"The Genotype-Phenotype Landscape of an Allosteric Protein" with David Ross, National Institute of Standards and Technology (Online Seminar)
Title: The Genotype-Phenotype Landscape of an Allosteric Protein
Speaker: David Ross, National Institute of Standards and Technology (NIST)
Hosted by: Arpita Upadhyaya & Jeffery Klauda
Allostery is a fundamental biophysical mechanism that underlies cellular sensing, signaling, and metabolism. Yet, because of its structurally distributed nature, a quantitative understanding of allosteric genotype-phenotype relationships remains elusive. To provide the large-scale data needed to inform predictive models of allosteric function, we have developed a method to quantitatively measure the dose-response curves and matching DNA sequences for every variant in a large library of allosteric sensor proteins. As a first demonstration of the method, we created a library of nearly 100,000 variants of the LacI genetic sensor in E. coli. We used an automated culture protocol and a combination of long- and short-read DNA sequencing to measure the dose-response curves and corresponding DNA sequences for every variant. With the resulting data, we can identify LacI genotypes with precisely targeted dose-response. For example, we engineered sensors with sensitivities (i.e. EC50) spanning 3 orders of magnitude with a 1.25-fold accuracy. In addition, the resulting data provide a quantitative map of the effect of amino acid substitutions on LacI allostery and reveal systematic sequence-structure-function relationships. We find that in many cases, allosteric phenotypes can be quantitatively predicted with additive or neural-network models, but unpredictable changes also occur. For example, we were surprised to find many LacI variants with phenotypes that differ qualitatively from the wild-type, including variants with an inverted phenotype and others with a never-before-seen band-stop (on-off-on) phenotype. These qualitative phenotypic changes are particularly interesting because they can provide specific insight into the biophysics of protein allostery and because they highlight the capability for evolutionary innovation that is inherent to allosteric biomolecules.
Seminars start at 4:00 pm and refreshments served at 3:45 pm. All seminars are held in the Conference Room (1116) of the Institute for Physical Science and Technology (IPST) Building, unless otherwise noted.