Developing and applying quantitative high-throughput microfluidics to elucidate enzyme functional architecture
Abstract/Contents
- Abstract
- Protein enzymes are Nature's most proficient catalysts, accelerating rates of chemical transformation to the timescales of life. They are also central to achieving clean and efficient industrial processes and to realizing next-generation precision health. Our ability to target and engineer enzymes, and predict how their variation alters biological function and dysfunction, ultimately requires that we describe them quantitatively and at massive scale. However, classical methods of enzymology are too laborious and time consuming to make measurements at the required scale. Here, I detail the development of High-Throughput Microfluidic Enzyme Kinetics (HT-MEK), a technology meeting this need through the integrated expression, purification, and quantitative measurement of enzymes at a hundred-fold the scale and at one-hundredth the time of classical methods. HT-MEK can be generally applied to any enzyme with a fluorescent readout of function. We used HT-MEK to deeply study mutations at each position in an efficient phosphatase, PafA, measuring > 6,000 kinetic and thermodynamic constants for > 1000 mutants using multiple substrates, inhibitors, and under varied conditions. We found that positions with mutational effects on different functions compose discrete regions, PafA's functional architecture, extending to surfaces that may be harnessed for rational engineering. We also discovered widespread misfolding to inactive states, a potentially-common feature among highly-stable enzymes. Fundamentally, HT-MEK provides ground truth physical constants, a basis for accurate next-generation enzyme function prediction algorithms. These constants are also needed to understand how enzyme disfunction manifests in human disease. Towards this, I present preliminary work extending HT-MEK to the large-scale characterization of variants of the human phosphatase SHP2, some of which can cause cancer. Measurements of biophysical constants describing catalysis, stability, and inhibition for more than 100 SHP2 variants (> 200 total mutants) revealed discrete regions of activation and destabilization relevant for explaining known in vivo effects and predicting new ones. HT-MEK and the described studies of PafA and SHP2 advance classical enzymology into the post-genomic era, and are a basis for new efforts to engineer enzymes, realize precision medicine by broadly describing allelic variation, and more deeply understand Nature.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Mokhtari, Daniel Alexander |
|---|---|
| Degree supervisor | Fordyce, Polly |
| Degree supervisor | Herschlag, Daniel |
| Thesis advisor | Fordyce, Polly |
| Thesis advisor | Herschlag, Daniel |
| Thesis advisor | Kobilka, Brian K |
| Thesis advisor | Li, Lingyin |
| Thesis advisor | Walsh, Christopher |
| Degree committee member | Kobilka, Brian K |
| Degree committee member | Li, Lingyin |
| Degree committee member | Walsh, Christopher |
| Associated with | Stanford University, Department of Biochemistry |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Daniel A. Mokhtari. |
|---|---|
| Note | Submitted to the Department of Biochemistry. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/ht233ft1963 |
Access conditions
- Copyright
- © 2021 by Daniel Alexander Mokhtari
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...