A method for detergent-free isolation of membrane proteins in their local lipid environment

TLDR: This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems and provides a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Abstract: Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Figures

Figure 1.

Figure 3. Forming SMALP from membrane preparations using SMA (a) Membranes are suspended in buffer at a concentration of 20-40 mg.ml-1 prior to the addition of SMA. (b) The membrane solution with SMA at first appears cloudy (c) After 2 hours the suspension has become clear. !

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University of Birmingham
A method for detergent-free isolation of membrane
protein with its local lipid environment
Lee, Sarah C; Knowles, Tim J; Postis, Vincent L G; Jamshad, Mohammed; Parslow,
Rosemary A; Lin, Yu-pin; Goldman, Adrian; Sridhar, Pooja; Overduin, Michael; Muench,
Stephen P; Dafforn, Timothy R
DOI:
10.1038/nprot.2016.070
License:
None: All rights reserved
Document Version
Peer reviewed version
Citation for published version (Harvard):
Lee, SC, Knowles, TJ, Postis, VLG, Jamshad, M, Parslow, RA, Lin, Y, Goldman, A, Sridhar, P, Overduin, M,
Muench, SP & Dafforn, TR 2016, 'A method for detergent-free isolation of membrane protein with its local lipid
environment', Nature protocols, vol. 11, no. 7, pp. 1149–1162. https://doi.org/10.1038/nprot.2016.070
Link to publication on Research at Birmingham portal
Publisher Rights Statement:
Checked for eligibility: 01/06/2016
Lee, S.C., Knowles, T.J., Postis, V.L., Jamshad, M., Parslow, R.A., Lin, Y.P., Goldman, A., Sridhar, P., Overduin, M., Muench, S.P. and
Dafforn, T.R., 2016. A method for detergent-free isolation of membrane proteins in their local lipid environment. Nature protocols, 11(7),
p.1149. https://doi.org/10.1038/nprot.2016.070
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A Method for Detergent-free isolation of Membrane Protein
with its Local Lipid Environment
!
Sarah C. Lee
ag
, Tim J. Knowles
bg
,
Vincent L.G. Postis
cdg
,
Mohammed Jamshad
a
, Rosemary A. Parslow
a
,
Yu-pin Lin
a
, Adrian Goldman
ce
, Pooja Sridhar
b
, Michael Overduin
ae
, Stephen P. Muench
c
, Timothy R.
Dafforn
a
.
a
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
b
School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
c
School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, LS2 9JT, U.K.
d
Biomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett University, LS1
3HE, U.K.
e
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta
T6G 2H7, Canada EC1R 0BE
f
Department of Biosciences, Division of Biochemistry, University of Helsinki, FIN-00014 Helsinki, Fin-
land
!
g
These authors contributed equally.
!
To whom correspondence should be addressed: Professor Timothy Dafforn, School of Biosciences, Uni-
versity of Birmingham, Birmingham, Edgbaston, B15 2TT U.K. Email: T.R.Dafforn@bham.ac.uk. Tel:
(+44) (0)121 414 3506.
KEYWORDS: Styrene Maleic Acid, SMA, SMALPs, Membrane proteins, Detergent free, Protein pu-
rification, biophysical studies
!
!
!
!
!
!
!
!
!
!
!

ABSTRACT
Despite the great importance of membrane proteins, structural and functional studies present ma-
jor challenges. A significant hurdle is the extraction of the functional protein from its natural
lipid membrane. Traditionally achieved with detergents, purification procedures can be costly
and time consuming. A critical flaw with detergent approaches is the removal of the protein from
the native lipid environment required to maintain functionally stable protein. This protocol de-
scribes the preparation of Styrene Maleic Anhydride Co-polymer (SMA) to extract membrane
proteins from prokaryotic and eukaryotic expression systems. Successful isolation into SMA
lipid particles (SMALPs) allows membrane proteins to remain with native lipid, surrounded by
SMA. We detail procedures for obtaining 25g of SMA (4 days), explain the preparation of both
SMALPs with (2 days) and without (1-2 hours) protein, SMALP protein identification and esti-
mation (4 hours), and detail biophysical methods to undertake initial structural studies to charac-
terise SMALPs (~2 days). Together these methods provide a practical tool-kit for those wanting
to use SMALPs to study membrane proteins.
!
!

