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A variable light domain fluorogen activating protein homodimerizes to activate dimethylindole red.

Senutovitch N, Stanfield RL, Bhattacharyya S, Rule GS, Wilson IA, Armitage BA, Waggoner AS, Berget PB
Biochemistry 51 2471-85 (2012)
Related entries: 3t0v, 3t0w

Cited: 15 times
EuropePMC logo PMID: 22390683

Abstract

Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (V(H)) and variable light (V(L)) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, V(H)-V(L) M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8V(L) domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8V(L) forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8V(L) homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both V(H)-V(L) M8 and M8V(L), led us to rationally design tandem, covalent homodimers of M8V(L) domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.

Articles - 3t0x mentioned but not cited (1)

  1. A variable light domain fluorogen activating protein homodimerizes to activate dimethylindole red. Senutovitch N, Stanfield RL, Bhattacharyya S, Rule GS, Wilson IA, Armitage BA, Waggoner AS, Berget PB. Biochemistry 51 2471-2485 (2012)


Reviews citing this publication (3)

  1. Linkers in the structural biology of protein-protein interactions. Reddy Chichili VP, Kumar V, Sivaraman J. Protein Sci. 22 153-167 (2013)
  2. From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules. Truong L, Ferré-D'Amaré AR. Protein Sci 28 1374-1386 (2019)
  3. Fluorogen-activating proteins: beyond classical fluorescent proteins. Xu S, Hu HY. Acta Pharm Sin B 8 339-348 (2018)

Articles citing this publication (11)

  1. Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces. Szent-Gyorgyi C, Stanfield RL, Andreko S, Dempsey A, Ahmed M, Capek S, Waggoner AS, Wilson IA, Bruchez MP. J. Mol. Biol. 425 4595-4613 (2013)
  2. Engineering fluorogen activating proteins into self-assembling materials. Saunders MJ, Liu W, Szent-Gyorgyi C, Wen Y, Drennen Z, Waggoner AS, Meng WS. Bioconjug. Chem. 24 803-810 (2013)
  3. Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging. Zhang M, Chakraborty SK, Sampath P, Rojas JJ, Hou W, Saurabh S, Thorne SH, Bruchez MP, Waggoner AS. J. Clin. Invest. 125 3915-3927 (2015)
  4. Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools. Schütz M, Batyuk A, Klenk C, Kummer L, de Picciotto S, Gülbakan B, Wu Y, Newby GA, Zosel F, Schöppe J, Sedlák E, Mittl PRE, Zenobi R, Wittrup KD, Plückthun A. J. Mol. Biol. 428 1272-1289 (2016)
  5. Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors. Wang Y, Telmer CA, Schmidt BF, Franke JD, Ort S, Arndt-Jovin DJ, Bruchez MP. Bioconjug. Chem. 26 137-144 (2015)
  6. Spectral fine tuning of cyanine dyes: electron donor-acceptor substituted analogues of thiazole orange. Rastede EE, Tanha M, Yaron D, Watkins SC, Waggoner AS, Armitage BA. Photochem. Photobiol. Sci. 14 1703-1712 (2015)
  7. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation. Magenau AJ, Saurabh S, Andreko SK, Telmer CA, Schmidt BF, Waggoner AS, Bruchez MP. Biomaterials 66 1-8 (2015)
  8. Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules. Pham HH, Szent-Gyorgyi C, Brotherton WL, Schmidt BF, Zanotti KJ, Waggoner AS, Armitage BA. Org. Biomol. Chem. 13 3699-3710 (2015)
  9. Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection. Gallo E, Snyder AC, Jarvik JW. Protein Eng. Des. Sel. 28 327-337 (2015)
  10. A cell surface display fluorescent biosensor for measuring MMP14 activity in real-time. Braun A, Farber MJ, Klase ZA, Berget PB, Myers KA. Sci Rep 8 5916 (2018)
  11. Self-Reporting Chemically Induced Protein Proximity System Based on a Malachite Green Derivative and the L5** Fluorogen Activating Protein. Zeng G, Wang Y, Bruchez MP, Liang FS. Bioconjug. Chem. 29 3010-3015 (2018)


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