From the test tube to the cell: exploring the folding and aggregation of a beta-clam protein

Abstract

A crucial challenge in present biomedical research is the elucidation of how fundamental processes like protein folding and aggregation occur in the complex environment of the cell. Many new physico-chemical factors like crowding and confinement must be considered, and immense technical hurdles must be overcome in order to explore these processes in vivo. Understanding protein misfolding and aggregation diseases and developing therapeutic strategies to these diseases demand that we gain mechanistic insight into behaviors and misbehaviors of proteins as they fold in vivo. We have developed a fluorescence approach using FlAsH labeling to study the thermodynamics of folding of a model beta-rich protein, cellular retinoic acid binding protein (CRABP) in Escherichia coli cells. The labeling approach has also enabled us to follow aggregation of a modified version of CRABP and chimeras between CRABP and huntingtin exon 1 with its glutamine repeat tract. In this article, we review our recent results using FlAsH labeling to study in-vivo folding and present new observations that hint at fundamental differences between the thermodynamics and kinetics of protein folding in vivo and in vitro.

FIGURE 1

Design of tetra-Cys CRABP. (A) Backbone structure of CRABP (PDB code 1CBI) showing the position of the tetra-Cys motif in the highly variable Ω-loop. (B) Fluorescence spectra of FlAsH-labeled tetra-Cys CRABP in its native state (folded) and denatured with 8M urea (unfolded). [Figure adapted from Ignatova, Z.; Gierasch, L. M. Proc Natl Acad Sci USA 2004, 101, 523–528.]

FIGURE 2

Monitoring aggregation in vivo. (A) Time course of in-cell expression of FlAsH-labeled tetra-Cys CRABP I, P39A tetra-Cys CRABP and chimeras of tetra-Cys CRABP and exon 1 of Huntingtin with 40 and 53 glutamine residues followed at 530 nm., Cells were pre-loaded with FlAsH before induction of protein expression at time zero. Tetra-Cys CRABP (red) is soluble, P39A tetra-Cys CRABP (purple) partitions 50% into the insoluble fraction, tetra-Cys CRABP HD40 (orange) is 20% in the insoluble fraction, and tetra-Cys CRABP Htt53 (light blue) is isolated only in an insoluble state. For comparison, the fluorescence in all the samples is normalized against the highest fluorescence measured (tetra-Cys CRABP Htt53) and the cell number. (B) Fluorescence microscopy images of E. coli cells expressing P39A tetra-Cys CRABP showing the conversion of the uniformly distributed fluorescence (arising from the soluble state) into hyperfluorescent aggregates localized at the poles of the cells. [Parts of this figure reproduced with permission from Ignatova, Z.; Gierasch, L. M. J Biol Chem 2006, 281, 12959–12967.]

FIGURE 3

Urea denaturation curves as a function of incubation time in vitro and in vivo. (A) Urea melt of FlAsH-labeled tetra-Cys CRABP in vitro after sample incubation for the indicated times, monitored by Trp fluorescence; (B) urea melt of FlAsH-labeled P39A tetra-Cys CRABP in vitro after sample incubation for the indicated times, monitored by Trp fluorescence; (C) urea melt of FlAsH-labeled tetra-Cys CRABP in vivo after sample incubation for the indicated times, monitored by FlAsH fluorescence; and (D) urea melt of FlAsH-labeled P39A tetra-Cys CRABP in vivo after sample incubation for the indicated times, monitored by FlAsH fluorescence.