!
!
!
!
INTRODUCTION
The lack of progress in the study of membrane protein structure and function remains a signifi-
cant frustration for academics and commercial organisations alike. Membrane proteins them-
selves represent some of the most important molecules in life sitting as they do either between
the cell and the outside world or between cellular compartments. As such they underpin a wide
range of fundamental cellular functions from cellular signalling, nutrient uptake, and secretion to
communication, motility and adhesion. The combination of these activities with the exposure of
many membranes to the extracellular milieu, make membrane proteins important targets for ther-
apeutic development. For instance, more than 40% of pharmaceutical agents interact with a sin-
gle class of membrane proteins, the G-protein coupled receptors
1
. However despite the acknowl-
edged importance of these molecules, and the efforts put into their study, research on them re-
mains a significant challenge.
This challenge centres on the need to disrupt the phospholipid bilayer in order to facilitate the
separation of the target membrane protein/protein complex from all others. The disruption of the
membrane is not a challenge per se as many agents are available (generally surface active agents
or detergents) that can fragment membranes. However, it has long been known that while the
membrane needs to be fragmented, the lipophilic character of the membrane protein means that
complete removal of the membrane from around the protein generally renders it unfolded and
inactive. This conundrum has challenged the biochemical world for a significant time, with the
choice of what membrane protein to study often not being made on the basis of importance but
more on which protein can be extracted from the membrane in the active, folded form. This re-
striction in a biochemist’s freedom to operate is even starker when it is recognised, that although
more than 30% of all transcribed proteins are membrane proteins only 2% of high resolution
structures are of membrane proteins
2
.
!
!

Existing approaches to membrane protein solubilisation
Since the discovery of membrane proteins as a distinct sub-class the majority of approaches to
their isolation have relied on the use of surface active agents (more commonly called
detergents)
3,4,5,6
. Simply, detergents provide an alternative solubilisation environment for mem-
brane proteins that, unlike a phospholipid membrane, is not a continuum. The outcome of any
detergent based method is a solution of micellar particles that contain individual membrane pro-
teins. These protein-micelle complexes can then be separated by virtue of their physico-chemical
properties. At first view, this seems like a perfect solution and indeed it has been used to produce
pure, active samples on essentially all membrane proteins previously studied or currently inves-
tigated. However there are a number of fundamental issues that exist with this approach that
make it less than perfect. The use of detergents in many ways neglects to consider the physico-
chemical complexity of the membrane environment and its importance in maintaining protein
structure and activity
7,8
. Even a simple phospholipid membrane made of a single lipid contains a
number of distinct environments that run within the membrane leaflet
9
. Perhaps the most obvious
of these are the hydrophilic outer surface and the hydrophobic interior. This becomes significant-
ly more complex in membranes containing mixed lipids where the interfaces between different
lipid types provide yet more new and distinct environments
10
. Membrane proteins have evolved
to exist in this complex environment and as such it has become increasingly clear that the struc-
ture of the membranes are adapted to provide optimum protein folding and hence activity in a
very specific lipidic context.
Given this complexity the use of detergent solubilisation with a single detergent will never effec-
tively replicate the native lipid environment and hence will always be a sub-optimal solution.
This issue has been encountered countless times with detergent solubilisation experiments failing
to produce active membrane proteins. Even a thermostable protein, such as Thermotoga mariti-
ma integral membrane pyrophosphatase, is stable and active in only a few detergents
11
but most
of these attempts are never published by virtue of the negative nature of the results. The sad con-
sequence is that multiple groups waste valuable time and resources attempting the same experi-
ments without knowing that the experiments have already been proven to be futile.

Frequently asked questions

Q1. What are the contributions in "University of birmingham a method for detergent-free isolation of membrane protein with its local lipid environment" ?

