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A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization.
J Mol Biol 2010; 401(5):776-91JM

Abstract

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells.

Authors+Show Affiliations

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20603133

Citation

Hoi, Hiofan, et al. "A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization." Journal of Molecular Biology, vol. 401, no. 5, 2010, pp. 776-91.
Hoi H, Shaner NC, Davidson MW, et al. A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. J Mol Biol. 2010;401(5):776-91.
Hoi, H., Shaner, N. C., Davidson, M. W., Cairo, C. W., Wang, J., & Campbell, R. E. (2010). A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. Journal of Molecular Biology, 401(5), pp. 776-91. doi:10.1016/j.jmb.2010.06.056.
Hoi H, et al. A Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization. J Mol Biol. 2010 Sep 3;401(5):776-91. PubMed PMID: 20603133.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A monomeric photoconvertible fluorescent protein for imaging of dynamic protein localization. AU - Hoi,Hiofan, AU - Shaner,Nathan C, AU - Davidson,Michael W, AU - Cairo,Christopher W, AU - Wang,Jiwu, AU - Campbell,Robert E, Y1 - 2010/07/13/ PY - 2010/02/20/received PY - 2010/06/15/revised PY - 2010/06/25/accepted PY - 2010/7/7/entrez PY - 2010/7/7/pubmed PY - 2010/9/14/medline SP - 776 EP - 91 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 401 IS - 5 N2 - The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and to image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and to overcome the limitations imposed by the oligomeric structure of natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Using monomeric versions of Clavularia sp. cyan FP as template, we employed sequence-alignment-guided design to create a chromophore environment analogous to that shared by known photoconvertible FPs. The designed gene was synthesized and, when expressed in Escherichia coli, found to produce green fluorescent colonies that gradually switched to red after exposure to white light. We subjected this first-generation FP [named mClavGR1 (monomeric Clavularia-derived green-to-red photoconvertible 1)] to a combination of random and targeted mutageneses and screened libraries for efficient photoconversion using a custom-built system for illuminating a 10-cm Petri plate with 405-nm light. Following more than 15 rounds of library creation and screening, we settled on an optimized version, known as mClavGR2, that has eight mutations relative to mClavGR1. Key improvements of mClavGR2 relative to mClavGR1 include a 1.4-fold brighter red species, 1.8-fold higher photoconversion contrast, and dramatically improved chromophore maturation in E. coli. The monomeric status of mClavGR2 has been demonstrated by gel-filtration chromatography and the functional expression of a variety of mClavGR2 chimeras in mammalian cells. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 both when the membrane is in contact with a T-lymphocyte expressing leukocyte-function-associated antigen 1 and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein subpopulations and subsequent tracking of dynamic changes in localization in living cells. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/20603133/A_monomeric_photoconvertible_fluorescent_protein_for_imaging_of_dynamic_protein_localization_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(10)00699-6 DB - PRIME DP - Unbound Medicine ER -