Unfolding and compaction in chaperonin-assisted protein folding followed by single molecule and ensemble FRET

To become biologically active, most proteins need to fold into
precise three dimensional structures. It has been well established
that all the folding information is contained within the primary
structure of a protein. However, the mechanisms utilized by
proteins to avoid sampling the extraordinarily large amount of
possible conformations during their folding process are just
beginning to be understood. Molecular chaperones assist the folding
of newly synthesized and denatured proteins in acquiring their
native state in the crowded intracellular environment. As a nascent
chain leaves the ribosome, it is captured first by the upstream
chaperones and then possibly transferred to the downstream
chaperonins. GroEL-GroES, the Hsp-60 of E.coli, is one of the best
studied chaperone systems. An appreciable amount of data is
available providing information regarding its structure and
function. GroEL encapsulates the substrate into the central cavity
where folding occurs unimpaired by aggregation and unwanted
inter-molecular interactions. Nevertheless, many important aspects
of the GroEL mechanism remain to be addressed. Some of the open
questions we have addressed in this study include: In what
conformation does a substrate protein bind to the apical domains of
GroEL; how is it that GroEL is able to accelerate the rate of
folding of certain proteins, and how do the conformational
properties of the substrate change as it undergoes repeated
cycling. By using ensemble FRET and Sp-FRET (Single
Pair-Fluorescence Resonance Energy Transfer), we have probed the
conformation of the model substrate DM-MBP (Double Mutant Maltose
Binding Protein) during different stages of the functional cycle of
GroEL. With Sp-FRET coupled to PIE (Pulsed Interleaved Excitation),
we have been able to explore the heterogeneity of the GroEL bound
substrate protein and observed a bimodal conformational
distribution. One of the two populations is as compact as the
native state, whereas the other is as extended as the unfolded
protein in denaturant. This unfolding is a local phenomena and can
also be observed when the substrate is transferred from DnaK/J
system (bacterial Hsp70) to GroEL, indicating the possibility of
the existence of this conformational heterogeneity in vivo as the
protein follows the cellular chaperone pathway. Subsequent to GroEL
binding, there is a transient expansion of the protein upon binding
of ATP to GroEL, followed by compaction when GroES triggers the
encapsulation of the protein inside the chaperonin cage. This
transient expansion is however found not to be a necessary event
for the rate acceleration of DM-MBP folding, since ADP-AlFx
(transition state analogue of ATP hydrolysis) results in a much
slower rate of expansion, which does not cause a change in the
folding rate. Anisotropy measurements, probing the freedom of
motion of different regions of the GroEL bound protein, revealed
that there is a segmental release of the substrate protein from the
GroEL surface upon binding of ATP and GroES. As a consequence, the
hydrophobic collapse of the protein upon encapsulation by GroES
follows a step-wise mechanism. In this process, less hydrophobic
regions are released upon binding of ATP, prior to more hydrophobic
ones which are released only by GroES binding. Thus, the order of
Hydrophobic collapse is reversed as compared to spontaneous folding
possibly resulting in conformationally different folding
intermediates. Evidence that the folding pathway inside the cage
differs from that of spontaneous folding was obtained by observing
the effect of external perturbations (e.g. mutations in substrate
protein and use of different solvent conditions) on the rate of
spontaneous and GroEL assisted folding reactions. These two folding
reactions respond differently to the introduced perturbations.
Kinetic data obtained from ensemble FRET measurements suggest that
the conformation of refolding intermediate is altered by the GroEL
cavity, which leads to a folding pathway that is different from the
spontaneous refolding pathway. In summary, this study revealed
significant novel aspects of the GroEL folding mechanism and
provided insights into the basis of rate acceleration of the
substrate protein by the chaperonin. This work may thus contribute
to advance our fundamental knowledge of the chaperonin system and
the basic mechanism of protein folding.