broad phase
380
transitions measured for the q=0.75 and q=0.5 DMPC/DHPC bicelles by the infrared
381
spectroscopy24. These measurements were made at 400 mM concentration, which corresponds to
382
ca. 20% w/w fraction and substantially exceeds all concentrations tested in the present work. 12 ACS Paragon Plus Environment
Page 12 of 25
Page 13 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
383
The concentration also appeared to affect the temperature-induced growth of IsoBs, which
384
is observed above the critical temperatures of lipids in some kinds of bicelles12,22. Gradual dilution
385
of initial 10% w/w DMPC/CHAPS q*=1.5 bicelle solution with 2.5 mM CHAPS revealed the decrease
386
in the magnitude of the specified effect. The radius of bicelles at 40 ºC decreased from 8 nm at 60
387
mM of DMPC to 4.34 nm at 1.8 mM, while below the phase transition (15 ºC) it remained almost
388
unchanged and equal to 3.7 nm (Figure 7 C,D). Thus, the 32-fold dilution allowed to weaken the
389
growth upon heating to 40 ºC from initial 116% to 19%. This implies that the temperature-induced
390
growth is also depending on the collision frequency of bicelles in solution. The consequences of
391
this conclusion and the nature of the growth are discussed below.
392
Summary and Conclusions. To understand the nature and reasons of the temperature-
393
induced growth of IsoBs, one needs to summarize all findings, reported in the present work and in
394
the literature. Now we know the following facts:
395 396 397 398
1) The growth of IsoBs upon heating is not observed in particles with radii less than 3.0 nm12,20,22. 2) The effect depends on the lipid composition of bicelles: it is at most expressed for the unsaturated lipids (POPC)12.
399
3) The magnitude of the effect depends on the rim-forming detergent, maximal growth is
400
observed for the CHAPS and Facade-EM and minimal growth is observed for the Facade-EPC and
401
DHPC. Detergents that provide the minimal temperature-induced growth reveal the more
402
pronounced phase transition of lipids in bicelles. Thus, growth is not related directly to the lipid
403
phase transition.
404 405
4) The effect depends on the concentration of bicelles, at high concentration bicelles grow much more rapidly with heating.
406
5) The growth of IsoBs is not likely to be the result of the mixing between the lipid and
407
detergent: 31P spectra of bicelles that experience the growth and of bicelles that do not grow upon
408
heating are almost identical, and the absence of growth at low concentrations of bicelles does not
409
agree with the mixing hypothesis.
410
Thinking of the phenomenon that could explain all listed facts, we can suggest only the
411
single hypothesis - IsoBs can merge upon collisions. Such fusion of bicelles should be reversible to
412
explain that at any given temperature the equilibrium size of bicelles is observed. Thus, the
413
process, that results in the temperature-induced growth may be considered as a reaction of bicelle
414
"oligomerization". Higher concentrations favor the fused state, which explains the concentration 13 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
415
dependence, and, at any given composition the propensity of fusion is governed by the
416
thermodynamics of the system - the free energy difference between the "fused" and "monomeric"
417
states. If the free energy of rim allows the long existence of such fused bicelles then the growth
418
occurs. This explains the absence of growth in small bicelles - the free energy pit is too narrow for
419
small bicelles, while in large bicelles the perforations and other rim irregularities may be formed to
420
stabilize the merged state of IsoBs. The reason for the enhanced temperature dependence of the
421
fusion is not clear and should be somehow related to the entropy gain or heat capacity changes of
422
the system upon the fusion. The propensity of fusion should also depend on the elasticity of
423
collisions and, therefore, the packing of lipids in bicelles. This explains the absence of growth in the
424
gel-phase bicelles and the effect of POPC. In addition, both the elasticity of collision and the
425
parameters of free energy profile of bicelles may depend on the type of particular rim-forming
426
agent. Facade-EPC and DHPC that possess charged zwitterionic headgroups may be less prone to
427
the bicelle fusion, while uncharged CHAPS and Facade-EM are more likely to interact across the
428
rims of two colliding particles. The proposed hypothesis may also explain several facts regarding
429
the behavior of large anisotropic bicelles. First of all, the spontaneous orientation of bicelles is
430
known to occur only at temperatures higher than phase transition temperature of bilayer lipid and
431
reproducible orientation is obtained only at relatively high concentrations of bicelles (25% w/w)32.
