Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

Invisible detergents for structure determination of membrane proteins by small-

angle neutron scattering

1

2

1,3

1†

Søren Roi Midtgaard *, Tamim A. Darwish *, Martin Cramer Pedersen , Pie Huda , Andreas Haahr

1

Larsen , Grethe Vestergaard Jensen , Søren Andreas Røssel Kynde , Nicholas Skar-Gislinge ,

Agnieszka Janina Zygadlo Nielsen , Claus Olesen , Mickael Blaise , Jerzy Józef Dorosz , Thor Seneca

Thorsen , Raminta Venskutonytė , Christian Krintel , Jesper V. Møller , Henrich Frielinghaus ,

Elliot Paul Gilbert , Anne Martel , Jette Sandholm Kastrup , Poul Erik Jensen , Poul Nissen

4

5

6,7

8

9,13

10

11

12

8

4

13,14*

, Lise

1

Arleth *

1

) Structural Biophysics, X-ray and Neutron Science, The Niels Bohr Institute, University of

Copenhagen, Denmark

2

) National Deuteration Facility, Australian Nuclear Science and Technology Organization, Lucas

Heights, Australia

3

) Department of Applied Mathematics, Research School of Physics and Engineering, Australian

National University, Canberra ACT 2601, Australia.

4

5

6

7

) Copenhagen Plant Science Center, University of Copenhagen, Denmark

) Department of Biomedicine, Aarhus University, Denmark

) Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, France

) Centre for Carbohydrate Recognition and Signaling, Department of Molecular Biology, Aarhus

University, Denmark

8

) Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University

of Copenhagen, Denmark

9

1

) Department of Biomedicine, Aarhus University, Denmark

0) Forschungszentrum Jülich GmbH, TUM FRM-2, Garching, Germany

1) Australian Centre for Neutron Scattering, Australian Nuclear Science and Technology

Organization, Lucas Heights, Australia

1

2) Institut Laue-Langevin, Grenoble, France

3) Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and

Disease – PUMPkin, Danish National Research Foundation, Aarhus University, Denmark

1

4) DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Denmark

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/febs.14345

Article type : Original Article

*

To whom correspondence should be addressed:

Dr. S. R. Midtgaard: soromi@nbi.ku.dk

Dr. T. Darwish: tde@ansto.gov.au

Prof. P. Nissen: pn@mbg.au.dk

Prof. L. Arleth: arleth@nbi.ku.dk

†

:

Current address: Centre for Advanced Imaging, The University of Queensland, Brisbane, Australia

Abbreviations:

SANS: Small-Angle Neutron Scattering

Cryo-EM: Cryo Electron Microscopy

DDM: n-dodecyl-β-D-maltopyranoside

OG: octyl β-D-glucopyranoside

bR: Bacteriorhodopsin

LamB: Maltoporin

PSI: Photosystem I

GluA2: Ionotropic glutamate receptor A2 ₂

+

SERCA: Sarco/endoplasmatic reticulum Ca ATPase

MW: Molecular Weight

g

R : Radius of Gyration

D

max: Maximum distance

SEC: Size

kDa: Kilo Dalton

SAXS: Small-Angle X-ray Scattering

FWHM: Full Width Half Maximum

SI: Supplemental Information

Keywords:

Small-Angle Neutron Scattering

SANS

Membrane Proteins

Deuteration

Contrast Matching

Abstract

A novel and generally applicable method for determining structures of membrane proteins in

solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing

membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic

neutron scattering length difference between hydrogen and deuterium. Individual

hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the

formed micelles became effectively invisible in heavy water (D O) when investigated by neutrons.

2

This way, only the signal from the membrane protein remained in the SANS data. We demonstrate

that the method is not only generally applicable on five very different membrane proteins but also

2+

reveals subtle structural details about the sarco/endoplasmatic reticulum Ca ATPase (SERCA). In all,

the synthesis of isotope-substituted detergents makes solution structure determination of

membrane proteins by SANS and subsequent data analysis available to non-specialists.

Introduction

Biological membranes form the barrier between internal and external environments of cells and

create the separation of compartments and organelles within the cell. They are functionalized by a

plethora of membrane proteins that include structural proteins, receptors, channels, active

transporters and proteolytic enzymes. Studies of the structure and conformational changes related

to the function of membrane proteins therefore represent very important problems in molecular cell

biology. Structural studies usually require that the membrane protein of interest is removed from

the native membrane, isolated and purified to homogeneity. This requires that the amphipathic

membrane protein is stabilized in solution. Multiple systems, including traditional detergents[1],

bicelles[2,3], peptide discs[4], amphiphilic polymers[5,6] and nanodiscs[7] are available for this

purpose. While detergents and bicelles have been useful for Cryo-EM[8], NMR[9] and crystallization

experiments[1], the comparatively monodisperse nanodisc particles are promising in combination

with small-angle scattering and allows low-resolution structural information of the membrane

protein to be obtained under solution conditions[10–12]. However, reconstitution of membrane

proteins in nanodiscs remains a challenge with respect to obtaining sufficiently homogeneous

samples for scattering experiments. In our attempt to find a more general and easily applicable

solution to these problems, the use of detergents was re-examined and combined with our

previously developed idea of a “stealth”-carrier for SANS studies of membrane proteins[10]. The

detergents, octyl β-D-glucopyranoside (OG) and n-dodecyl-β-D-maltopyranoside (DDM) that are

both compatible with membrane protein reconstitution were deuterated through a new synthesis

method that allowed for selective deuteration of the two detergents to different predetermined

levels at the hydrophilic headgroups and hydrophobic tails. To obtain the best possible SANS signal-

to-noise of the membrane protein structure, the different parts of the detergents were deuterated

such that they had the same neutron scattering length density as 100% deuterium oxide (D

