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An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.

ABSTRACT: Protein evolution proceeds by a complex response of organismal fitness to mutations that can simultaneously affect protein stability, structure, and enzymatic activity. To probe the relationship between genotype and phenotype, we chose a fundamental paradigm for protein evolution, folding, and design, the (??)₈ TIM barrel fold. Here, we demonstrate the role of long-range allosteric interactions in the adaptation of an essential hyperthermophilic TIM barrel enzyme to mesophilic conditions in a yeast host. Beneficial fitness effects observed with single and double mutations of the canonical ??-hairpin clamps and the ?-helical shell distal to the active site revealed an underlying energy network between opposite faces of the cylindrical ?-barrel. We experimentally determined the fitness of multiple mutants in the energetic phase plane, contrasting the energy barrier of the chemical reaction and the folding free energy of the protein. For the system studied, the reaction energy barrier was the primary determinant of organism fitness. Our observations of long-range epistatic interactions uncovered an allosteric pathway in an ancient and ubiquitous enzyme that may provide a novel way of designing proteins with a desired activity and stability profile.

SUBMITTER: Chan YH

PROVIDER: S-EPMC7454521 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.

Chan Yvonne H YH Zeldovich Konstantin B KB Matthews Charles R CR

Protein science : a publication of the Protein Society 20200720 9

Protein evolution proceeds by a complex response of organismal fitness to mutations that can simultaneously affect protein stability, structure, and enzymatic activity. To probe the relationship between genotype and phenotype, we chose a fundamental paradigm for protein evolution, folding, and design, the (βα)<sub>8</sub> TIM barrel fold. Here, we demonstrate the role of long-range allosteric interactions in the adaptation of an essential hyperthermophilic TIM barrel enzyme to mesophilic conditi ...[more]

PMID: 32643222

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Project description:Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (?/?)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (?G°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ?G° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.

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An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.

Publications

An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.

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