Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2014;43:119-40.
doi: 10.1146/annurev-biophys-051013-022811.

Mechanisms of Cellular Proteostasis: Insights From Single-Molecule Approaches

Affiliations
Free PMC article
Review

Mechanisms of Cellular Proteostasis: Insights From Single-Molecule Approaches

Carlos J Bustamante et al. Annu Rev Biophys. .
Free PMC article

Abstract

Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state. Polypeptides that are no longer needed or pose a threat to the cell, such as misfolded proteins and aggregates, are removed in an efficient and timely fashion by ATP-dependent proteases. In this review, we describe how applications of single-molecule manipulation methods, in particular optical tweezers, are shedding new light on the molecular mechanisms of quality control during the life cycles of proteins.

Keywords: chaperones; force spectroscopy; proteases; protein folding; ribosome; worm-like chain.

Figures

Figure 1
Figure 1
Energetic coupling of subdomain folding in T4 lysozyme revealed by nonequilibrium single-molecule force spectroscopy. (a) Experimental setup. A T4 lysozyme protein is attached to polystyrene beads by means of DNA handles. The protein contains cysteines that permit covalent attachment to two 500–base pair double-stranded DNA molecular handles through disulfide linkages in only one domain of the protein. These DNA handles are derivatized on their 5′ ends with digoxigenin and biotin to bind the respective beads (antidigoxigenin-coated beads and streptavidin-coated beads, respectively). The streptavidin bead is attached to a micropipette by suction and has a diameter of 2.1 μm. This system is inside a laminar flow camera. Modified with permission from Reference . (b) Force-extension curves obtained by stretching (red ) and relaxing (blue) a single domain of the T4 lysozyme; DNA handles were attached in the N domain of the protein. The data shown were collected at a 50-Hz sampling rate and a pulling speed of 60 nm/s. Reprinted with permission from Reference . (c) The normalized probability curves of unfolding and refolding work for 16,61 WT*T4L. The normalized probability curves of the work required for the unfolding (red ) and refolding (blue) were obtained from the unfolding events in panel b. The Crooks fluctuation theorem (CFT) was used to calculate the free energy from the single-molecule experiments. The calculated free energy for 16,61 WT*T4L, obtained by the crossing point between the refolding and unfolding work (ΔG, CFT = 12.3 ± 0.6 kcal/mol), agrees well with the free energy measured in bulk solution unfolding experiments (ΔG, bulk = 14.1 ± 0.6 kcal/mol). Reprinted with permission from Reference . Abbreviations: pN, piconewton; T4L, T4 lysozyme; WT, wild type.
Figure 2
Figure 2
Real-time observation of folding transitions in apomyoglobin. (a) Constant trap position experiments performed across the N and C termini of apomyoglobin H36Q. One-second trace of a constant trap position experiment at 1,000 Hz sampling frequency (blue) for the N- to C-terminal attachment points. The inferred trajectory of the molecule at 500 Hz is shown (red ). The plot shows the lifetimes in each state at a particular force, and the protein “hops” between each state. (b) Linear fits of the natural logarithm (ln) of the rate constants as a function of force are shown for apomyoglobin. The distance to the transition state is determined from the slope of the lines using Bell’s model [see the equation in the sidebar, Dependence of the Rate Constant on Force (Bell Equation)]. The Δxunf = 3.4 ± 1.2 nm, the Δxfold = 7.6 ± 3.3 nm, the Δxtotal (sum of Δx) = 11.0 ± 3.5 nm, and the Δxtotal (measured in the optical tweezers instrument) = 12 ± 1 nm. Adapted with permission from Reference . Abbreviation: pN, piconewton.
Figure 3
Figure 3
