Studying protein folding in health and disease using biophysical approaches

Abstract

Protein folding is crucial for normal physiology including development and healthy aging, and failure of this process is related to the pathology of diseases including neurodegeneration and cancer. Early thermodynamic and kinetic studies based on the unfolding and refolding equilibrium of individual proteins in the test tube have provided insight into the fundamental principles of protein folding, although the problem of predicting how any given protein will fold remains unsolved. Protein folding within cells is a more complex issue than folding of purified protein in isolation, due to the complex interactions within the cellular environment, including post-translational modifications of proteins, the presence of macromolecular crowding in cells, and variations in the cellular environment, for example in cancer versus normal cells. Development of biophysical approaches including fluorescence resonance energy transfer (FRET) and nuclear magnetic resonance (NMR) techniques and cellular manipulations including microinjection and insertion of noncanonical amino acids has allowed the study of protein folding in living cells. Furthermore, biophysical techniques such as single-molecule fluorescence spectroscopy and optical tweezers allows studies of simplified systems at the single molecular level. Combining in-cell techniques with the powerful detail that can be achieved from single-molecule studies allows the effects of different cellular components including molecular chaperones to be monitored, providing us with comprehensive understanding of the protein folding process. The application of biophysical techniques to the study of protein folding is arming us with knowledge that is fundamental to the battle against cancer and other diseases related to protein conformation or protein-protein interactions.

Keywords: biophysical techniques; cancer; neurodegeneration; protein conformation; proteostasis; single molecule detection.

Figure 1.. In-cell and in vitro study of protein folding has been significantly advanced by using biophysical approaches including FRET, NMR, CEST-MRI and optical tweezers.

Figure 2.. SmFRET using alternative laser excitation (ALEX) is applied to detect the structural distribution and changes of cytosolic proteins in live cells.

(A) Experimental setup. (B) Single-molecule detection of freely diffusing cytosolic molecules in a live cell. (C) Typical fluorescence burst signals. X → Y denotes the fluorescence signal from dye Y with the excitation of dye X, where X and Y are the donor (D) or the acceptor (A), respectively. FG: function generator; λ/4: quarter wave plate; AOM: acousto-optic modulator; M: mirror; DF: dichroic filter; OL: objective lens; MH: metal holder; CS: coverslip; EF: emission filter; TL: tube lens; L: lens; BPF: band-pass filter; APD: avalanche photodiode; CB: counter board; PC: personal computer. Figure reproduced from ref. [25] with permission.

Figure 3.. In-cell NMR is applied to detect the conformational changes of cytosolic proteins in live cells.

(A) The vector containing a gene of interest (green arrow) is delivered by transient transfection in labeled medium. Cells expressing the protein of interest (green) are collected and placed in a 3 mm Shigemi NMR tube and applied for in-cell NMR analysis. (B–D) ¹H−¹⁵N NMR spectra of SOD1 in the apo and reduced state (B), the one-zinc-ion (cyan)-per-monomer bound and dimerized state (C) and the fully mature, copper (salmon)-zinc (cyan)-bound and disulfide-oxidized state (D) in human cells. Figure adapted from ref. [36] with permission. (Further permissions related to this figure should be directed to the ACS. Original figures at: https://pubs.acs.org/doi/10.1021/acs.accounts.8b00147).

Figure 4.. Single-molecule force experiments of the SBD of the Hsp70 chaperone performed with optical tweezers.

(A) Optical tweezers assay. The SBD (green/red surfaces) is tethered to the beads (gray spheres) by two DNA handles, and beads are trapped in highly focused laser beams (red cones). The connection between the DNA and protein is realized by the modification of the two cysteine residues of the protein by the single-stranded DNA-maleimide oligonucleotide complementary to the DNA-handle overhang. One of the beams is reflected by a steerable mirror, which enables pulling and stretching of a single protein. (B) Force-extension curves of a single SBD domain show the order of the individual unfolding events varies. Pathway I corresponds to unfolding of the larger fragment first, followed by the shorter SBD fragment. For pathway II, the order of unfolding events is reversed. (C) Summary of bifurcating unfolding pathways of the SBD. Figure adapted from ref. [56] with permission.