2

O). This

way the obtained SANS data of membrane proteins stabilized in these detergents in D

consists of the single contrast signal stemming from the purified membrane protein.

2

O only

The general idea to use the contrast match-point of a detergent or surfactant to study membrane

protein structures has previously been investigated[13–29]. However, a common feature for all

previous studies is that they rely on commercially available hydrogenated detergents or fully

deuterated detergents developed for different purposes. This is reflected in the resulting neutron

scattering data, which include scattering cross-terms from the entire detergent-membrane protein

complex due to the differences in excess scattering length density of the hydrophobic and

hydrophilic parts of the detergents present on length scales of 10-20 Å. This effect is difficult to

disentangle from the signal from the membrane protein and significantly limits the resolution that

can be obtained. In addition, the overall match-point of the commercially available detergents is

generally different from that of 100% D O, hence yielding additional incoherent scattering

2

background from the hydrogen in the buffer. While the studies have provided overall shape

parameters of studied membrane proteins[14,20,21,24,27,28], their finer structure could not be

extracted from the data due to these factors.

In this study, two commonly used detergents, OG and DDM, were synthesized with varying

deuteration levels in head- and tailgroups such that they were fully match-out deuterated in D O

2

buffer when investigated by SANS. This yielded SANS data where only the membrane proteins were

visible. The practical workflow is outlined in figure 1.

Initially, the membrane protein is purified in hydrogenated buffer and hydrogenated detergent. This

step ensures that as many impurities as possible are removed before the more expensive deuterated

buffers are introduced. Afterwards, a size exclusion purification in the equivalent deuterated buffer

and match-out deuterated detergent is performed. This step ensures that the hydrogenated

detergent and buffer are fully exchanged to the deuterated counterparts (see SI 3 for details).

Furthermore, any aggregated protein that might be present in the sample arising from e.g. freezing,

transport, etc., is also removed to ensure optimal data quality. The peak fraction from the buffer and

detergent exchange is collected and, without further manipulation, measured by SANS. Scattering

data obtained this way can be reduced and subsequently analyzed by a number of available

programs for small-angle scattering (such as ATSAS[30], WillItFit[31], SASSIE[32], IRENA[33],

FoXS[34] etc.) due to the simplified contrast situation achieved through the of use of de facto

invisible detergents.

Results

Detergent deuteration.

As described above, the chosen deuterated detergents were two commonly used sugar based

detergents, namely OG and DDM. The partial specific molecular volumes of hydrophilic head and

hydrophobic tail groups were determined by densitometry (see SI 1 for details) such that the

o

deuteration level for complete match-out at 100% D

2

O at 20 C could be calculated (see table 1).

Custom syntheses were then developed to produce the desired partially deuterated versions of both

detergents.

Synthesis of detergents with controlled deuteration levels in hydrophilic headgroups and

hydrophobic tails

Each of OG and DDM is made of two parts; the C8 or C12 alkyl chain tail and the corresponding sugar

heads of glucose and maltose, respectively. In the case of OG, the required deuteration levels in the

octyl chain and the glucose sugar head (7 non-exchangeable positions) groups were 94% D and 52%

D, respectively. In DDM, the required deuteration levels in the dodecyl chain and the maltose sugar

head (14 non-exchangeable positions) groups were 89% D and 57% D, respectively. Deuterating the

detergent molecules directly using hydrothermal reactions was not possible as the harsh conditions

of high temperature and pressure were incompatible with the delicate nature of the molecules. The

deuteration of methylene units in alkyl hydrocarbons can only be achieved using forced conditions

of a hydrothermal catalytic exchange reaction[35], while the deuteration of the sugar groups can be

achieved using milder reaction conditions[36]. An alternative approach of deuterating the

corresponding sugar molecule and the alkyl chains separately before attaching them together would

not be feasible since the sugar groups, glucose and maltose, are reducing sugars, which means they

would quickly decompose in the presence of reducing reagents due to the hydrolysis of their hemi-

acetal moieties. Reducing sugars must be preceded by conversion into glycoside to prevent

reduction at the anomeric carbon when exposed to Raney Nickel[36]. Therefore, to achieve specific

deuteration levels in the two different parts of any of the two detergent molecules, it was necessary

to follow a multiple-step synthesis approach and two sequential deuteration steps. This involved

(illustrated in figure 2 for OG): A) deuterating the alkyl chain at the required deuteration level

starting from the corresponding fatty acid and using hydrothermal Pt/C catalyzed H/D exchange

reactions at 220°C in the appropriate molar ratio of deuterium and hydrogen atoms in the mixture;

