JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

 JoVE Biology

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

1, 1, 1

1Department of Chemistry and Biochemistry, University of Colorado at Boulder

Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.226.235.222, User IP: 54.226.235.222, User IP Hex: 920841182

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    An efficient procedure to assess the oligomerization propensity of single-pass transmembrane domains (TMDs) is described. Chimeric proteins consisting of the TMD fused to ToxR are expressed in an E. coli reporter strain. TMD-induced oligomerization causes dimerization of ToxR, activation of transcription and production of the reporter protein, -galactosidase.

    Date Published: 5/26/2011, Issue 51; doi: 10.3791/2721

    Cite this Article

    Joce, C., Wiener, A., Yin, H. Transmembrane Domain Oligomerization Propensity determined by ToxR Assay. J. Vis. Exp. (51), e2721, doi:10.3791/2721 (2011).

    Abstract

    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).

    Protocol

    1. Cloning Considerations

    1. Commercially prepared oligonucleotides representing the TMD of interest flanked by NheI and BamHI restriction sites and 5'-phosphorylated can be ligated into pTox7 (modified in our laboratory by insertion of one base pair directly after the BamHI restriction site14) (Figure 3) digested sequentially with BamHI and NheI. An example oligonucleotide is shown below:
      5'ctagcTMDSEQUENCEg3'
      3' gTMDSEQUENCEcctag5'

    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

    1. Gently thaw FHK12 competent cells (200 μl) on ice and transfer into a 15 ml culture tube. Add plasmid DNA (200 ng) and incubate the cells on ice for 30 min.
    2. Heat-shock cells by incubation for 90 s at 42 °C, followed by incubation on ice for 2 min.
    3. Add SOC media (800 μl) and incubate the samples at 37 °C with shaking (300 rpm) for one h.
    4. Inoculate 5 ml LB media with chloramphenicol (30 μg/ml) and arabinose (0.0025% w/v) with 50 μl of the transformation mixture in 15 ml culture tubes in triplicate. Incubate samples at 37 °C with shaking (300 rpm) for 20 h. (Alternatively 5 μl of culture can be used to inoculate 100 μl of medium in a 96-well plate This method is useful when dealing with large numbers of samples, although errors will be slightly higher. In order to avoid evaporation, which would lead to error, fill the outermost wells with media, but do not use them for samples. Finally, double-wrap the joint between the lid and the plate with parafilm).

    3. Measurement of β-galactosidase Activity

    1. Preheat the plate-reader to 28 °C.
    2. Transfer the Z-buffer into a reservoir with a large pipette tip, making sure to only take up the upper (aqueous) layer. Transfer 100 μl of freshly prepared Z-buffer/chloroform to the wells of a 96-well plate. Transfer 5 μl of each culture into the wells of the plate in quadruplicate. Omit the culture from four wells which will serve as the blank.
    3. Measure the OD595 of the plate to determine cell density.
    4. Add 50 μl of Z-buffer/SDS to all wells of the plate. Shake the plate in the plate-reader for 10 min to lyze the cells. Make sure the cell suspensions are clear after lysis and repeat the shaking step if required. Incomplete lysis suggests the Z-buffer/chloroform was not freshly prepared.
    5. Add 50 μl freshly prepared Z-buffer/ONPG to all wells and return the plate to the plate reader and measure OD405 every 30 s for 20 min.
    6. Calculate β-galactosidase activity using the following equation (remembering to subtract the blank). The ratio of OD405/min should be calculated using all data points in the OD405 range 0.0 to 1.0 using a linear model fit.
      Equation 1
      Miller units differ sometimes when recorded on different days. Therefore, a reference construct like GpA should be measured in each test. Its values can be used for normalization of ToxR values.

    4. Control for Protein Expression

    1. Perform Western blotting to verify even protein expression between constructs. Combine 50 μl of the triplicate cultures and centrifuge (2000 rpm, 4 min) in a microcentrifuge. Remove the supernatant by pipetting and resuspend the residual pellet in 2 x sample loading buffer.
    2. Load 7.5 μl on a standard 8% gel and carry out electrophoreses at 125 V for 1 hour 5 min. After transfer, incubate with anti-MBP HRP-conjugated antibody and visualize; the chimeric protein is observed at approximately 70 kDa with some degradation products sometimes seen around 48 kDa. Endogenous MBP is also observed at 45 kDa (see Figure 5).

    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.

