Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

Abstract

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin Legend. The GroEL/ES-mediated folding of the model substrate protein DM-MBP is monitored by single-molecule FRET. Folding intermediate bound to GroEL shows a low FRET efficiency distribution. Upon refolding, DM-MBP converts to its compact native structure that exhibits a high FRET efficiency distribution. Featured ArƟcle 0022-2836

In vivo aspects of protein folding and quality control

“…This step is accompanied by extensive conformational changes of the GroEL subunits that enlarge the ring cavity and alter its physical properties from hydrophobic to hydrophilic (80,82). Proteins up to~60 kDa in size are now free to fold in the chaperonin nanocage for the time it takes GroEL to hydrolyze its bound ATP to ADP (~2 s at 37°C) (85). Binding of ATP to the opposite GroEL ring then induces an allosteric signal that causes ADP to dissociate and GroES to unbind.…”

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…This model also implies that the substrate protein is capable of reaching native state in a biologically relevant timescale provided that the aggregation is prevented and that the folding energy landscape of the substrate protein remains unaltered in the presence of GroEL. However, the second model, known as active caging model proposed by Ulrich Hartl and colleagues, contradicts passive caging model by proposing that encapsulation by chaperonin accelerates folding of the substrate proteins in addition to preventing aggregation (Brinker et al 2001;Tang et al 2006;Chakraborty et al 2010;Gupta et al 2014). This model suggests that by accelerating folding of certain substrate proteins, GroEL can modulate their folding energy landscape.…”

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

“…The mechanism underlying GroE-assisted protein folding remains controversial, particularly whether GroE acts as a passive antiaggregation cage (8,9) or actively accelerates protein folding (10,11); however, both of these are known to require efficient substrate protein encapsulation. The C-terminal tails of GroEL have recently been suggested to play a key role in efficient protein encapsulation (12).…”