B) reducing the fatty acid molecule to the corresponding alcohol and attaching the deuterated

alcohol to the corresponding acetylated bromo-sugar head group (i.e., 2,3,4,6-tetra-O-acetyl-α-D-

glucopyranosyl bromide and 2,3,6,2',3',4',6'-hepta-O-acetyl-a-D-maltosyl bromide) according to

standard procedures; C) deacetylation of the sugar head group followed by deuterating it to achieve

the required deuteration level by using mild conditions of Raney Nickel as a catalyst in D

2

O/H O

2

mixture at 80°C for 18 h. The latter step allows the incorporation of deuterium atoms on carbons

adjacent to free hydroxyl groups (α positions) in the sugar head group with retention of

configuration, but it does not affect any H/D back-exchange at the more inert alkyl chain sites[36–

3

8] (see SI 2 for details).

SANS contrast variation on empty micelles

The deuteration levels obtained from the custom synthesis were close to the desired values (Table

1

), implying that the scattering length density from the detergents should theoretically be close to

that of 100% D O. To experimentally verify this, micelles of the match-out deuterated detergents

were measured by SANS in a set of buffers with D O content ranging from 60% to 100%. Experiments

2

were performed at ANSTO and FRMII (See SI 4). Background subtracted data are plotted in figure 3.

The SANS intensity from both detergents is reduced by more than two orders of magnitude when

changing the percentage of D

2

O in the buffer from 60% to 100%. Indeed, at 100% D O, the signal

2

from the match-out deuterated detergents is effectively at the noise-level over the measured q-

range and exhibiting no significant q-dependence. I(0) values (figure 3C) were determined by indirect

Fourier transformation[39]. The expected parabolic behavior of the I(0) as a function of D

was confirmed (figure 3C) and the match-points were found to be at 102% D O for DDM and 103%

O for OG.

2

O contents

2

D

2

In figures 3D and E the obtained contrast-matched data are compared to previously obtained and

published SANS data from the commercially available tail-deuterated counterparts to OG and DDM:

d17-OG and d25-DDM[28]. These data, (obtained and plotted with permission from the authors), are

measured at the D22 instrument at ILL, France and plotted at their respective match-points which is

9

0% D

2

O for d17-OG and 85% for d-25-DDM [28].

The data from the tail-deuterated detergent d17-OG exhibit the expected q-dependence with an

-

1

oscillation having a maximum at around 0.15 Å , but overall a very low intensity of the I(q) at the

match-point. Note however, that the d17-OG data had a small experimental discrepancy between

the background levels of the measured buffer and sample. Due to the low signal to background

ratio, this had relatively large impact on the obtained scattering intensity as illustrated in fig 3D

which plots both the d17-OG data with background subtracted using the theoretical normalization

light grey curve) and the same data with a small renormalization of the background (dark grey

(

curve). As it is seen, this causes a relatively large shift in the overall scattering intensity, which

unfortunately makes a direct quantitative comparison to our data difficult (figure 3D). In any case,

the density fluctuations expected in the d17-OG are systematic and clearly visible in the I(q)-data

regardless of the buffer level, while there is no indication of this feature in the data from the match-

out deuterated OG.

In the comparison between the d25-DDM at 85% D

E) the above mentioned experimental discrepancy between the buffer and sample background

2

O and the match-out d-DDM at 100% D O (figure

2

3

levels is not present to the same extent. Furthermore, a much stronger difference between the

custom-synthesized and commercial variants is observed which clearly demonstrates the added

value of the custom synthesis of these match-out detergents. Indeed, the scattering intensity of the

-

1

d25-DDM is 5-6 times as strong around 0.1 Å as compared to match-out d-DDM and its q-

dependence is, as expected, very significant due to the internal core-shell contrast.

Figure 3F shows the simulated scattering intensity of bR reconstituted in, respectively, a match-out

deuterated d-DDM micelle in 100% D

the overall scattering intensity is lower than for d-DDM. This is a trivial consequence of the different

O concentrations. More importantly, the q-dependence differs significantly in the two systems.

2

O and a d25-DDM micelle at 85% D O. Note that for d25-DDM

2

D

2

-

1

The oscillation around 0.1 Å arising from the core-shell contrast of the d25-DDM is still visible in the

bR-micelle complex, but already from q>0.02 Å the two I(q)-functions start differing (see dashed

-

1

line in figure 3F) as a result of the internal contrast of the d25-DDM micelles. This is already visible in

the Guinier range and show that not even a reliable value for the R of bR can be obtained with the

g

d25-DDM as reconstitution system. Similar effects, though less pronounced are to be expected if

using d17-OG as the reconstitution system.

Exchange of protonated to deuterated detergents

The extent of exchange from hydrogenated to deuterated detergents in samples of membrane

proteins outlined in figure 1, was evaluated by monitoring the background SANS signal at high q as a

function of SEC flowrate, as the hydrogen from any non-exchanged buffer or detergent would

generate an increase in incoherent background scattering. In initial experiments, the background

scattering was found to be very high due to incomplete exchange of the detergent. The Agilent Bio

SEC-3-300 column, used in combination with a high flowrate (0.7 ml/min) was found to be the cause

and a lowering of the flowrate solved to 0.4 ml/min solved the problem. Hence, for this method to

perform as intended, it is of paramount importance to use a sufficiently low flowrate in the SEC to

enable an enhanced H/D exchange of detergent and solvent (see further details in SI 3).

