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An Analysis of the Stability, Aggregation Propensity, and Negative Cooperativity of the Escherichia coli Chaperonin GroEL

dc.contributor.advisorLorimer, George Hen_US
dc.contributor.authorWehri, Sarahen_US
dc.description.abstractSince the discovery of chaperonin GroEL and co-chaperonin GroES, there has been a deluge of literature investigating many aspects of the system. Substrate proteins are protected from aggregation through a cycle of capture, encapsulation, and release made possible through rigid body motions of the GroE system driven by a combination of allosteric controls influenced by nucleotide, potassium and denatured protein termed substrate protein (SP). This dissertation first explores the sequential transition of GroEL that maintains the rings operating in an alternating fashion. To do this, an intra-subunit, inter-domain mutant, GroEL<sub>D83A</sub., was created that lacks the salt-bridge that stabilizes the <bold>T</bold> state. Steady state ATPase assays, stopped-flow fluorescence, and gel filtration chromatography were all used to demonstrate that the <italics>trans</italics> ring must access the <bold>T</bold> state before ligands can be discharged from the <italics>cis</italics> ring. The dual-heptameric ring structure of GroEL and the post-translational assembly of the protein make creating mutants with a mutation within a single subunit of a ring almost impossible, however the ability to do so opens the opportunity for a myriad of experiments that explore the allosteric transitions of GroEL. Two potential recombination methods, acetone treatment and heat treatment, were investigated. Förster resonance energy transfer (FRET) and electrospray ionization mass spectrometry (ESI-MS) were used to study recombination facilitated by such treatments. Recombination using the acetone method resulted in a one-in-one-out subunit exchange, however aggregation complicated the exchange. Heat treatment resulted in exchange of rings. Finally, dynamic light scattering (DLS) was used to investigate stability and aggregation on the chaperonin. It was observed that the chaperonin is stable for over 30 days while incubated continuously at 37°C in sterile buffered solution, however interesting aggregation kinetics are observed upon addition of acetone, the solvent used to strip SP from GroEL during the standard lab purification procedure. GroEL partitions into 10nm and 100nm species that are extremely stable before the appearance of macromolecular aggregates and precipitation is observed.en_US
dc.titleAn Analysis of the Stability, Aggregation Propensity, and Negative Cooperativity of the Escherichia coli Chaperonin GroELen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.subject.pquncontrolledGro ELen_US
dc.subject.pquncontrolledprotein kineticsen_US

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