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Labeling for quantitative comparison of imaging measurements in vitro and in cells Caitlin M Davis, and Martin Gruebele Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00141 • Publication Date (Web): 16 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018
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Biochemistry
Labeling for quantitative comparison of imaging measurements in vitro and in cells Caitlin M. Davis*† and Martin Gruebele*†,‡ †
Department of Chemistry, Department of Physics, and ‡Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
* Corresponding Authors (C.M.D) Phone: (217) 244-5062, E-mail:
[email protected] (M.G.) Phone: (217) 333-1624, E-mail:
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ABBREVIATIONS 2-AP, 2-aminopurine; FlAsH, fluorescein arsenical helix binder; FRET, Förster resonance energy transfer; GFP, Green fluorescent protein; hPin1, human Pin1; PEG, polyethylene glycol; PGK, phosphoglyerate kinase; ReAsH, resorufin arsenical hairpin binder; revSL2, reverse stem loop 2; SL2, stem loop 2; SOD1, superoxide dismutase 1; U-2 OS, human osteosarcoma; Yfh1, yeast frataxin
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Biochemistry
ABSTRACT Qualitative imaging of biomolecular localization and distribution inside cells has revolutionized cell biology. Most of these powerful techniques require modifications to the target biomolecule. Over the past 10 years, these techniques have been extended to quantitative measurements, from in-cell protein folding rates, to complex dissociation constants, to RNA lifetimes. Such measurements can be affected even when a target molecules is just mildly perturbed by its labels. Here, the impact of labeling on protein (and RNA) structure, stability, and function in cells are discussed via practical examples from recent literature. General guidelines for selecting and validating modification sites are provided to bring the best from cell biology and imaging to quantitative biophysical experiments inside cells.
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INTRODUCTION Pioneering fluorescent labeling experiments sought to image proteins inside cells to answer qualitative questions about cellular localization, movement and interactions for the first time.1 Many of these studies were enabled by the discovery of Green Fluorescent Protein (GFP) in 1962 and its subsequent development as a fluorescent protein label.2–5 Dye labeling techniques for proteins and nucleic acids advanced at the same time, ranging from fluorescent dyes for visible microscopy imaging to infrared probes in the ‘water free’ infrared window around 2500 cm-1. In parallel, Dexter transfer emerged to study contact formation between biomolecules,6 and Förster resonance energy transfer (FRET) emerged as a powerful molecular distance measure.7–10 Dexter transfer relies on short-range electron transfer to sense contacts on a