Membrane protein data and analysis.

To evaluate this method, five structurally different membrane proteins were investigated;

bacteriorhodopsin (bR), maltoporin (LamB), photosystem I (PSI), the ionotropic glutamate receptor

2+

A2 (GluA2) and the sarco/endoplasmatic reticulum Ca ATPase (SERCA). The first three proteins, bR,

LamB and PSI are all examples of membrane proteins that have either no or small domains outside

the cell membrane. These three proteins were also chosen as they are relatively stable and at the

same time representative of the wide range of molecular weights found in membrane proteins viz.

~27 kDa (bR), ~47 kDa (LamB) and ~650 kDa (PSI). Two additional membrane proteins, GluA2 (~385

kDa) and SERCA (~110 kDa), were selected as examples of more unstable membrane proteins with

complex structures and large protein domains outside the membrane spanning region that may be

captured in distinct functional states. SANS data on the LamB protein was obtained on the QUOKKA

instrument at ANSTO, GluA2 was measured on KWS1 at FRM-II and data from bR, PSI and SERCA

were obtained on the D22 instrument at ILL. The SANS data and results obtained using the invisible

detergents in 100% D O are presented in figure 4 (See SI 4 for further details).

2

Crystal structures were available for all proteins, and these were used as a basis for modeling the

theoretical scattering patterns to be compared to the experimental data (figure 4). bR was found

agree with a dimer structure, LamB with a combination of homo-trimers and higher oligomers of

these, PSI data with a monomer structure, GluA2 with a homo-tetramer and SERCA with a

combination of two structural states combined with a minor aggregated fraction of SERCA using a

previously developed approach[40] (see SI 4 for more details). Hydration layers were added to all

water-exposed surfaces. Instrumental resolution effects on the SANS data were taken into account

in the analysis[41] and the required values for the Δq(q) for the resolution function calculations for

the different instrumental settings were provided by the beamlines. The resolution effect is most

clearly visible for the SERCA data where an (expected) discontinuity of the fitted model is observed

around q=0.03 1/Å. A constant background and a scale factor were fitted to obtain the best

agreement between the data and model, shown in figure 4. The figure also plots the theoretical pair

distance distribution functions p(r) calculated from the fitted models together with the ones

obtained from the data by indirect Fourier transformation[39]. The values of radius of gyration, R

and maximum internal distances, Dmax, obtained from the p(r) functions, are listed in table 2

g

,

together with molecular masses estimated from the estimated porod volume and the partial specific

molecular volume found by Mylonas et al.[42]. Table 2 compares these values to the theoretical R

max and MW-values based on the available crystal-structures. Note that the experimental estimates

for the Porod volume are not expected to have a precision better than ±20%[30]. Finally, Ab initio

g

,

D

bead modelling was performed for bR, PSI and GluA2 and compared with the known structures (See

Fig 4 and SI 4).

bR with a monomeric molecular weight of ~27 kDa, is a relatively small membrane protein with

seven transmembrane helices. Due to its small size, it scatters weakly and any interfering signal from

the detergent should be visible in the data. As is evident from figure 4, the experimental data

correspond well with a dimer of bR (PDB ID: 1PY6). While bR is traditionally found as a monomer or

trimer, a dimeric form is well documented[43]. A closer look into the structural parameters in table

2

, show that the experimentally obtained R , Dmax and MW-values are generally slightly larger than

g

what would be expected from the crystal structures of the dimer. This larger size is also indicated in

the obtained ab initio structure (figure 4) and can both be indications of slightly more loose or open

dimer-structures in solution than in the crystals, but may also indicate the presence of small

populations of higher order oligomers. These questions could be pursued further in a future work.

However, the ability to discern the oligomeric state show that the method can probe the structure of

relatively small membrane proteins in solution without any significant contribution from the match-

out deuterated detergent. bR has the same size and general fold as many G-protein coupled

receptors (GPCRs)[44]. The data shows that this interesting class of membrane proteins, typically too

small for cryo-EM fall well within the range of this SANS-based method.

The LamB protein is a simple beta barrel porin structure with a monomer weight of ~47 kDa[45],

hence a membrane protein with intermediate molecular weight. The SANS data presented in figure 4

were found to agree with an oligomeric mixture of monomer, trimer and heptameric states of the

underlying LamB homo-trimer (PDB ID: 1MAL, See SI 4 for further details), making an ab initio

g

structure irrelevant. While it is clear from the data and the values for R , Dmax and MW listed in table

2

that the data must arise from, on average, larger particles than the basic LamB homo-trimeric

structure, another base set of structures derived from the homo-trimer could likely also have

provided a good representation of the measured data. A small deviation between the model and

-

1

data is visible at 0.11 Å , this hints towards the possibility of investigating finer structural details of

the LamB oligomerization with this method. However, a larger set of data would be required for this

purpose.

