Linked Life Data

21673634

Source:http://linkedlifedata.com/resource/pubmed/id/21673634

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
51
pubmed:dateCreated
2011-6-15
pubmed:abstractText
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 channels, ion pumps, and transporters. 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 labeling. Furthermore, this method provides an approach to determine distance constraints between specific residues. 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 signal. 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 proteins. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein. 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 (<5%) 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 protein. 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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1940-087X
pubmed:author
pubmed:issnType
Electronic
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:dateRevised
2011-11-17
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.
pubmed:affiliation
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute.
pubmed:publicationType
Journal Article, Video-Audio Media