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Sharma, Shruti (2007): Unfolding and compaction in chaperonin-assisted protein folding followed by single molecule and ensemble FRET. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



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.