Plant PSI is a large protein complex with a diameter of around 20 nm that comprises multiple, mostly

alpha-helical subunits[46,47]. It is evident from the SANS data in figure 4 that the overall structure of

PSI in solution is highly similar to the one recently found by X-ray crystallography (PDB ID: 4RKU).

-

1

Interestingly, a small discrepancy is found around 0.1 Å and the experimental values for R and Dmax

g

are slightly higher than the comparable value for the crystal structure. The slightly larger size

observed by SANS is also reflected in the plotted ab initio structure (fig 4). The observed differences

likely stem from the fact that ~10% of the amino acid residues are not found in the crystal structure.

Other contributing factors could be that the solution structure, due to increased flexibility, is slightly

different from the one confined in the crystal or that the crystal structure is from pea PSI and the

SANS data was obtained from barley PSI. In either case, it is interesting that this method is able to

demonstrate minor discrepancies between the known model and the obtained SANS data for this

otherwise highly studied membrane protein complex.

GluA2 is a neurotransmitter activated receptor with a structure consisting of a large, extended

homo-tetramer[48]. The scattering data correspond very well with the known structure of GluA2

(

PDB ID: 4U2P), which is also seen from the resemblance between the crystal structure and the ab

inito model (see figure 4) and from the good agreement (within the experimental accuracy) between

the experimental and expected values for the R , Dmax and MW listed in table 2. The values found in

g

table 2 also indicate a good agreement with the expected structure within the expected accuracy.

This demonstrates that the method can readily probe the structure of large and complicated

membrane proteins and, in this case, confirm that the crystal structure is representative of the

solution structure.

SERCA is a calcium transporting ATPase and more difficult to stabilize in solution than the other

proteins in this study. It has a complex fold with three major cytoplasmic domains outside the

membrane spanning region that rearrange with respect to each other during the calcium transport

cycle[49]. Several of the structural states relating to the pumping cycle of SERCA have been revealed

by crystallography via stabilization with co-factors and inhibitors[50]. However, some states and

dynamic transitions are still debated. Here the structure in the absence of calcium and stabilized by

the non-hydrolysable ATP analog AMPPCP has been investigated, representing the so-called E1 and

E2 states. High quality SANS data has been collected as evidenced by the relatively small error bars

and the presence of significant features in the high-q region. The data correspond to monomeric

SERCA together with a minor fraction of oligomeric SERCA (~2% of the molecules, see SI 4 for

details). The data clearly delineate calcium-free SERCA structures, as is also evident from the

enhanced view in figure 4 where the structure with calcium (PDB ID: 1T5S) completely fails to

describe the finer details observed in the experimental data. The structural state probed in this study

is expected to be dynamic and potentially switch between the E2 (PDB ID: 4UU1) and E1 (PDB

ID:4H1W) calcium-free states[50]. Fitting the individual E2 and E1 states and a superposition of the

two structures to the data indeed reveals that while both the calcium-free states represent the

features in the data better than the calcium-bound form, the best description of the data is obtained

by a linear combination of the E2 and E1 states with a distribution of 50% E2 and 50% E1 (see SI 4 for

details). Not only does this confirm what has been previously suggested in the literature but also

provides a result that could not have been obtained using a traditional crystallographic approach.

Additionally, this result demonstrates that the workflow and method presented here is compatible

with probing the finer structure of this challenging and unstable type of membrane proteins. There

was aggregation in the SERCA sample, as clearly seen from the experimental data in figure 4, and

reflected in the R , Dmax and MW listed in table 2. This effect was included in the modelling as a

g

structure factor and using a previously developed approach[40]. However, since the two structures

of interest, E1 and E2, had approximately the same size, and hence the same scattering intensity at

low-q, the aggregation did not affect the assessment of the equilibrium between these states. A

detailed description of the data analysis, the structure factor as well as a more thorough discussion

of this matter can be found in the supplemental information (SI 4).

Discussion

The feasibility of using custom synthesized match-out deuterated detergents for elucidating the

structure of membrane proteins by solution SANS has been demonstrated. Firstly, the synthesis of

partly deuterated versions of DDM and OG were developed and the desired match-out deuteration

confirmed by SANS. Secondly, the detergents were successfully used for stabilizing membrane

proteins while performing SANS experiments. Thirdly, by analysis of the obtained SANS data, it was

demonstrated that this method does indeed perform as well as expected and hence provides a novel

and easy-to-use tool for low-resolution structural studies of membrane proteins. For the past

decade, performing a SAXS experiment to complement high-resolution structural work by

crystallography has become a standard approach when investigating soluble proteins[51]. This is not

least due to the easy analysis made possible by the ATSAS suite[30] and development of stable and

high throughput SAXS beamlines[52]. This approach is now directly transferable to membrane

proteins by using the approach of deuterated detergents and SANS.

