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Proc Natl Acad Sci U S A. 2017 Mar 28;114(13):E2624-E2633. doi: 10.1073/pnas.1700269114. Epub 2017 Mar 10.

Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins.

Author information

1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
2
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
3
Twist Bioscience, San Francisco, CA 94158.
4
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125; fha@cheme.caltech.edu.

Abstract

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.

KEYWORDS:

channelrhodopsin; chimeragenesis; membrane proteins; structure-guided recombination

PMID:
28283661
PMCID:
PMC5380088
DOI:
10.1073/pnas.1700269114
[Indexed for MEDLINE]
Free PMC Article

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