432
Apparently, below the critical temperatures and at low concentrations anisotropic bicelles are too
433
small to orient spontaneously, while the fusion of bicelles at high temperatures and in
434
concentrated samples results in the formation of very large particles that undergo the orientation
435
at strong magnetic fields. Another fact is the ability of Facade-EPC detergent to support the bicelle
436
formation at extremely high q*, while the other rim-forming surfactants at high q* fail to form
437
bicelles and the other phase is observed19. As we show here, Facade-EPC is resistant to bicelle
438
fusion upon collision and supports the least distorted packing of lipids in the bilayer area, which
439
explains the phenomenon.
440
Thus, we state that the present work and suggested hypothesis allow to explain the
441
phenomenon of temperature-induced growth of IsoBs with no need in bringing into consideration
442
the altered bicelle morphology or mixing between the lipids and detergents.
443
The problem of mixing between the detergent and lipid in the "bilayer" area of bicelles is
444
also crucial for the structural biology. One would like to mimic the properties of cell membranes,
445
and if the detergents are present in the bilayer in high amounts, they can distort its structure. This
446
would decrease the extent of identity between the bicelles and bilayer membranes and challenge 14 ACS Paragon Plus Environment
Page 14 of 25
Page 15 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
447
the relevance of the experimental data, obtained in such an environment. To get the complete
448
understanding of the bicelle structure, one needs to summarize the whole set of data regarding
449
the IsoBs.
450
To proceed, we need to define what is implied by the "substantial extent of mixing". Such
451
mixing is quite difficult to detect, and it is also hardly possible to put forward any kind of
452
quantitative cut-off for the majority of bicelle systems. Therefore, we propose that the presence of
453
substantial mixing can be postulated if the behavior of bicelles fails to reproduce any aspect of the
454
behavior of lipid bilayers or particles that were shown to contain the patch of the lipid bilayer, such
455
as the LPNs. These aspects may include the lipid packing properties, lipid phase transitions,
456
behavior of membrane proteins inside the bicelles. The presence of lipid bilayer in the large IsoBs
457
(q>0.75 for DMPC/DHPC) was shown by a variety of approaches. Direct observation of particles
458
with cryoelectron microscopy revealed the discoidal shape of the bicelles in solution5,38, large
459
bicelles were shown to undergo the gel to liquid-crystalline phase transitions at temperatures,
460
close to the observed for bilayer lipids24, and, finally, most membrane proteins are able to sustain
461
the native fold and activity in the bicelle environment39,40. Thus, the main question that needs to
462
be answered is: do the small bicelles, used in structural studies behave in the same way as the
463
large bicelles and LPNs?
464
Until recently, there were two major effects that suggested the mixing of lipids and
465
detergents in IsoBs. First, several studies stated, that the size of small bicelles does not fit the so-
466
called "ideal bicelle model"5,39. However, the refined models that take into account the shape of
467
the bicelle rim12,23, revealed that size of small bicelles perfectly follow the theoretical predictions.
468
Actually, the large IsoBs demonstrated the "deviant" behavior, the so-called temperature-induced
469
growth12. However, according to the current work, this effect has a pronounced concentration
470
dependence. Therefore, we suggested that the growth is a result of the reversible fusion of
471
bicelles. Second, the phase transition of lipids was thought to be absent in small IsoBs24. The phase
472
transition is a fundamental property of lipid bilayer, therefore the absence of such transition was
473
considered as the evidence in favor of the absence of the bilayer itself in IsoBs. This problem
474
seems to be also resolved in the current work - we showed that the phase transitions of lipids are
475
observed even in the smallest bicelles, which are applied in the structural studies. On the other
476
hand, the properties of the phase transitions in small IsoBs are peculiar: these transitions are
477
"fractional" and reveal the reduced critical temperature, which depends on the size of the
478
particles. When gel and fluid phase particles coexist in solution, the size of gel phase bicelles is 15 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
479
much greater, than the size of the liquid-crystalline particles, which stands against the lipid packing
480
properties in gel and liquid-crystalline bilayers. These phenomena may be caused by the presence
481
of lipid/detergent mixing, however, as we show above, another explanation is more likely to be
482
relevant. The mixing hypothesis fails to account for some of the reported data: the concentration
483
dependence of the phase transition properties in small IsoBs and the greater size of liquid-
484
crystalline IsoBs, when the whole population of bicelles is considered above the critical
485
temperature. Above, we provide several possible explanations of the phase transition properties in
486
small IsoBs, based on the broad bicelle size distribution, destabilizing effects of the rim detergent
487
and bicelle collisions. Therefore, we can state that there is no data up to date, that does not agree
488
with the presence of lipid bilayer in the planar area of small IsoBs.
489
This certainly does not mean that there is at all no mixing between the lipids and detergents
490
in IsoBs. However, there is some indirect data that suggests, that if such mixing is present, it is not
491
strong enough to alter the lipid packing properties in IsoBs, in comparison to large bicelles, LPNs,
492
and lipid bilayers. First of all, we show here, that detergents always remain "liquid", regardless the
493
state of lipids in bicelles. When two types of particles are observed, there is no additional peak of
494
detergent found, which would correspond to the detergent molecules, "trapped" inside the gel
495
bilayer, due to the mixing. The intensity ratio between the detergent and lipid peaks is always
496
retained. Therefore, lipids and detergents are not mixed substantially in the gel phase IsoBs.
497
Moreover, we observe that 31P chemical shifts behave almost identically in large and small bicelles
498
of the same composition above the critical temperature. According to our data, 31P chemical shift
499
difference is much greater between the bicelles formed by various rim detergents, than between
500
the small and large liquid-crystalline bicelles of the same composition (Figure S6 A,B). This suggests
501
that if lipid/detergent mixing is present it is similar in large and small bicelle particles of the same
502
contents.
503
In addition, we studied the behavior of other lipid/detergent mixtures, e.g. DMPC/SDS and
504
found no sign of phase transition even for very large particles (5.5 nm). Moreover, the size of such
505
mixed micelles does not fit to the ideal bicelle model with reasonable parameters, the rim
506
thickness appeared to be equal to 3.1 ±0.1 nm (Figure S6C), which exceeds even the length of
507
DMPC molecule (2.0-2.2 nm). For comparison, the thickness of CHAPS rim is as small as 1.1 nm12.
508
This experiment may be considered as a negative control, demonstrating that when an indeed
509
substantial lipid/detergent mixing is present in bicelles or mixed micelles, it could be detected
510
following the size of particles and 31P chemical shifts of the phospholipid. 16 ACS Paragon Plus Environment
Page 16 of 25
Page 17 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
511
The main idea that we would like to contradict in the current work is that small IsoBs that
512
are used in solution NMR studies are somehow different from LPNs and large bicelles. We think
513
that our data demonstrates clearly that properties of large and small bicelles are similar. We do
514
not observe any abrupt change in the behavior of low-q and high-q mixtures, which would allow to
515
postulate the distinct morphology of small and large particles. All bicelle parameters change
516
gradually and do not contradict the "ideal bicelle model". If lipid-detergent mixing is present, it is
517
similar in very large and smallest particles and permits to preserve the patch of lipid bilayer that
518
undergoes the phase transition.
17 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
519
FIGURES
520 521 522 523 524
Figure 1. 31P NMR may be used to detect the lipid phase transitions in small bicelles. 31P NMR spectra of DPPC/CHAPS bicelles prepared with q*=1.5, 1.0 and 0.8 recorded at 323 (blue), 303 (red), 293 (green) and 283 (magenta) K. Peaks, corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively.