    1. Transform PD28 cells (as described for FHK12 cells) and inoculate 2 ml of LB medium. Grow the cells at 37 °C with shaking (300 rpm) overnight.
    2. Pellet the cells by centrifugation at 3500 rpm, 10 min, 4 °C and wash by resuspension in PBS (2 ml) by gentle pipetting with a large tip or gentle vortexing. Pellet the cells (as above), wash with PBS for a second time, pellet and finally resuspend in PBS (1 ml).
    3. Use 25 μl of resuspended cells to inoculate 5 ml minimal media in triplicate and incubate at 37 °C with shaking (300 rpm). Take OD595 readings between 15-25 h, approximately every 2 hours by transferring 200 μl of each sample into a 96-well plate and reading using the plate-reader.

    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
    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
    Figure 2. The hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP).

    Figure 3
    Figure 3. Plasmid map of pToxR7.

    Figure 4
    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
    Figure 5. Western blot for protein expression.

    Figure 6
    Figure 6. PD28 complementation assay to control for correct membrane insertion to the periplasm. Negative control represents a construct deficient in maltose binding protein.

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    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.

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    No conflicts of interest declared.

    Acknowledgements

    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).

    Materials

    Name Company Catalog Number Comments
    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
    Na2HPO4 Sigma-Aldrich S9390
    NaH2PO4 Sigma-Aldrich S9638
    KCl Mallinckrodt Baker Inc. 6858-06
    MgSO4.7H2O Sigma-Aldrich 63138
    Sodium dodecylsulfate (SDS) Sigma-Aldrich L6026
    2-Nitrophenyl β-D-galactopyranoside (ONPG) Sigma-Aldrich 73660
    Z-buffer 16.1 g Na2HPO4
    5.5g NaH2PO4
    0.75g KCl
    0.246g MgSO4
    Make up to 1 l, pH 7.0
    Z-buffer/chloroform 200 mL β-mercapt–thanol, 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
    β-mercapt–thanol Calbiochem 444203
    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

    References

    1. Parker, M.S.et al. Oligomerization of the heptahelical G protein coupling receptors: a case for association using transmembrane helices. Mini Rev Med Chem 9, 329-39 (2009).
    2. Mo, W. & Zhang, J.T. Oligomerization of human ATP-binding cassette transporters and its potential significance in human disease. Expert Opin Drug Metab Toxicol 5, 1049-63 (2009).
    3. von Heijne, G. Membrane-protein topology. Nat Rev Mol Cell Biol 7, 909-18 (2006).
    4. Van Horn, W.D.et al. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324, 1726-9 (2009).
    5. Hattori, M.et al. Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis. Embo J 28, 3602-12 (2009).
    6. Ulmschneider, M.B. & Sansom, M.S. Amino acid distributions in integral membrane protein structures. Biochim Biophys Acta 1512, 1-14 (2001).
    7. Deber, C.M. & Goto, N.K. Folding proteins into membranes. Nat Struct Biol 3, 815-8 (1996).
    8. Gerber, D. & Shai, Y. In vivo detection of hetero-association of glycophorin-A and its mutants within the membrane. J Biol Chem 276, 31229-32 (2001).
    9. Langosch, D., Brosig, B., Kolmar, H. & Fritz, H.J. Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. J Mol Biol 263, 525-30 (1996).
    10. Russ, W.P. & Engelman, D.M. TOXCAT: a measure of transmembrane helix association in a biological membrane. Proc Natl Acad Sci U S A 96, 863-8 (1999).
    11. Berger, B.W.et al. Consensus motif for integrin transmembrane helix association. Proc Natl Acad Sci U S A 107, 703-8 (2010).
    12. Oates, J., King, G. & Dixon, A.M. Strong oligomerization behavior of PDGFbeta receptor transmembrane domain and its regulation by the juxtamembrane regions. Biochim Biophys Acta 1798, 605-15 (2010).
    13. Sal-Man, N., Gerber, D. & Shai, Y. Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins. J Mol Biol 344, 855-64 (2004).
    14. Sammond, D.W.et al. Transmembrane peptides used to investigate the homo-oligomeric interface and binding hot-spot of the oncogene, latent membrane protein 1. submitted (2010).
    15. Li, R.et al. Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes. J Biol Chem 279, 26666-73 (2004).
    16. Ruan, W., Becker, V., Klingmuller, U. & Langosch, D. The interface between self-assembling erythropoietin receptor transmembrane segments corresponds to a membrane-spanning leucine zipper. J Biol Chem 279, 3273-9 (2004).
    17. Finger, C., Escher, C. & Schneider, D. The single transmembrane domains of human receptor tyrosine kinases encode self-interactions. Sci Signal 2, ra56 (2009).

    Comments

    0 Comments

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter