Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins
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.
Additional Information
© 2017 National Academy of Sciences. Contributed by Frances H. Arnold, February 13, 2017 (sent for review January 6, 2017; reviewed by Hagan Bayley and David Drew) Published online before print March 10, 2017. We thank Dr. John Bedbrook for critical reading of the manuscript. Imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. This work is funded by the National Institute for Mental Health Grant R21MH103824 (to V.G. and F.H.A.); the Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research for technology development and broad dissemination: www.beckmaninstitute.caltech.edu/clover.shtml (V.G.); and the Institute for Collaborative Biotechnologies through Grant W911F-09-0001 from the US Army Research Office (to F.H.A.). C.N.B. and A.J.R. are funded by Ruth L. Kirschstein National Research Service Awards F31MH102913 and F32GM116319. K.K.Y. is a trainee in the Caltech Biotechnology Leadership Program and has received financial support from the Donna and Benjamin M. Rosen Bioengineering Center. The content is solely the responsibility of the authors and does not necessarily reflect the position or policy of the National Center for Research Resources, the National Institutes of Health, or the Government, and no official endorsement should be inferred. C.N.B. and A.J.R. contributed equally to this work. Author contributions: C.N.B., A.J.R., V.G., and F.H.A. designed research; C.N.B., A.J.R., and X.D. performed research; S.C. and E.M.L. contributed synthesized ChR genes; C.N.B. and A.J.R. analyzed data; and C.N.B., A.J.R., K.K.Y., and F.H.A. wrote the paper. Reviewers: H.B., University of Oxford; and D.D., Stockholm University. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1700269114/-/DCSupplemental.Attached Files
Published - PNAS-2017-Bedbrook-E2624-33.pdf
Supplemental Material - pnas.1700269114.sd01.csv
Supplemental Material - pnas.1700269114.sd02.csv
Supplemental Material - pnas.1700269114.sd03.txt
Supplemental Material - pnas.1700269114.sd04.txt
Supplemental Material - pnas.1700269114.sd05.txt
Supplemental Material - pnas.201700269SI.pdf
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Additional details
- PMCID
- PMC5380088
- Eprint ID
- 75032
- DOI
- 10.1073/pnas.1700269114
- Resolver ID
- CaltechAUTHORS:20170310-130542741
- Caltech Beckman Institute
- Arnold and Mabel Beckman Foundation
- NIH
- R21MH103824
- Army Research Office (ARO)
- W911F-09-0001
- NIH Predoctoral Fellowship
- F31MH102913
- NIH Predoctoral Fellowship
- F32GM116319
- Donna and Benjamin M. Rosen Bioengineering Center
- Created
-
2017-03-10Created from EPrint's datestamp field
- Updated
-
2022-03-29Created from EPrint's last_modified field
- Caltech groups
- Caltech Center for Environmental Microbial Interactions (CEMI), Rosen Bioengineering Center