Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics
| Autor: | Polly M. Fordyce, Chiara Sabatti, Daniel A. Mokhtari, Scott A. Longwell, Daniel Herschlag, Mason J. Appel, Eyal Akiva, Fanny Sunden, Craig J. Markin |
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| Rok vydání: | 2020 |
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Models
Molecular Protein Folding Protein Conformation Microfluidics Mutant Kinetics Computational biology Flavobacterium Phosphates 03 medical and health sciences Protein structure 0302 clinical medicine Catalytic Domain Enzyme kinetics Throughput (business) 030304 developmental biology chemistry.chemical_classification 0303 health sciences Multidisciplinary Hydrolysis Alkaline Phosphatase Oxygen Enzyme chemistry Mutation Biocatalysis Biophysics Thermodynamics Alkaline phosphatase Protein folding 030217 neurology & neurosurgery Function (biology) |
| Popis: | Go big or you'll get lost Rational mutagenesis is a common approach to investigating or engineering enzyme function in vitro, but the ease with which one can manipulate protein sequences belies many pitfalls in connecting sparse activity data to an enzyme's true functional landscape. Using a high-throughput platform, Markin et al. expressed, purified, and performed an array of kinetic measurements on a target esterase, collecting data from >1000 mutations spanning the entire protein (see the Perspective by Baumer and Whitehead). Protein misfolding into an inactive state, rather than decreased equilibrium stability, was a crucial factor in negatively affected variants spread throughout the protein. When combined with prior mechanistic understanding and structures, four “functional components” help to rationalize the otherwise complex spatial pattern of effects of mutations on different aspects of enzyme function, all of which would be invisible from mutagenesis of just a few residues. Science , abf8761, this issue p. eabf8761 ; see also abj8346, p. 391 |
| Databáze: | OpenAIRE |
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