The authors detail procedures for obtaining 25g of SMA ( 4 days ), explain the preparation of both SMALPs with ( 2 days ) and without ( 1-2 hours ) protein, SMALP protein identification and estimation ( 4 hours ), and detail biophysical methods to undertake initial structural studies to characterise SMALPs ( ~2 days ). Together these methods provide a practical tool-kit for those wanting to use SMALPs to study membrane proteins. The lack of progress in the study of membrane protein structure and function remains a significant frustration for academics and commercial organisations alike. This conundrum has challenged the biochemical world for a significant time, with the choice of what membrane protein to study often not being made on the basis of importance but more on which protein can be extracted from the membrane in the active, folded form. The outcome of any detergent based method is a solution of micellar particles that contain individual membrane proteins. At first view, this seems like a perfect solution and indeed it has been used to produce pure, active samples on essentially all membrane proteins previously studied or currently investigated. This becomes significantly more complex in membranes containing mixed lipids where the interfaces between different lipid types provide yet more new and distinct environments10. Membrane proteins have evolved to exist in this complex environment and as such it has become increasingly clear that the structure of the membranes are adapted to provide optimum protein folding and hence activity in a very specific lipidic context. The lack of a definitive indication that the project is fruitless also wastes time and resources. More recently scientists have acknowledged the failings of detergents and have begun to develop other moieties aimed at stabilising membrane proteins. Overall detergent solubilisation, has been the method of choice for the past forty years its significant limitations have placed constraints on the study of membrane proteins. Early pioneers in this approach were the groups of Sligar and co-workers who showed that amphipathic peptides could be used to stabilise nanoscale disc-like structures that contained a lipid bilayer17. However, neither provides the perfect solution, as both required that the protein was pre-solubilised in detergent before insertion into the new lipid containing nanoparticle. In 2009, the authors published data that showed that a simple organic polymer ( Styrene Maleic Acid Co-polymer, SMA, ( Fig. 1a ) could be used to directly extract proteins from membranes into self-contained styrene maleic acid lipid particles ( SMALPs ) 21,22. This work built on earlier work by Tighe and colleagues on the conformational transitions of SMA and its resultant physical properties23,24. The authors have also shown that the encapsulated bilayer retains many of the physical properties of the parent membrane including the lipid mixture26, structural organisation and phase behaviour25. Since the publication of this work the authors have worked with a number of collaborators to examine whether the method is widely applicable and if the resulting preparations are appropriate for analysis using a range of biophysical and biochemical methods. As part of these studies the authors have also shown that SMALP encapsulated proteins are amenable to study using a range of techniques including Circular Dichroism ( CD ) 34, Analytical Ultracentrifugation ( AUC ) 34, Differential Scanning Calorimetry ( DSC ) 25, Negative stain27 and cryo Transmission Electron Microscopy ( TEM ) 28, and small angle Neutron ( SANS ) scattering25 demonstrating the general utility of the method. Limitations of the SMALP method The Styrene Maleic Acid Lipid Particle ( SMALP ) method detailed in this protocol solves a number of issues that have historically afflicted detergent based systems. The disc shaped nanoparticle that forms the basis of the method has a nominal maximal diameter that is close to 15 nm corresponding to a molecular mass of less than approximately 400 kDa25. In their own studies the authors have solubilised more than 30 membrane proteins and have shown that proteins that contain up to 36 transmembrane helical elements can be solubilised. Here the authors describe a comprehensive set of protocols required to prepare the relevant reagents, use these reagents to purify membrane proteins in SMALPs and carry out initial biophysical characterisation of the resulting preparation. The authors also describe how to prepare the SMA polymer and SMALPs without encapsulated membrane proteins. The authors demonstrate how this SMALP protocol has been used to prepare a variety of active proteins from various sources including bacteria, insect cells, mammalian cells and the yeast. The precipitate is washed three times with water followed by separation using centrifugation. In this section the authors discuss how to produce a protein free SMALP. Here, the authors give a protocol to prepare protein-free SMALPs using E. coli polar lipid extract, which provides a relevant control for experiments using SMALPs containing protein from E. coli membrane. In the example, the authors show how a SMALP containing a protein with a histidine affinity tag is purified by Nickel affinity chromatography. The authors have success either with adding powdered polymer directly ( described here ) to membrane solutions, or by adding a 5 % solution to an equal quantity of resuspended membranes. It is outside the scope of the protocol to describe all the downstream characterisation methods that are employed for the study of membrane proteins. However the authors have found significant utility in performing three analyses with all the proteins that they have produced. Circular Dichroism ( CD ) spectroscopy provides invaluable information on the secondary structure of the protein in the SMALP allowing a rapid confirmation that the protein is folded within the particle. A quick method of studying the quality of the SMALP protein sample is provided by negative stain microscopy as exemplified in 27. Subsequent data collection and processing can provide further structural insight to a modest resolution but are outside the scope of this report. SMALP protein can be treated like a globular protein and studied in many downstream applications including characterisation using AUC and CD analysis. Analyse data using the program SEDFIT38 using the c ( S ) and c ( M ) routines to provide estimations sedimentation coefficient and mass of the particle. The following steps must be carried out in the fume hood. The concentration of lipid in the extract should be provided by the supplier. 56. Remove the cuvette and clean with 3 washes of MilliQ water followed by ethanol and dry in a flow of dry nitrogen. Detailed protocols from grid preparation, data collection and processing are beyond the scope of this article however the authors would like to make the reader aware of the following research papers which provide more in depth protocols and advice ; Negative stain grid preparation and initial data collection 48, cryo-EM sample preparation and data collection49,50 data processing47,51,52. Here, the authors detail the purification of membrane proteins over-expressed in E. coli as an example, but the same protocol can be used in other systems such as, insect cells, mammalian cells and yeast. The authors therefore suggest that an initial small scale binding trial is carried out to determine which is optimal for the protein being purified. In some cases the authors observe very tight binding to resins, meaning that a more conventional column format binding step can be used. The authors have also found that the relatively high negative charge on the SMALP leads to significant nonspecific binding to the resin. At this stage of the SMALP experiment, it is not necessary to add any further SMA. It is also further possible to continue with activity assays and structural studies as you would with a membrane protein that has been purified with a detergent method. The suggested volume is 135 ml. Balance bottles by further addition of water if necessary. Balance bottles by further addition of water if necessary. 13. Repeat steps 10 to 12 two further times. 

Q2. How many transmembrane helical elements can be solubilised?

In their own studies the authors have solubilised more than 30 membrane proteins and have shown that proteins that contain up to 36 transmembrane helical elements can be solubilised. 

Q3. What are the other surface active agents being trialed?

A range of other surface active agents including fluorinated detergents 14 are being trialed, longer polymeric materials (termed amphipols) are also showing some success. 

Q4. What is the role of the G-protein coupled receptors in the study of membrane proteins?

For instance, more than 40% of pharmaceutical agents interact with a single class of membrane proteins, the G-protein coupled receptors1. 

Q5. What are the physical properties of the encapsulated bilayer?

The authors have also shown that the encapsulated bilayer retains many of the physical properties of the parent membrane including the lipid mixture26, structural organisation and phase behaviour25. 

Q6. What is the recent development of the lipid moieties?

More recently scientists have acknowledged the failings of detergents and have begun to develop other moieties aimed at stabilising membrane proteins. 

Q7. What does this mean for comparative experiments?

This means that comparative experiments between samples often suffer from uncertainty in terms of the specific activity of the preparation. 

Q8. How many attempts are published by virtue of the negative nature of the results?

Even a thermostable protein, such as Thermotoga maritima integral membrane pyrophosphatase, is stable and active in only a few detergents11 but most of these attempts are never published by virtue of the negative nature of the results. 

Q9. How do you determine the sedimentation coefficients and molecular masses?

48. Analyse the data with the continuous c(s) analysis method to determine sedimentation coefficients and molecular masses using the SEDFIT software using the method of Schuck38.