Overcoming the Cellular Crowding Effect: VlsE-FRET is Destabilized Inside Cells

Abstract

The understanding of protein folding dynamics inside cells is crucial for the development of new therapies against protein folding and aggregation diseases. In addition, many processes inside cells such as gene expression, association and dissociation processes, as well as protein degradation are controlled by protein stability and kinetics. Fluorescence Relaxation Imaging (FReI) instrument combines temperature jump, FRET and cell imaging to study dynamic processes inside cells from the microseconds to the seconds time scales. using FReI, we have carried out experiment to calculate the melting temperature (Tm) and relaxation time (τ) of a constructed FRET pair protein of the Variable Lyme Disease Surface Antigen E (VlsE). VlsE-FRET has an in vitro Tm= 38 ± 1 °C and τ = 0.5 ± 0.1 s. In contrast, VlsE-FRET has an in cell Tm = 35 ± 1 °C and τ = 2 ± 2 s. Also, the in vitro data support a single exponential unfolding process with β = 1.0 ± 0.1, but the in cell data does not with β = 0.8 ± 0.2. Interestingly, VlsE-FRET has two distinct populations in cell with melting temperatures of 33 ± 1 °C and 36 ± 1 °C. These data support that some proteins can overcome the crowding effect inside cells due to the contribution of chemical/electrostatic forces. VlsE has different regions that undergo antigenic variation in order to evade the immune system of the host. Probably, the VlsE-FRET destabilization in cell is part of the trigger mechanism for the extensive genetic and antigenic variation that allows VlsE to evolve.