Folding of ribosome-bound nascent polypeptides. (a) Experimental setup for optical tweezers measurements of ribosome-bound nascent proteins. A ribosome–nascent chain complex is tethered between two polystyrene microspheres via DNA handles. Attachment points are located on the large subunit of the ribosome and the N terminus of the nascent protein. The force applied to the assembly can be varied by moving the optical trap. (b) Representative force-clamp trace for the folding of a single ribosome-bound T4 lysozyme molecule. At the beginning, the force is lowered to 3.6 pN, and the extension is monitored over time. In this example, the protein folds after 25 s. Equilibrium “hopping” between the unfolded state and an intermediate state is also observed. (c) Apparent refolding rates for three constructs studied in the optical tweezers, which are referred to as +41, +60, and free, for the ribosome-bound T4 lysozyme with a 41–amino acid C-terminal linker, the ribosome-bound T4 lysozyme with a 60–amino acid C-terminal linker, and the T4 lysozyme in the absence of the ribosome, respectively. (d ) Schematic energy landscape based on single-molecule experiments, illustrating how the ribosome affects folding. The height of the barrier between the intermediate and native state is affected by the ribosome. Figure adapted with permission from Reference . Abbreviation: pN, piconewton.
Figure 4
Figure 4
Unfolding and translocation of protein substrates by ATP-dependent proteases monitored at the single-molecule level. (a) ClpXP binds, unfolds, and translocates ssrA-tagged protein substrates in an ATP-dependent manner. (b) Dual-trap optical tweezers assay to study protein unfolding and polypeptide translocation by individual ClpXP molecules. In this assay, ClpX is immobilized on the surface of a streptavidin (SA)-coated bead via a biotin-SA interaction. ClpP binds ClpX in trans. The DNA-tethered protein substrate has a digoxigenin (dig) molecule in one 5′ end of the DNA that binds to antidig (AD)-coated beads. The ssrA-tagged substrate consists of two green fluorescent protein (GFP) molecules separated by two permanently unfolded I27 titin domains (59). (c) Examples of molecular trajectories averaged down to 50 Hz showing protein unfolding and polypeptide translocation by ClpXP. Sudden gains in extension correspond to GFP unfolding events (red arrowheads). After unfolding, the gradual decrease in extension reflects the translocation of the unfolded polypeptide through the ClpX pore into ClpP. Occasionally, polypeptide translocation is interrupted by regions of near-zero velocity that were identified as pauses (blue trajectory). (d ) Cartoon illustrating the sequence of events occurring in the molecular trajectories shown in panel c. Except for the green trajectory, ClpXP successfully unfolded both GFP molecules (59). Abbreviations: ATP, adenosine triphosphate; ssrA, small stable RNA A.
Figure 5
Figure 5
Unfolding trajectories of protein substrates by ClpXP. (a) Rips corresponding to the cooperative unfolding of filamin A domains. The black arrow indicates the presence of a transient unfolding intermediate (7). (b) Green fluorescent protein (GFP) unfolding events were observed as a rip-transition-rip sequence, indicating the presence of an unfolding intermediate (black arrow). Numbers correspond to the sequence of events during GFP unfolding illustrated in panel c. (c) Model of GFP unfolding by ClpXP. ❶ After several unfolding attempts, ClpXP successfully extracts β-strands 11 through 7, generating an unfolding GFP intermediate. ❷ The remaining metastable intermediate unfolds spontaneously after ~180 ms. During the lifetime of the GFP intermediate, it is possible to monitor polypeptide translocation by ClpXP. ❸ After the complete unfolding of GFP, ClpXP continues translocating the unraveled polypeptide (59). Abbreviation: pN, piconewton; τ, lifetime.
Figure 6
Figure 6
Translocation velocity and stepping behavior of ClpXP. (a) Pause-free translocation velocity in nanometers per second as a function of force. Red and blue squares correspond to velocities for ClpX and ClpXP, respectively, here and in panel b. (b) Pause-free translocation velocity in amino acids (aa) per second as a function of force. (c,d ) High-resolution data clearly displayed 2-nm or 3-nm steps during polypeptide translocation by ClpXP (c and d, respectively). Raw data ( gray) were obtained at 2 kHz. The raw data were filtered down to 50 Hz (blue) and were fitted using a t-test step-detection algorithm (solid black lines). Figure adapted with permission from Reference .

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback