Revisiting allostery in lac repressor / Matthew Alexander Stetz.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Stetz, Matthew Alexander, author.

Contributor:

Wand, A. Joshua (Andrew Joshua), degree supervisor.

Lewis, Mitchell, degree committee member.

Lynch, Kristen W., degree committee member.

Murakami, Kenji, degree committee member.

Sharp, Kim A., degree committee member.

Van Duyne, Gregory D., degree committee member.

Zuiderweg, Erik R. P., degree committee member.

University of Pennsylvania. Department of Biochemistry and Molecular Biophysics, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Biochemistry and Molecular Biophysics.

Biochemistry and Molecular Biophysics--Penn dissertations.

Local Subjects:

Penn dissertations--Biochemistry and Molecular Biophysics.

Biochemistry and Molecular Biophysics--Penn dissertations.

Physical Description:

xiv, 225 leaves : illustrations ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2016.

Summary:

Lac repressor (LacI) is an allosterically regulated transcription factor which controls expression of the lac operon in bacteria. LacI consists of a DNA-binding domain (DBD) and regulatory domain (RD), connected by a linker called the "hinge". Binding of a small molecule inducer to the RD relieves repression through what is presumed to be a series of conformational changes mediated through the hinge. Despite decades of study, our understanding of this allosteric transition remains incomplete-mostly inferred from partial crystal structures and low-resolution scattering studies. In principle, solution-NMR could provide structural and dynamical information unobtainable by X-ray methods. However, due to LacI's high molecular weight, low solubility, and transient stability, such studies have been limited to the non-allosteric, isolated DBD. Here, we present a solution-NMR study of the changes in structure and dynamics that underlie the allosteric transition of intact LacI. First, an optimized expression system is presented which enables characterization of LacI using NMR methodologies for high molecular weight proteins. Next, alternative NMR data sampling methods are implemented and further extended to overcome the low-solubility and transient stability limitations. Finally, these developments are combined to characterize LacI in each of its functional states. It is shown that the RD but not the DBD of apo LacI exists in an equilibrium between induced and repressed states with exchange occurring on the µs-ms timescale. Inducer binding in the absence of operator mostly quenches exchange but does not result in structural changes in the hinge or DBD. Conformational dynamics detected in the induced state are shown to be localized to a "network" of RD residues previously characterized to be critical for allostery. These dynamics are shown to be quenched in non-allosteric mutants which suggests functional relevance. Operator binding results in globally quenched dynamics and dramatic changes to the structure of the hinge. Inducer binding in the presence of operator results in only minor structural perturbation in the hinge and DBD. However, dynamics are shown to be activated in the RD. These results suggest that conformational dynamics may be critical to the allosteric transition of LacI.

Notes:

Ph. D. University of Pennsylvania 2016.

Department: Biochemistry and Molecular Biophysics.

Supervisor: A. Joshua Wand.

Includes bibliographical references.