The method of using match-out deuterated detergents to obtain solution structures of membrane

proteins fills an important gap in the toolbox for their detailed investigation. While traditional

protein crystallography has undeniably produced fantastic new insights into biological processes

over several decades, obtaining crystals of membrane proteins diffracting to high resolution remains

a bottleneck[53]. Indeed, larger flexible membrane proteins are under-represented in the protein

data bank due to this obstacle. The method presented here allows for probing the structure in detail

early in the process when a robust expression and purification protocol has been established. Recent

advances in both the methodology and hardware have matured Cryo-EM to a technique that has

become a major game changer in structural biology[8]. However, a major drawback of Cryo-EM is

the requirement of proteins with a molecular mass above ~100-150 kDa to obtain sufficiently high

signal-to-noise ratios to allow for the alignment and averaging of the individual frames required for

obtaining the desired high resolution data[8]. This means that many important membrane proteins

are too small for Cryo-EM. NMR is also a popular method for determining the high resolution

structure of membrane proteins[54], but due to the overcrowding of the spectra, only relatively

small proteins (~20-30 kDa) may be solved. Hence, the combined SANS and invisible detergent

method described in this paper provides an easily applicable alternative that allows for analyzing

protein structures with a resolution down to about 10 Å, and that importantly contributes to

bridging the gap between existing methods.

Several other strategies have been proposed in the literature which utilize some of the aspects of

the method presented here. The use of small-angle X-ray scattering (SAXS) for elucidating the

structure of membrane proteins in detergent micelles has been thoroughly tested[55–58] but thus

far mainly revealed information on the detergent rim around the membrane proteins. Furthermore,

using so-called nanodiscs coupled with small-angle scattering has also been shown to be possible but

the sample preparation remains challenging[11,59]. This is contrasted with the approach presented

here, where the sample preparation is uncomplicated and the signal almost exclusively comes from

the membrane protein, significantly simplifying the data analysis and increasing the information that

can be extracted about the structure of the investigated membrane protein. Importantly, this

method allows for probing the structure of un-crystalizable dynamic structural states of proteins.

Recent technical developments in SEC-SANS sample environments will furthermore allow for

performing the whole workflow outlined in figure 1 in situ at the neutron instrument, together with

removing any oligomeric species caused by unstable proteins[60].

Five different membrane proteins and two detergents were investigated in this study. The data

demonstrate the general applicability of the approach and provide promising perspectives for future

use. The basic idea of match-out deuteration may easily be generalized to other detergent types to

accommodate special needs for particular membrane proteins or in combination with selective

deuteration of different subunits.

Acknowledgements

The authors acknowledge Dr. Robert Knott at ANSTO and Dr. Adam Round at ESRF for their

assistance in obtaining supporting SAXS data. We thank ANSTO, ILL and FRM II for awarding us

beamtime for this project at, respectively, the QUOKKA, D22 and KWS-1 instruments. R.V., C.K.,

T.S.T., J.J.D and J.S.K. thank Eric Gouaux for providing the GluA2cryst construct and protein

purification protocol. Dr. Frank Gabel and Dr. Christine Ebel is thanked for kindly providing data on

the tail-deuterated detergents previously published in [28]. PhD student Nicolai Tidemand Johansen

is thanked for helping with the SANS measurements of GluA2. We thank the Danish Agency for

Science, Technology and Innovation funding agency for making this study possible through a Sapere

Aude grant to L.A.. R.V., C.K., and J.S.K. acknowledge the support from the Lundbeck Foundation,

The Danish Council for Independent Research – Medical Sciences and GluTarget. The authors also

acknowledge financial support from the Center for Synthetic Biology (bioSYNergy) funded by the

UCPH Excellence Programme for Interdisciplinary Research and to the Lundbeck foundation

“BRAINSTRUC” center.

Author Contributions

S.R.M., T.D. and L.A. conceived the project. T.D. developed methods and synthesized the deuterated

detergents. S.R.M., A.J.Z.N., P.E.J., M.B., C.O., P.N., R.V., T.S.T., J.J.D., C.K., J.S.K. produced and

provided proteins. S.R.M., M.C.P., S.A.R.K., P.H. A.H.L. performed the experiments. A.H.L., N.S.G.,

G.V.J., M.C.P., S.A.R.K performed the data analysis. H.F., E.G. and A.M. were SANS instrument

responsible and participated in the SANS measurements. S.R.M., T.D. and L.A. co-wrote the paper.

Competing Financial Interests

The authors declare no competing financial interests.

Methods

General Materials

Hydrogenated Octyl-β-D-glucopyranoside (OG) was purchased from Applichem while hydrogenated

n-dodecyl-β-D-maltopyranoside (DDM) was purchased from Sigma Aldrich. Match-out deuterated

detergents were custom synthesized as described briefly below and in detail in the supplemental

information. The membrane proteins used in the present study were expressed and purified

according to standard protocols as described the supplemental information. All pH values have been

measured using a standard glass electrode and all reported values are from the direct

measurement[61].

Densitometry

Densitometry was performed using a DMA 5000 density meter (Anton Paar). Octyl-β-D-

glucopyranoside (OG) (Sigma-Aldrich) was solubilized in 20 mM Tris/HCl, pH 7.5 and 100 mM NaCl in

concentrations of 25 and 100 mM. n-dodecyl-β-D-maltopyranoside (DDM) (Sigma-Aldrich) was

solubilized in identical buffer at concentrations of 15 and 60 mM. All samples were prepared in

o

triplicates and measured at 4 C intervals between 4 C and 28 C. See further details in the

supplemental information.

