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Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer.

Abstract：

:Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Basak S,Sakia N,Dougherty L,Guo Z,Wu F,Mindlin F,Lary JW,Cole JL,Ding F,Bowen ME

doi

10.1016/j.jmb.2020.166793

subject

Has Abstract

pub_date

2021-01-01 00:00:00

pages

166793

issue

5

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(20)30718-X

journal_volume

433

pub_type

杂志文章

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