Department of Chemistry and Biochemistry, University of Colorado at Boulder
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Joce, C., Wiener, A., Yin, H. Transmembrane Domain Oligomerization Propensity determined by ToxR Assay. J. Vis. Exp. (51), e2721, doi:10.3791/2721 (2011).
The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).
In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).
1. Cloning Considerations
The TMD sequence should be 12-24 residues (shorter sequences will presumably be elongated by vector encoded hydrophobic residues). In order to investigate the interface, four variants of the TMD design should be created where sequential residue insertions and concomitant residue deletions result in rotation of the TMD relative to ToxR.15,16 Finally, the arabinose concentration should be varied between 0.001 and 0.01% (w/v) to identify the concentration where maximum differences in β-galactosidase signals between different TMD sequences are observed; testing different expression levels is recommended to identify conditions under which different affinities can be distinguished best. In addition to arabinose and antibiotics, 0.4 mM IPTG can be used to enhance differences of affinities between different TMDs. The ToxR measurement should be performed at least in quadruplicate. The whole procedure should be repeated at least three times with different plasmid transformations.
2. Growth of Bacterial Cultures
3. Measurement of β-galactosidase Activity
4. Control for Protein Expression
5. Control for Proper Membrane Insertion
A cell line deficient in maltose binding protein is used to assess proper membrane insertion of the chimeric TMD construct. When grown on minimal media with maltose as the sole carbon source, only cells expressing a membrane-integral expression product with maltose binding protein correctly located to the periplasm are able to grow.
6. Representative Results:
An example of the use of the ToxR transcriptional reporter assay to analyze the oligomerization propensity of transmembrane domains in shown in Figure 4. Previously we have investigated the oligomerization of transmembrane domains from the multispanning membrane-integral protein latent membrane protein-1 (LMP-1) by various techniques, including ToxR.14 Transmembrane domain five (TM5) was shown to exhibit a strong propensity to oligomerize; this is demonstrated by high Miller Units, comparable to the positive control, GpA, a well-established dimerizing sequence. A deleterious mutation in TM5, D150A, reduces the ability of the sequence to oligomerize. LMP-1 TM1 does not significantly oligomerize and exhibits a very low Miller Unit signal, just above the signal for blank, non-transformed FHK12 cells.
Figure 1. Cartoon depicting the ToxR reporter assay. Transmembrane domain (TMD) driven oligomerization results in dimerization of ToxR and activation of LacZ transcription. The gene product of LacZ, β-galactosidase can be quantified as a measure of the propensity of a TMD to oligomerize.
Figure 2. The hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP).
Figure 3. Plasmid map of pToxR7.
Figure 4. Representative ToxR transcriptional reporter assay analysing the oligomerization propensity of latent membrane protein-1 transmembrane domains. Transmembrane domain 5 (TM5) oligomerizes strongly, whilst transmembrane domain 1 (TM1) exhibits only a weak interaction. Mutation D150A in TM5 significantly reduces its ability to oligomerize. GpA is included as a positive control sequence for strong dimerization. Blank represents untransformed FHK12 cells.
Figure 5. Western blot for protein expression.
Figure 6. PD28 complementation assay to control for correct membrane insertion to the periplasm. Negative control represents a construct deficient in maltose binding protein.
The ToxR transcriptional reporter assay is a facile way to identify transmembrane sequences with the potential to oligomerize. Since the interactions are occurring within the bacterial inner membrane, this assay circumvents the issues associated with the validity of studying systems in membrane-mimetic environments. Given that cloning of multiple TMDs into a single plasmid can readily be done in parallel and the entire assay can be carried out in 96-well plate format, this assay can be used for high throughput analysis of large numbers of protein sequences.17 Once an interaction has been detected, the key functional residues can be interrogated by mutational analysis, allowing mapping of the structural features involved. In many cases, crystallographic analysis of transmembrane proteins is problematic, requiring alternative tools such as the ToxR assay to establish the molecular basis of function.
No conflicts of interest declared.
We thank the National Institutes of Health (1R21CA138373 and Stand Up to Cancer (SU2C) for financial supports of this work. H.Y. is grateful for the 2009 Elion Award from the American Association of Cancer Research, a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research (SKF-08-101), and the National Science Foundation Faculty Early Career Award (NSF0954819).
|BamHI restriction enzyme||Invitrogen||15201023||Invitrogen enzymes were found to be more efficient than alternative suppliers|
|NheI restriction enzyme||Invitrogen||15444011||Invitrogen enzymes were found to be more efficient than alternative suppliers|
|15 mL culture tubes||Fisher Scientific||14-956-1J|
|SOC media||TEKnova, Inc.||S0225||Made up to the appropriate volume and sterilized by autoclaving.|
|LB media||Sigma-Aldrich||L7275||Made up to the appropriate volume and sterilized by autoclaving.|
|Chloramphenicol||Sigma-Aldrich||CO378||Stock solution of 30 mg/ mL in ethanol stored in freezer|
|Arabinose||Fluka||10839||Stock solution of 2.5% (w/v) in water stored in freezer|
|KCl||Mallinckrodt Baker Inc.||6858-06|
|Sodium dodecylsulfate (SDS)||Sigma-Aldrich||L6026|
|2-Nitrophenyl Î²-D-galactopyranoside (ONPG)||Sigma-Aldrich||73660|
|Z-buffer||16.1 g Na2HPO4
Make up to 1 l, pH 7.0
|Z-buffer/chloroform||200 mL Î²-mercaptoethanol, 2 mL chloroform, make up to 20 mL with Z-buffer. Vortex for 1 min, centrifuge for 1 min at 800 rpm. Make fresh for each plate.|
|Z-buffer/SDS||160 mg SDS dissolved in 10 mL Z-buffer|
|Z-buffer/ONPG||40 mg ONPG in 10 mL Z-buffer. Make fresh for each plate|
|Anti-MBP monoclonal antibody (HRP conjugated)||New England Biolabs||E8038S|
|Minimal media with maltose||1 x M9 salts, 0.4% maltose, 1 mg/ mL thiamin, 2 mM MgSO4|
|96-well flat bottom plate||Sarstedt Ltd||83.1835.300|
|Plate-reader||Beckman Coulter Inc.||DTX880 Multimode Detector|
|Water bath||VWR international||89032-204|
|Shaking incubator||Forma Scientific|