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. 2014 Jan 9;426(1):11-20.
doi: 10.1016/j.jmb.2013.08.024. Epub 2013 Sep 4.

The extracellular protein VlsE is destabilized inside cells

Irisbel Guzman  1 , Hannah Gelman  2 , Jonathan Tai  1 , Martin Gruebele  3
Affiliations

The extracellular protein VlsE is destabilized inside cells

Irisbel Guzman et al. J Mol Biol. .

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.

Keywords: extracellular protein; fast relaxation imaging (FReI); fluorescence resonance energy transfer (FRET); protein folding; variable major protein-like sequence expressed (VlsE).

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Figures

Figure 1
Figure 1. VlsE protein
(a) VlsE crystal structure. (b) Simplified scheme of VlsE protein translocation across the Borrelia bugdolferi double membrane. ⓐ VlsE is synthesized by the ribosome and folds in the cytoplasm. ⓑ The molecular chaperone SecB recognizes the signal sequence in the N-terminal region of VlsE and transfers VlsE to the Sec machinery that translocates it inside the periplasmic space using ATP energy. ⓒ Phosphatidylglycerol/prolipoprotein diacylglyceryl transferase (Lgt), lipoprotein signal peptidase (LspA) and phospholipid/apolipoprotein transacylase (Lnt) process the lipoprotein precursor into a mature form. ⓓ The transporter LolCDE removes the lipoprotein from the inner membrane, and forms a hydrophilic complex with LolA. ⓔ The periplasmic molecular chaperone LolA removes the lipoprotein from LolCDE. ⓕ LolA transfers the lipoprotein to LolB. ⓖ The lipoprotein is incorporated into the outer membrane by LolB. For our experiments we truncated the lipidation signal sequence and transfected a DNA plasmid containing the VlsE gene into U2OS cells, where it remains in the cytoplasm after translation.
Figure 2
Figure 2
(a) Representative cell demonstrating uniform VlsE-FRET dispersion inside a U2OS cell. The darker area is the location of the cell nucleus, where the optical pathlength through the cytoplasm is shorter. (b) The same area with pixel values (brightness) multiplied by 20×. The original cell is saturated, and the red arrows indicate the position of a non-transfected cell that shows very low autofluorescence emission background (≈ 2% of the transfected cell).
Figure 3
Figure 3
Melting temperature Tm of VlsE-FRET vs. Ficoll-70 concentration (0 to 150 mg/ml) fitted to a line.
Figure 4
Figure 4
(a) Temperature titration traces of D/A in cells (red) and in aqueous buffer (blue). (b) Histogram of melting temperatures in cells (red) and in aqueous buffer (blue). (c) Averaged traces of in cell (red) and in buffer (blue) experiments.
Figure 5
Figure 5
Sample kinetics traces. (a) in cells (red). (b) in aqueous buffer (blue). The colored temperatures indicate the temperature after the temperature jump, and black arrows indicate the cycle of temperatures at which successive kinetic data points were collected.
Figure 6
Figure 6
Distinct populations inside cells: (a) T-jump relaxation time τ vs. melting temperature Tm. (b) Negative correlation between D/A ratio at 25 °C vs. Tm (red dots: in cell; blue dots: in aqueous buffer).
Figure 7
Figure 7
Simplified energy landscapes demonstrating how (a) the long-range electrostatic scenario, and (b) the shape-dependent crowding scenario can rationalize VlsE destabilization inside cells and VlsE stabilization in Ficoll, relative to aqueous buffer experiments.
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