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An experimental study of GFP-based FRET, with application to intrinsically unstructured proteins.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Ohashi, Tomoo Galiacy, Stephane D. Erickson, Harold P. Briscoe, Gina
Description	Country affiliation: United States Author Affiliation: Ohashi T ( Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. t.ohashi@cellbio.duke.edu)
Abstract	We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	7
Volume Number	16
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	2007-07-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Fluorescence Resonance Energy Transfer Green Fluorescent Proteins Chemistry Proteins Algorithms Electrophoresis, Polyacrylamide Gel Luminescent Proteins Models, Molecular Protein Conformation Research Support, N.i.h., Extramural Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
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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