| The term allostery describes indirect interactions between distant binding sites that are modulated by allosteric ligands. Allostery plays a fundamental role in many cellular processes such as cell signaling, metabolism and replication. Allosteric regulation is often described by the classical concerted (MWC) and sequential (KNF) models. Both models, however, are unable to provide insight into the communication pathways between sites and it is usually difficult to distinguish between these models experimentally. In my thesis, I developed several approaches to study pathways of allosteric communication and applied them to chaperonins that are well-known examples for multimeric allosteric proteins. Chaperonins play an essential role in protein folding in both prokaryotic and eukaryotic organisms. I studied the allosteric mechanisms of two members of the chaperonin family -- the prokaryotic homo-oligomeric GroEL and the eukaryotic hetero-oligomeric CCT (cytosolic chaperonin containing t-complex polypeptide 1). The allosteric mechanisms of GroEL and CCT were studied using structural mass-spectrometry (MS), steady state kinetics and single particle reconstruction electron-microscopy (EM).;Chapter 1 focuses on the allosteric mechanism of the prokaryotic chaperonin GroEL. In this part, I present a new approach, based on structural MS and data analysis, to distinguish between possible allosteric models using ensemble measurements in bulk protein solution. The structural MS data provided the required resolution to identify the co-existing populations of GroEL with different numbers of bound ATPs in an ATP-dependent manner. From these distributions it was possible to determine all the binding constants and the ATP loading pathway. Moreover, knowing the values of the 14 ATP binding constants (K i) of GroEL provided a way to discriminate between various allosteric mechanisms, which supported the nested model. The results obtained in this study were summarized in an article entitled: "Allosteric mechanisms can be distinguished using structural mass spectrometry". PNAS (2013) 110, 7235-9..;Chapter 2 focuses on the eukaryotic CCT chaperonin. Our main goal was to identify the way its conformational change propagates around the ring. The conformational wave is believed to be important for the function of the chaperonin. In order to address this question, we utilized two experimental approaches: Arrhenius analyses and single-particle EM. In both cases, we utilized purified WT CCT and mutants with an identical mutation in the ATP binding site introduced in each subunit in turn (Amit et al. JMB 401, 532-43 (2010)). The Arrhenius analyses showed that we can rank the mutants according to their activation energies (MA6 > MA3 > MA5 > MA2, MA7, MA8, WT) and suggested a sequential clockwise pathway. The second experimental approach we utilized was single-particle EM, which captured the conformational effects of the ATP binding and hydrolysis in WT CCT and in the mutants. Comparing between CCT's, with and without ATP, revealed distinct effects of the mutation on the conformational wave. The extent and directionality of the conformational wave seems to depend on the identity of the mutated subunit. For example, when a mutation was introduced into subunit 6, a conformational change was observed in five out of the total eight subunits while in the WT, a change was observed in seven of the subunits. As a whole, the two approaches seem to indicate that CCT6 has an important role in signal propagation in the CCT complex. |
