Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins
| Autor: | Kevin K. Yang, Xiaozhe Ding, Austin J. Rice, Viviana Gradinaru, Emily M LeProust, Claire N. Bedbrook, Siyuan Chen, Frances H. Arnold |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Genetics Rhodopsin Multidisciplinary Recombinant Fusion Proteins fungi Spectral properties Channelrhodopsin Computational biology Biology Embryonic stem cell Schema (genetic algorithms) 03 medical and health sciences HEK293 Cells 030104 developmental biology 0302 clinical medicine Protein sequencing PNAS Plus Channelrhodopsins Membrane protein Humans Integral membrane protein 030217 neurology & neurosurgery Recombination |
| Zdroj: | Proceedings of the National Academy of Sciences. 114 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1700269114 |
| Popis: | Integral membrane proteins (MPs) are key engineering targets due to their critical roles in regulating cell function. In engineering MPs, it can be extremely challenging to retain membrane localization capability while changing other desired properties. We have used structure-guided SCHEMA recombination to create a large set of functionally diverse chimeras from three sequence-diverse channelrhodopsins (ChRs). We chose 218 ChR chimeras from two SCHEMA libraries and assayed them for expression and plasma membrane localization in human embryonic kidney cells. The majority of the chimeras express, with 89% of the tested chimeras outperforming the lowest-expressing parent; 12% of the tested chimeras express at even higher levels than any of the parents. A significant fraction (23%) also localize to the membrane better than the lowest-performing parent ChR. Most (93%) of these well-localizing chimeras are also functional light-gated channels. Many chimeras have stronger light-activated inward currents than the three parents, and some have unique off-kinetics and spectral properties relative to the parents. An effective method for generating protein sequence and functional diversity, SCHEMA recombination can be used to gain insights into sequence-function relationships in MPs. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|