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FRET assessment of CFTR molecular assembly
Author(s) -
Berdiev Bakhrom K,
Tousson Albert,
OosterveldHut Henderika M.J.,
Qadri Yawar J.,
CormetBoyaka Estelle,
Hong Jeong S.,
Fuller Cathy M.,
Sorscher Eric J.,
Lukacs Gergely L.,
Benos Dale J.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.934.17
Subject(s) - förster resonance energy transfer , chemistry , yellow fluorescent protein , biophysics , fluorescence recovery after photobleaching , fluorescence , cytoplasm , microbiology and biotechnology , biochemistry , biology , gene , membrane , physics , quantum mechanics
In spite of extensive studies, uncertainty exists regarding the number of CFTR molecules that come together to form a conducting pore. At present, findings point both to monomeric and multimeric assembly of CFTR protein. We evaluated the CFTR assembly by fluorescence resonance energy transfer imaging. FRET exploits the exquisite sensitivity of fluorescence measurements to detect molecular complexes with near angstrom resolution. We assayed for FRET between (i) N‐terminally tagged CFTRs (ECFP‐CFTR and EYFP‐CFTR); (ii) C‐terminally tagged CFTRs (CFTR‐ECFP and CFTR‐EYFP); and (iii) both N‐terminally and C‐terminally tagged CFTRs (ECFP‐CFTR and CFTR‐EYFP). We found no appreciable increase in CFP fluorescence after selective photobleaching of YFP, indicative of no FRET occurrence. Our findings show that the cytoplasmic tails of CFTR are not in sufficient proximity for the occurrence of FRET, suggestive of monomeric organization of CFTR. Supported by NIH 2RO1‐DK37206‐15, NIH P50 DK53090‐05, NIH RO1‐ DK075302 , CFF/CFFT.