एकल पास transmembrane डोमेन की oligomerization प्रवृत्ति (TMDs) का आकलन करने के लिए एक कुशल प्रक्रिया में वर्णित है. Chimeric ToxR के लिए इनकार TMD से मिलकर प्रोटीन ई. कोलाई संवाददाता तनाव में व्यक्त कर रहे हैं. TMD प्रेरित oligomerization ToxR, प्रतिलेखन और रिपोर्टर प्रोटीन के उत्पादन, galactosidase के सक्रियण के dimerization का कारण बनता है.
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).
ToxR transcriptional संवाददाता परख सतही oligomerize क्षमता के साथ transmembrane दृश्यों की पहचान का तरीका है. चूंकि बातचीत बैक्टीरियल भीतरी झिल्ली के भीतर उत्पन्न कर रहे हैं, इस परख झिल्ली अनुकरण करनेवाला वातावरण में अध्ययन प्रण?…
The authors have nothing to disclose.
हम स्वास्थ्य के राष्ट्रीय संस्थान (1R21CA138373 धन्यवाद और इस काम के वित्तीय समर्थन के लिए कैंसर (SU2C) करने के लिए स्टैंड अप हरियाणा के लिए कैंसर, Sidney Kimmel फाउंडेशन से एक Kimmel विद्वान पुरस्कार अनुसंधान के अमेरिकन एसोसिएशन से 2009 Elion पुरस्कार के लिए आभारी है. कैंसर रिसर्च (SKF-08-101), और राष्ट्रीय विज्ञान फाउंडेशन संकाय जल्दी कैरियर पुरस्कार (NSF0954819).
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
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 | 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 Chemicals | 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 |
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Z-buffer/chloroform | 200 mL β-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 | ||
β-mercaptoethanol | Calbiochem | 444203 | |
Anti-MBP monoclonal antibody (HRP conjugated) | NEB | 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 | 83.1835.300 | |
Plate-reader | Beckman Coulter | DTX880 Multimode Detector | |
Water bath | VWRI | 89032-204 | |
Shaking incubator | FormaScientific |