525 526 527 528 529 530
Figure 2. Effect of q*. A - chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DPPC/CHAPS bicelles, which were prepared with various q* (see the legend). Average linear approximation of temperature dependencies is plotted by the dashed (liquid-crystalline lipids) and dotted (gel lipids) lines. For q=0.6 the peak, corresponding to gel lipids, is too broad to get the chemical shifts with necessary precision. B - the fraction of gel phase 18 ACS Paragon Plus Environment
Page 18 of 25
Page 19 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
531 532 533
DPPC/CHAPS bicelles (relative intensity of the broad peak in 31P spectra) is plotted as a function of temperature for various q*.
534 535 536
Figure 3. Radii of DPPC/CHAPS q*=1.50 and 1.35 bicelles measured for the narrow (A) and broad (B) peaks in 31P NMR spectrum at 298 and 293 K based on the NMR diffusion coefficients. pvalues were calculated using the Student test.
537 538 539 540 541 542 543 544 545
Figure 4. Effect of the lipid chain length and saturation. A - 31P NMR spectra of DMPC/CHAPS q*=1.5 (left panel) and POPC/CHAPS q*=1.2 (right panel) bicelles recorded at various temperatures, as indicated. Peaks, corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively. B - chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DPPC/CHAPS q*=1.5 (blue, red), DMPC/CHAPS q*=1.5 (green, brown) and POPC/CHAPS q*=1.2 bicelles (yellow). Average linear approximation of temperature dependencies is plotted by the dashed lines.
19 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
546 547 548 549 550 551 552 553 554 555 556 557 558 559 560
Figure 5. Effect of the rim-forming detergent. A - 31P NMR spectra of DMPC/DHPC q*=0.75 (left panel) and DMPC/Facade-EPC q*=1.5 (right panel) bicelles recorded at various temperatures, as indicated. Peaks, corresponding to liquid-crystalline and gel lipids are indicated by * and **, respectively. Peak from DHPC is indicated by D, peaks from Facade-EPC phosphate moieties are indicated by P1 and P2. B - chemical shift temperature dependencies recorded for narrow and broad peaks observed in 31P spectra of DMPC/Facade-EPC q*=1.5 (red/blue), DMPC/CHAPS q*=1.5 (yellow, green), DMPC/FACADE-EM q*=1.5 (brown, cyan) and DMPC/DHPC q*=1 bicelles (darkgreen, phase transition in these bicelles is not fractional). Additionally plotted are the chemical shifts of Facade-EPC group P1 in DMPC/Facade-EPC q*=1.5 bicelles (light green). Average linear approximation of temperature dependencies is plotted by the dashed lines. C - the fraction of gel phase DMPC in bicelles (relative intensity of the broad peak in 31P spectra) is plotted for 283 (blue), 288 (red) and 293 (green) K for four kinds of bicelles with the same radius of 3.4 nm (below the phase transition temperature).
20 ACS Paragon Plus Environment
Page 20 of 25
Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
561 562 563 564 565 566 567 568 569
Figure 6. Lipid-protein nanodiscs. A - Superposition of 31P NMR spectra recorded at 283, 293 and 303 K for DMPC/MSPD1 LPNs (one signal) and DMPC/Facade-EPC q*=3.0 bicelles (three signals). B - chemical shift temperature dependencies recorded for peaks observed in 31P spectra of DMPC/Facade-EPC q*=3.0 (blue squares), DMPC/FACADE-EM q*=3 (red rhombuses), DMPC/DHPC q*=1 bicelles (yellow triangles) and DMPC/MSPD1 LPNs (green triangles). The linear approximation of data for LPNs below and above the critical temperature of DMPC is shown by dashed and dotted lines.
21 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
570 571 572 573 574 575 576 577 578 579 580 581 582 583
Figure 7. Concentration affects the phase transition and temperature-induced growth of isotropic bicelles. A - chemical shift temperature dependencies recorded for wide and narrow peaks observed in 31P spectra of DPPC/CHAPS q*=1.5 bicelles prepared at concentrations of 4% (red, blue), 1.3% (green, yellow) and 0.3 % w/w (cyan, brown). Lines represent the result of 3rd order polynomial approximation of the experimental data. B - the fraction of gel phase DPPC in q*=1.5 DPPC/CHAPS bicelles, prepared at 4% and 0.3% w/w. С - the radius of DMPC/CHAPS q*=1.5 bicelles is plotted as a function of DMPC concentration at 40 ºC, errors do not exceed the size of points on the plot. D - temperature dependence of the radius R of DMPC/CHAPS q*=1.5 bicelles measured at 29.5 mM DMPC and at 5.8 mM DMPC, errors do not exceed the size of points on the plot. The behavior of bicelles at 5.8 mM of DMPC below the phase transition is related to the variation of the free CHAPS concentration with temperature.
