Role of Denatured-State Properties in Chaperonin Action Probed by Single-Molecule Spectroscopy

Abstract

The bacterial chaperonin GroEL/GroES assists folding of a broad spectrum of denatured and misfolded proteins. Here, we explore the limits of this remarkable promiscuity by mapping two denatured proteins with very different conformational properties, rhodanese and cyclophilin A, during binding and encapsulation by GroEL/GroES with single-molecule spectroscopy, microfluidic mixing, and ensemble kinetics. We find that both proteins bind to GroEL with high affinity in a reaction involving substantial conformational adaptation. However, whereas the compact denatured state of rhodanese is encapsulated efficiently upon addition of GroES and ATP, the more expanded and unstructured denatured cyclophilin A is not encapsulated but is expelled into solution. The origin of this surprising disparity is the weaker interactions of cyclophilin A with a transiently formed GroEL-GroES complex, which may serve as a crucial checkpoint for substrate discrimination.

Integrated View of Internal Friction in Unfolded Proteins from Single-Molecule FRET, Contact Quenching, Theory, and Simulations

“…2B, Inset and SI Appendix). With increasing denaturant concentration, τ r first decreases from 95 ± 15 ns at 0.5 M GdmCl to 45 ± 7 ns at 4 M GdmCl, and then increases again, reaching 60 ± 9 ns at 7 M GdmCl, a behavior similar to that of other proteins of comparable size and sequence composition (10,12,16,27). Remarkably, τ r calculated directly from the MD trajectory of unfolded protein L yields a value of ∼110 ns for the dominant dynamic component ( Fig.…”

“…FRET-FCS reports on protein dynamics that result in distance fluctuations between the dyes (10,12,16,27). The correlation curves for unfolded protein L (Fig.…”

“…Förster resonance energy transfer (FRET) combined with nanosecond fluorescence correlation spectroscopy (nsFCS) on several unfolded proteins have revealed reconfiguration dynamics of 30-to 100-residue chain segments on time scales between about 10 ns and 100 ns (10,12,16,27). In contrast, loop formation detected in contact-based measurements between Cys and Trp in a 10-residue segment within unfolded protein L was reported to occur on much slower time scales, between 1 μs and 10 μs (18).…”

Quantifying kinetics from time series of single-molecule Förster resonance energy transfer efficiency histograms

“…A method that is frequently used for the analysis of multivariate experimental data and that is largely model-free is singular value decomposition (SVD) [36]. It has recently also started to be employed for the analysis of single-molecule experiments [9,19,20]…”

“…A method that is frequently used for the analysis of multivariate experimental data and that is largely model-free is SVD [36]. It has recently also started to be employed for the analysis of single-molecule experiments [9,19,20]. The procedures for decomposing H F and C presented above require a priori knowledge of the number of species present in the reaction and, additionally, information on either the shapes of their individual E histograms or on the time courses of their relative concentrations.…”