The Extracellular Protein VIsE Is Destabilized Inside Cells

JOURNAL OF MOLECULAR BIOLOGY

卷:426

Article

Guzman, Irisbel¹  Gelman, Hannah⁴  Tai, Jonathan¹  Gruebele, Martin^2,3,4 

[1] Univ Illinois, Dept Biochem, Urbana, IL 61801 USA

[2] Univ Illinois, Ctr Biophys & Computat Biol, Urbana, IL 61801 USA

[3] Univ Illinois, Dept Chem, Urbana, IL 61801 USA

[4] Univ Illinois, Dept Phys, Urbana, IL 61801 USA

关键词: protein folding;    extracellular protein;    variable major protein-like sequence expressed (VIsE);    fast relaxation imaging (FReI);    fluorescence resonance energy transfer (FRET);   

DOI  :  10.1016/j.jmb.2013.08.024

来源: Elsevier

【 摘 要 】

We use U2OS cells as in vivo test tubes to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by fast relaxation imaging, which combines a temperature jump with fluorescence microscopy of FRET (Forster resonance energy transfer)-labeled proteins. While the stability of the cytoplasmic enzyme PGK (phosphoglycerate kinase) increases in cells, the stability of the cell surface antigen VIsE, which presumably did not evolve for stability inside cells, decreases. VIsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VIsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VIsE is stabilized by 150 mg/mL of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VIsE in U2OS cells: long-range interactions competing with crowding or shape-dependent crowding favoring more compact states inside the cell over the elongated aqueous buffer native state. (C) 2013 Elsevier Ltd. All rights reserved.

【 授权许可】

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