| Chapter title |
Construction of Allosteric Protein Switches by Alternate Frame Folding and Intermolecular Fragment Exchange
|
|---|---|
| Chapter number | 2 |
| Book title |
Synthetic Protein Switches
|
| Published in |
Methods in molecular biology, March 2017
|
| DOI | 10.1007/978-1-4939-6940-1_2 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6938-8, 978-1-4939-6940-1
|
| Authors |
Jeung-Hoi Ha, Stewart N. Loh |
| Editors |
Viktor Stein |
| Abstract |
Alternate frame folding (AFF) and protein/fragment exchange (FREX) are related technologies for engineering allosteric conformational changes into proteins that have no pre-existing allosteric properties. One of their chief purposes is to turn an ordinary protein into a biomolecular switch capable of transforming an input event into an optical or functional readout. Here, we present a guide for converting an arbitrary binding protein into a fluorescent biosensor with Förster resonance energy transfer output. Because the AFF and FREX mechanisms are founded on general principles of protein structure and stability rather than a property that is idiosyncratic to the target protein, the basic design steps-choice of permutation/cleavage sites, molecular biology, and construct optimization-remain the same for any target protein. We highlight effective strategies as well as common pitfalls based on our experience with multiple AFF and FREX constructs. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 10 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 4 | 40% |
| Student > Postgraduate | 2 | 20% |
| Professor | 1 | 10% |
| Unspecified | 1 | 10% |
| Student > Ph. D. Student | 1 | 10% |
| Other | 1 | 10% |
| Readers by discipline | Count | As % |
|---|---|---|
| Agricultural and Biological Sciences | 3 | 30% |
| Biochemistry, Genetics and Molecular Biology | 3 | 30% |
| Unspecified | 1 | 10% |
| Environmental Science | 1 | 10% |
| Unknown | 2 | 20% |