title: The extracellular protein VlsE is destabilized inside cells.
abstract: 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 (Förster 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 VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE 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 VlsE is stabilized by 150mg/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 VlsE 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.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Irisbel
authors.initials: I
authors.lastname: Guzman
authors.affiliation: Department of Physics, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Gelman
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tai
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA; Department of Chemistry, University of Illinois, Urbana, IL 61801, USA; Department of Physics, University of Illinois, Urbana, IL 61801, USA. Electronic address: mgruebel@illinois.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008074:Lipoproteins / Q000737:chemistry* / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding / Q000528:radiation effects; D055550:Protein Stability; D013696:Temperature
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D008074:Lipoproteins; C105546:VlsE protein, Borrelia burgdorferi; D010735:Phosphoglycerate Kinase
keywords: extracellular protein; fast relaxation imaging (FReI); fluorescence resonance energy transfer (FRET); protein folding; variable major protein-like sequence expressed (VlsE)