A thermodynamic and structural dissection of cooperativity in natural and designed tetratricopeptide repeat proteins
Embargo until
2019-08-01
Date
2015-07-23
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Johns Hopkins University
Abstract
A major goal in modern biophysics has been to thermodynamically characterize macromolecular systems to enable an energetic description of biological processes. Despite considerable effort, the thermodynamic nature of cooperativity in protein folding is not fully understood. The primary reason for this is due to the apparent “two-state” folding behavior at equilibrium, lacking intermediates. To grasp cooperativity in protein folding, one needs to thermodynamically quantify intermediates. Repeat proteins have proven to be excellent systems to thermodynamically describe these intermediates.
My work focuses on developing a thermodynamic description of protein folding cooperativity using two experimental systems of tetratricopeptide repeat proteins (TPRs/nPRs). nPRs consist of a repetitive n-residue motif, which forms antiparallel A- and B-helices. While our lab and others have had similar objectives on other repeat systems, my contributions have been 1) to develop and apply a statistical framework for analyzing heterogeneous systems, 2) to thermodynamically characterize units of structure smaller than whole repeats, 3) to ascribe structural bases to measured energetics, and 4) to understand mechanisms of stabilization by consensus design by studying a natural repeat protein system.
Consensus ankyrin and leucine rich repeat proteins are characterized by very unfavorable intrinsic folding free energies and strong interfacial interactions. In contrast, isolated c34PRs have a Keq ~1 for folding, while interactions between helices are more modest. To determine the molecular origins of cooperativity in c34PRs, in Chapter 2, I develop and present a single helix heteropolymeric Ising model capable of resolving energies of half repeat units in nPR systems. I applied this model to consensus TPRs (c34PRs), and quantified energetics of single α-helices, as well as inter- and intra-repeat interfaces. While c34PR helices have different intrinsic energies, inter- and intra-repeat interfaces are similar in energy, despite structural differences.
In Chapters 3 and 4, I studied a naturally occurring 42PR with a longer sequence motif. I solved the X-ray crystal structure of five tandem 42PRs, and determined the longer sequence motif to result in helical extensions of the canonical helices of 34PRs. I quantified folding cooperativity in this system by using nearest-neighbor models in Chapter 4. 42PRs are more cooperative than 34PRs, due to increased magnitudes of both intrinsic and interfacial energies. Point substitutions suggest a single hydrogen bond in Pa 42PRs to contribute significantly to interfacial stability.
Description
Keywords
protein folding, biophysics, thermodynamics, Ising, nearest-neighbor, structure