NDLI: Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Gahl, Robert F. Tekle, Ephrem Tjandra, Nico
Description	Country affiliation: United States Author Affiliation: Gahl RF ( Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart Lung and Blood Institute - National Institutes of Health, Bethesda, MD 20892, United States.); Tekle E ( Laboratory of Biochemistry, Biochemistry and Biophysics Center, National Heart Lung and Blood Institute - National Institutes of Health, Bethesda, MD 20892, United States.); Tjandra N ( Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart Lung and Blood Institute - National Institutes of Health, Bethesda, MD 20892, United States. Electronic address: tjandran@nhlbi.nih.gov.)
Abstract	Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented.
File Format	HTM / HTML
ISSN	10462023
e-ISSN	10959130
DOI	10.1016/j.ymeth.2013.07.011
Journal	Methods
Issue Number	2
Volume Number	66
Language	English
Publisher	Elsevier
Publisher Date	2014-03-15
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Life sciences, Biochemistry Single-cell Analysis Bcl-2-associated X Protein Chemistry Animals Cells, Cultured Fluorescence Resonance Energy Transfer Fluorescent Dyes Mice Microinjections Microscopy, Confocal Microscopy, Fluorescence Protein Conformation Protein Transport Reference Standards Metabolism Research Support, N.i.h., Intramural
Content Type	Text
Resource Type	Article
Subject	Molecular Biology

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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