General detergent synthesis

Chemicals and reagents of the highest grade were purchased from Sigma-Aldrich and Carbosynth

Limited (Berkshire, UK) and were used without further purification. Solvents were purchased from

Sigma-Aldrich and Merck and were purified by established methods[62]. NMR solvents were

purchased from Cambridge Isotope Laboratories Inc. and Sigma-Aldrich and were used without

further purification. D

was performed on Fluka Analytical silica gel aluminium sheets (25 F254). Davisil® silica gel (LC60Å 40-

3 micron) was used for bench-top column chromatography.

2

O (99.8 %) was supplied by Sigma-Aldrich. Thin-layer chromatography (TLC)

6

Deuteration of octanoic acid and dodecanoic acid was performed using hydrothermal H/D exchange

reactions in D O at 220°C by mixing the appropriate amount of fatty acid with NaOD and Pt/C (10 %

w/w) in a Mini Benchtop 4560 Parr Reactor (600 mL vessel capacity, 3000 psi maximum pressure,

50 °C maximum temperature). This was followed by filtering the catalyst, acidifying the solution

2

3

and then extracting the aqueous phase with ethyl acetate. Thin layer chromatography was used

referenced with the protonated compound) to estimate the purity and to develop separation

(

1

13

2

protocols. H NMR (400 MHz), C NMR (100.6 MHz) and H NMR (61.4 MHz) spectra were recorded

on a Bruker 400 MHz spectrometer at 298 K. Chemical shifts, in ppm, were referenced to the

residual signal of the corresponding NMR solvent. Deuterium NMR was performed using the probe’s

lock channel for direct observation. Electrospray ionization mass spectra (ESI-MS) were recorded on

a 4000 QTrap AB Sciex spectrometer. The overall percentage deuteration of the molecules was

calculated by MS using the isotope distribution analysis of the different isotopologues. This was

13

calculated taking into consideration the C natural abundance, whose contribution was subtracted

from the peak area of each M+1 isotopologue to allow for accurate estimation of the percentage

deuteration of each isotopologue.

Transfer of membrane proteins into deuterated detergents

The transfer was performed using an Agilent Bio SEC-3-300 column with a flowrate of 0.4 ml/min.

The flow through from the column was collected, measured and used for background subtraction.

Buffers used for PSI, GluA2 and bR contained 0.5 mM match-out deuterated DDM, 20 mM Tris/DCl

pH 7.5 and 100 mM NaCl in D O. For LamB, 40 mM match-out deuterated OG was used instead of

2

DDM. For SERCA, the buffer used consisted of 20 mM MOPS pH 6.8, 100 mM KCl and 0.5 mM DDM.

SANS measurements

SANS measurements of the deuterated OG were performed at KWS 1, Forschungs Neutronenquelle

Heinz Maier-Leibnitz, Munich (FRM II). A neutron wavelength of 4.5 Å (±10% FWHM) was used at

-

1

-1

two sample-to-detector distances covering q-ranges of 0.012–0.16 Å and 0.0057–0.077 Å . Pre-

calibrated plexiglass was used as a standard for absolute calibration of the scattered intensity I(q) in

units of 1/cm, where q = (4π/λ)sin(θ) and 2θ is the scattering angle and λ is the wavelength. GluA2

was measured at the same instrument using three sample-to-detector distances: 1.5 m and 4.0 m

with 4 m collimation and 8.0 m with 8 m collimation were used.

SANS measurements of the LamB protein in deuterated OG and empty deuterated DDM micelles

were performed at QUOKKA[63], Australian Nuclear Science and Technology Organization (ANSTO),

Sydney. A neutron wavelength of 5.0 Å (±10% FWHM) was used at two sample-to-detector

-

1

-1

distances, covering q-ranges of 0.017-0.51 Å and 0.0076-0.098 Å . Data were absolutely calibrated

by an attenuated direct beam transmission measurement of the scattered intensity.

SANS measurements of the bR, SERCA and PSI in deuterated DDM were performed at D22, Institut

Laue-Langevin (ILL), Grenoble. A neutron wavelength of 6.0 Å (±10% FWHM) was used at two

-

1

-1

sample-to-detector distances covering q-ranges of 0.022–0.48 Å and 0.0087–0.12 Å . H O was used

2

as a standard for absolute calibration of the scattered intensity.

SANS data analysis

Pair distance distribution functions, p(r) and the associated values for the radius of gyration, R , and

g

maximum particle diameter, Dmax, were obtained from the scattering data using a Bayesian indirect

Fourier transform[39]. Porod volume based values for the protein molar mass was estimated by the

GNOM program of the ATSAS package [64]. Ab initio models of the proteins were obtained using the

DAMMIF program of the ATSAS package[64]. Theoretical scattering patterns for the relevant protein

structures were calculated using the program WillItFit[31]. Hydrogen and deuterium was added to

the structures by the Phenix program ReadySet![65], exchanging all labile hydrogens for deuterium.