584
Corresponding Author
585
*
[email protected] 586
Author Contributions
587
The manuscript was written through contributions of all authors. All authors have given
588
approval to the final version of the manuscript. 22 ACS Paragon Plus Environment
Page 22 of 25
Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Langmuir
589
Acknowledgment
590
The work is supported by the Russian Science Foundation, grant #14-14-00573. Experiments
591
were partially carried out using the equipment provided by the IBCH core facility (CKP IBCH,
592
supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018).
593 594 595
Supporting Information Available. Supporting Information includes six additional figures. This information is available free of charge via the Internet at http://pubs.acs.org/.
596 597
ABBREVIATIONS
598
NMR, nuclear magnetic resonance; LPN, lipid-protein nanodisc; IsoB, isotropic bicelle;
599
DMPC,
600
phosphocholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; DHPC, 1,2-dihexanoyl-
601
sn-glycero-3-phosphocholine;
602
propanesulfonate; SDS, sodium dodecyl sulfate.
603
1,2-dimyristoyl-sn-glycero-3-phosphocholine;
CHAPS,
DPPC,
1,2-dipalmitoyl-sn-glycero-3-
3-[(3-cholamidopropyl)dimethylammonio]-1-
References. (1) Sanders, C. R.; Prestegard, J. H. Magnetically Orientable Phospholipid Bilayers Containing Small Amounts of a Bile Salt Analogue, CHAPSO. Biophys. J. 1990, 58 (2), 447–460. (2) Gabriel, N. E.; Roberts, M. F. Interaction of Short-Chain Lecithin with Long-Chain Phospholipids: Characterization of Vesicles That Form Spontaneously. Biochemistry 1986, 25 (10), 2812–2821. (3) Sanders, C. R.; Schwonek, J. P. Characterization of Magnetically Orientable Bilayers in Mixtures of Dihexanoylphosphatidylcholine and Dimyristoylphosphatidylcholine by SolidState NMR. Biochemistry 1992, 31 (37), 8898–8905. (4) Gabriel, N. E.; Roberts, M. F. Spontaneous Formation of Stable Unilamellar Vesicles. Biochemistry 1984, 23 (18), 4011–4015. (5) Glover, K. J.; Whiles, J. A.; Wu, G.; Yu, N.; Deems, R.; Struppe, J. O.; Stark, R. E.; Komives, E. A.; Vold, R. R. Structural Evaluation of Phospholipid Bicelles for Solution-State Studies of Membrane-Associated Biomolecules. Biophys. J. 2001, 81 (4), 2163–2171. (6) De Angelis, A. A.; Opella, S. J. Bicelle Samples for Solid-State NMR of Membrane Proteins. Nat Protoc 2007, 2 (10), 2332–2338. (7) Ottiger, M.; Bax, A. Bicelle-Based Liquid Crystals for NMR-Measurement of Dipolar Couplings at Acidic and Basic PH Values. J. Biomol. NMR 1999, 13 (2), 187–191. (8) Mineev, K. S.; Nadezhdin, K. D. Membrane Mimetics for Solution NMR Studies of Membrane Proteins. Nanotechnology Reviews 2017, 6 (1). (9) Dürr, U. H. N.; Gildenberg, M.; Ramamoorthy, A. The Magic of Bicelles Lights Up Membrane Protein Structure. Chemical Reviews 2012, 112 (11), 6054–6074. (10) Warschawski, D. E.; Arnold, A. A.; Beaugrand, M.; Gravel, A.; Chartrand, É.; Marcotte, I. Choosing Membrane Mimetics for NMR Structural Studies of Transmembrane Proteins. Biochim. Biophys. Acta 2011, 1808 (8), 1957–1974. 23 ACS Paragon Plus Environment
Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(11) Lee, D.; Walter, K. F. A.; Brückner, A.-K.; Hilty, C.; Becker, S.; Griesinger, C. Bilayer in Small Bicelles Revealed by Lipid-Protein Interactions Using NMR Spectroscopy. J. Am. Chem. Soc. 2008, 130 (42), 13822–13823. (12) Mineev, K. S.; Nadezhdin, K. D.; Goncharuk, S. A.; Arseniev, A. S. Characterization of Small Isotropic Bicelles with Various Compositions. Langmuir 2016, 32 (26), 6624–6637. (13) Liebau, J.; Pettersson, P.; Zuber, P.; Ariöz, C.; Mäler, L. Fast-Tumbling Bicelles Constructed from Native Escherichia Coli Lipids. Biochim. Biophys. Acta 2016, 1858 (9), 2097–2105. (14) Struppe, J.; Whiles, J. A.; Vold, R. R. Acidic Phospholipid Bicelles: A Versatile Model Membrane System. Biophys. J. 2000, 78 (1), 281–289. (15) Marcotte, I.; Dufourc, E. J.; Ouellet, M.; Auger, M. Interaction of the Neuropeptide MetEnkephalin with Zwitterionic and Negatively Charged Bicelles as Viewed by 31P and 2H SolidState NMR. Biophys. J. 2003, 85 (1), 328–339. (16) Sasaki, H.; Fukuzawa, S.; Kikuchi, J.; Yokoyama, S.; Hirota, H.; Tachibana, K. Cholesterol Doping Induced Enhanced Stability of Bicelles. Langmuir 2003, 19 (23), 9841–9844. (17) Barbosa-Barros, L.; de la Maza, A.; López-Iglesias, C.; López, O. Ceramide Effects in the Bicelle Structure. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2008, 317 (1–3), 576–584. (18) Gayen, A.; Mukhopadhyay, C. Evidence for Effect of GM1 on Opioid Peptide Conformation: NMR Study on Leucine Enkephalin in Ganglioside-Containing Isotropic Phospholipid Bicelles. Langmuir 2008, 24 (10), 5422–5432. (19) Lee, S. C.; Bennett, B. C.; Hong, W.-X.; Fu, Y.; Baker, K. A.; Marcoux, J.; Robinson, C. V.; Ward, A. B.; Halpert, J. R.; Stevens, R. C.; et al. Steroid-Based Facial Amphiphiles for Stabilization and Crystallization of Membrane Proteins. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (13), E12031211. (20) Mineev, K. S.; Nadezhdin, K. D.; Goncharuk, S. A.; Arseniev, A. S. Façade Detergents as Bicelle Rim-Forming Agents for Solution NMR Spectroscopy. Nanotechnology Reviews 2017, 6 (1). (21) Ujwal, R.; Bowie, J. U. Crystallizing Membrane Proteins Using Lipidic Bicelles. Methods 2011, 55 (4), 337–341. (22) Ye, W.; Lind, J.; Eriksson, J.; Mäler, L. Characterization of the Morphology of Fast-Tumbling Bicelles with Varying Composition. Langmuir 2014, 30 (19), 5488–5496. (23) Triba, M. N.; Warschawski, D. E.; Devaux, P. F. Reinvestigation by Phosphorus NMR of Lipid Distribution in Bicelles. Biophys. J. 2005, 88 (3), 1887–1901. (24) Beaugrand, M.; Arnold, A. A.; Hénin, J.; Warschawski, D. E.; Williamson, P. T. F.; Marcotte, I. Lipid Concentration and Molar Ratio Boundaries for the Use of Isotropic Bicelles. Langmuir 2014, 30 (21), 6162–6170. (25) Schmidt, T.; Situ, A. J.; Ulmer, T. S. Direct Evaluation of Protein-Lipid Contacts Reveals Protein Membrane Immersion and Isotropic Bicelle Structure. J Phys Chem Lett 2016, 7 (21), 4420– 4426. (26) Denisov, I. G.; Grinkova, Y. V.; Lazarides, A. A.; Sligar, S. G. Directed Self-Assembly of Monodisperse Phospholipid Bilayer Nanodiscs with Controlled Size. J. Am. Chem. Soc. 2004, 126 (11), 3477–3487. (27) Jerschow, A.; Müller, N. Suppression of Convection Artifacts in Stimulated-Echo Diffusion Experiments. Double-Stimulated-Echo Experiments. Journal of Magnetic Resonance 1997, 125 (2), 372–375. (28) Zheng, G.; Price, W. S. Simultaneous Convection Compensation and Solvent Suppression in Biomolecular NMR Diffusion Experiments. Journal of Biomolecular NMR 2009, 45 (3), 295– 299. 24 ACS Paragon Plus Environment
Page 24 of 25
Page 25 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
604
Langmuir
(29) Tokuyama, null; Oppenheim, null. Dynamics of Hard-Sphere Suspensions. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 1994, 50 (1), R16–R19. (30) Ortega, A.; Garcı ́a de la Torre, J. Hydrodynamic Properties of Rodlike and Disklike Particles in Dilute Solution. The Journal of Chemical Physics 2003, 119 (18), 9914. (31) Li, M.; Morales, H. H.; Katsaras, J.; Kučerka, N.; Yang, Y.; Macdonald, P. M.; Nieh, M.-P. Morphological Characterization of DMPC/CHAPSO Bicellar Mixtures: A Combined SANS and NMR Study. Langmuir 2013, 29 (51), 15943–15957. (32) Morales, H. H.; Saleem, Q.; Macdonald, P. M. Thermal Stabilization of Bicelles by a Bile-SaltDerived Detergent: A Combined 31 P and 2 H Nuclear Magnetic Resonance Study. Langmuir 2014, 30 (50), 15219–15228. (33) Nagle, J. F.; Tristram-Nagle, S. Structure of Lipid Bilayers. Biochim. Biophys. Acta 2000, 1469 (3), 159–195. (34) Bocharov, E. V.; Mineev, K. S.; Volynsky, P. E.; Ermolyuk, Y. S.; Tkach, E. N.; Sobol, A. G.; Chupin, V. V.; Kirpichnikov, M. P.; Efremov, R. G.; Arseniev, A. S. Spatial Structure of the Dimeric Transmembrane Domain of the Growth Factor Receptor ErbB2 Presumably Corresponding to the Receptor Active State. J. Biol. Chem 2008, 283 (11), 6950–6956. (35) Denisov, I. G.; Sligar, S. G. Nanodiscs for Structural and Functional Studies of Membrane Proteins. Nature Structural & Molecular Biology 2016, 23 (6), 481–486. (36) Denisov, I. G.; McLean, M. A.; Shaw, A. W.; Grinkova, Y. V.; Sligar, S. G. Thermotropic Phase Transition in Soluble Nanoscale Lipid Bilayers. The Journal of Physical Chemistry B 2005, 109 (32), 15580–15588. (37) Nakano, M.; Fukuda, M.; Kudo, T.; Miyazaki, M.; Wada, Y.; Matsuzaki, N.; Endo, H.; Handa, T. Static and Dynamic Properties of Phospholipid Bilayer Nanodiscs. J. Am. Chem. Soc. 2009, 131 (23), 8308–8312. (38) van Dam, L.; Karlsson, G.; Edwards, K. Direct Observation and Characterization of DMPC/DHPC Aggregates under Conditions Relevant for Biological Solution NMR. Biochim. Biophys. Acta 2004, 1664 (2), 241–256. (39) Czerski, L.; Sanders, C. R. Functionality of a Membrane Protein in Bicelles. Anal. Biochem. 2000, 284 (2), 327–333. (40) Sanders, C. R.; Landis, G. C. Reconstitution of Membrane Proteins into Lipid-Rich Bilayered Mixed Micelles for NMR Studies. Biochemistry 1995, 34 (12), 4030–4040. Table of Contents Graphics:
605
25 ACS Paragon Plus Environment
