Name: examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling
Uploaded: 2011-05-29T14:00:00
Duration: 11 min 55 s
Description: Watch this Scientific Journal Video about Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling at JoVE.com

<ol>
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ब्याज की कोई संघर्ष की घोषणा की.

प्रयोगात्मक दृष्टिकोण है कि हम का वर्णन साइट विशेष fluorophore लेबलिंग और दो इलेक्ट्रोड वोल्टेज और झिल्ली प्रोटीन की संरचना और समारोह के बीच संबंधों की जांच करने के लिए क्लैंप को जोड़ती है. इस तकनीक झिल्ली प्रोटीन के गठनात्मक गतिशीलता पर आयन परिवहन के दौरान समय - हल जानकारी प्राप्त करने के लिए इस्तेमाल किया जा सकता है. इसके अलावा, इस दृष्टिकोण आयन पंप, आयन चैनलों और ट्रांसपोर्टरों के रूप में विभिन्न प्रोटीन के साथ काम करने के लिए अनुरूप किया जा सकता है. एक विशिष्ट अवशेषों पर गठनात्मक गतिशीलता की जांच करने के लिए इसके अलावा, यह भी संभव है प्रतिदीप्ति प्रतिध्वनि ऊर्जा हस्तांतरण का उपयोग करने के लिए एक holoenzyme भीतर दूरी बाधाओं को निर्धारित है. अवशेष के रूप में के रूप में अच्छी तरह से सब यूनिटों के बीच रिश्तेदार आंदोलन को मापने के बीच दूरी का निर्धारण में मदद कुंजी gating तंत्र से जुड़े सवालों को हल कर सकते हैं.

<table border="1"><tbody><tr><th> / अभिकर्मक और उपकरण </th><th> कंपनी </th><th> / अभिकर्मक और उपकरण </th><th> कंपनी </th></tr><tr><td> 100W टंगस्टन प्रकाश स्रोत </td><td> कार्ल Zeiss MicroImaging </td><td> fluorescein-5-maleimide </td><td> Invitrogen </td></tr><tr><td> 1B150F - 4 </td><td> विश्व परिशुद्धता उपकरण </td><td> उच्च शुद्ध पीसीआर निकालना किट </td><td> रॉश </td></tr><tr><td> 3.0-4.0 Ethicon Vicryl </td><td> जॉनसन एंड जॉनसन </td><td> mMessage mMachine किट </td><td> Ambion </td></tr><tr><td> 475AF40 उत्तेजना फिल्टर </td><td> ओमेगा ऑप्टिकल </td><td> MS-222 </td><td> सिग्मा Aldrich </td></tr><tr><td> 505DRLP dichroic दर्पण </td><td> ओमेगा ऑप्टिकल </td><td> Nucleospin प्लाज्मिड किट </td><td> Macherey - Nagel </td></tr><tr><td> Macherey - Nagel </td><td> ओमेगा ऑप्टिकल </td><td> Nanodrop 2000C Sprectrophotometer </td><td> थर्मो वैज्ञानिक </td></tr><tr><td> 535DF50 उत्तेजना फिल्टर </td><td> ओमेगा ऑप्टिकल </td><td> PC-10 micropipette डांड़ी </td><td> Narishige </td></tr><tr><td> 560DRLP dichroic दर्पण </td><td> ओमेगा ऑप्टिकल </td><td> 10 सॉफ्टवेयर pCLAMP </td><td> Axon उपकरण </td></tr><tr><td> 565ALP उत्सर्जन फिल्टर </td><td> ओमेगा ऑप्टिकल </td><td> PfuTurbo पोलीमरेज़ डीएनए </td><td> Strategene </td></tr><tr><td> 565EFLP उत्सर्जन फिल्टर </td><td> ओमेगा ऑप्टिकल </td><td> पिन-022A photodiode </td><td> संयुक्त वेक्षक टेक्नोलॉजीज </td></tr><tr><td> 570DRLP dichroic दर्पण </td><td> ओमेगा ऑप्टिकल </td><td> Polarized फ़िल्टर </td><td> Linos Phtonics इंक </td></tr><tr><td> Linos Phtonics इंक </td><td> ओमेगा ऑप्टिकल </td><td> QuickChange निर्देशित - साइट Mutagenisis किट </td><td> Stratagene </td></tr><tr><td> Axio परीक्षक प्रतिदीप्ति माइक्रोस्कोप </td><td> कार्ल Zeiss MicroImaging GMBH </td><td> RC-10 प्रतिदीप्ति चैंबर </td><td> वार्नर उपकरण </td></tr><tr><td> वार्नर उपकरण </td><td> जीवन टेक्नोलॉजीज </td><td> tetramethylrhodamine 6 - maleimide </td><td> Invitrogen </td></tr><tr><td> Digidata 1440A डाटा अधिग्रहण प्रणाली </td><td> Axon उपकरण </td><td> Top10 Electrocompetent कोशिकाओं </td><td> Invitrogen </td></tr><tr><td> DPN मैं </td><td> न्यू इंग्लैंड Biolabs </td><td> टर्बो प्रवर्धक Tec-05X </td><td> NPI </td></tr></tbody></table>

examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Watch this Scientific Journal Video about Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling at JoVE.com

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

हम एक विधि है जो साइट विशिष्ट गठनात्मक परिवर्तन एकल कक्षों पर प्रतिदीप्ति का उपयोग का विश्लेषण के साथ - साथ झिल्ली प्रोटीन आयन परिवहन के कैनेटीक्स उपायों का वर्णन करेंगे. इस तकनीक आयन चैनल, ट्रांसपोर्टरों और आयन पंप के लिए अनुकूलनीय है और प्रोटीन सब यूनिटों के बीच की दूरी बाधाओं का निर्धारण करने के लिए उपयोग किया जा सकता है.

झिल्ली प्रोटीन के गठनात्मक गतिशीलता की जांच बगल में साइट - निर्देश प्रतिदीप्ति लेबल के साथ

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

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An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

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Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

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प्रतिदीप्ति का प्रयोग mRNA अणुओं के दृश्य और विश्लेषण<em&gt; स्वस्थानी</emमें&gt; संकरण<em&gt; Saccharomyces cerevisiae</em

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