A surface hydration layer was added to the hydrophilic areas of the structures using an in-house

routine which identifies surface residues and places beads at a distance of 5 Å from the C of the

surface residues. This routine allows for not adding a water layer at the hydrophobic portion of the

protein surface as identified by the Orientations of proteins in Membranes’ database (OPM)[66].

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OG

head group

DDM

head group

tail group

Chemical composition of the detergent

component

C

H O

2 11 6

C

H

8 17

C

H O

12 21 11

C

H

12 25

Exchangeable hydrogens

4

0

7

0

Theoretical level of deuteration needed for

C

6

D

7.6

H

3.4

O

6

C

8

D

15.9

H

1.1

C

12

D

15.2

H

5.8

O

11

C

12

D

22.4

H

2.6

match-out at 100% D

Experimentally obtained level of deuteration

in 100% D

2

O

C

6

D

7.64

H

3.36

O

6

C

8

D

15.98

H

1.12

C

12

D

14.98

H

6.02

O

11

C

12

D

22.25

H

2.75

2

O

Table 1: Deuteration levels needed and obtained for n-octyl β-D-glucopyranoside (OG) and n-dodecyl-β-D-

maltopyranoside (DDM).

R

g

exp. [Å]

R

g

calc. [Å]

17.4

33.4

51.3

56.2

D

max exp. [Å]

Dmax calc. [Å] MW exp. [kDa] MW calc. [kDa]

bR

LamB

PSI

GluA2

21.6 ± 0.04

57.9 ± 0.2

55.2 ± 0.2

60.0 ± 0.4

56.4 ± 0.1

60.2 ± 0.9

201 ± 1

53

87

44.6

210

473

278

460

27

47

199 ± 5

166 ± 1

159

173

123

650

385

110

SERCA E1 + E2

38.4

204.5 ± 0.6

Table 2: Structural parameters deduced from the experimental data and the calculated theoretical values from the fitted

models as obtained through Indirect Fourier Transform. R : Radius of gyration. Dmax: Maximum distance present in the

g

scattering particle. MW: Molecular weight. Experimental values obtained from SANS data, theoretical values calculated

from the available crystal structures.

Figure 1: Process outline of the experiment. Initially, the membrane protein is in H O based buffer and hydrogenated

2

detergents. This sample is then applied to a size exclusion column equilibrated in D O based buffer and the match-out

deuterated detergents, allowing the hydrogenated buffer and detergent to be exchanged to their deuterated

counterparts. The obtained sample is used directly for SANS measurements to obtain a single contrast dataset yielding

only the scattering from the membrane protein. Data from the experiment can then be analyzed by generally available

software developed for determining the low resolution structure of proteins in solution.

Figure 2. Reaction scheme for the synthesis of specifically deuterated levels of OG (the corresponding scheme for DDM

can be found in Figure S1). Step A) The fatty acid is deuterated by Pt/C catalyzed H/D exchange reactions at 220°C to

produce the correctly deuterated version and then it is reduced to the corresponding alcohol (1) using the specified D:H

ratio. Step B) The deuterated alcohol (1) is coupled to the acetylated bromo-sugar head-group, producing the tail

deuterated version of the detergent, which is then deacetylated to produce (2). Step C) The sugar head group is

deuterated via Raney Nickel catalysis to produce the final partially deuterated detergent (3) with different levels of

deuteration in the head and the tail groups.

Figure 3: (A) Background subtracted SANS data from match-out deuterated OG in increasing percentage of D

2

O in the

buffer. (B) SANS data from match-out deuterated DDM in increasing percentage of D O in the buffer. (C) Experimental

2

I(0) values plotted (points) and the corresponding quadratic power law fits (lines). (D) Comparison of I(q) from match-

out d-OG (yellow) and d17-OG (light and dark grey), both measured at their match-point. Light-grey curve plots d17-OG

with the usual background subtraction. In the dark grey curve the d17-OG background has been empirically increased by

a factor of 1.039. (E) Comparison of match-out d-DDM (turquoise) and d25-DDM (grey) measured at its match-point.

Data from d17-OG and d25-DDM are reproduced from [28]. (A)-(E) Errorbars are the standard deviation (SD), obtained

from counting statistics and standard error propagation. (F) Simulation of a Bacteriorhodopsin monomer measured by

SANS in respectively match-out d-DDM (black) and in d25-DDM (grey and dashed) micelles.

Figure 4: Experimental SANS data (colored points) and the resulting model fits (black full and dashed lines) for the five

investigated membrane proteins. Errorbars are the standard deviation (SD), obtained from counting statistics and

standard error propagation. Note that instrumental resolution effects are included in the models, which then exhibit

small discontinuities at medium q-values. Inserts: The corresponding pair-distance distribution functions as determined

by Indirect Fourier transform[39] with identical color scheme. Figures are the known structures from crystallography

together with the ab initio bead models where appropriate. Note that SERCA (data, model and p(r)) contain a fraction of

aggregated protein.

Article Doi

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

DOI: 10.1111/febs.14345

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Article abstract of DOI:10.1111/febs.14345

Full text of DOI:10.1111/febs.14345

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