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Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

Bedbrook, Claire N. and Rice, Austin J. and Yang, Kevin K. and Ding, Xiaozhe and Chen, Siyuan and LeProust, Emily M. and Gradinaru, Viviana and Arnold, Frances H. (2017) Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins. Proceedings of the National Academy of Sciences of the United States of America, 114 (13). E2624-E2633. ISSN 0027-8424. PMCID PMC5380088. http://resolver.caltech.edu/CaltechAUTHORS:20170310-130542741

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://dx.doi.org/10.1073/pnas.1700269114DOIArticle
http://www.pnas.org/content/114/13/E2624PublisherArticle
http://www.pnas.org/content/114/13/E2624?tab=dsPublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380088/PubMed CentralArticle
ORCID:
AuthorORCID
Gradinaru, Viviana0000-0001-5868-348X
Arnold, Frances H.0000-0002-4027-364X
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.
Group:Caltech Center for Environmental Microbial Interactions (CEMI), Rosen Bioengineering Center
Funders:
Funding AgencyGrant Number
Caltech Beckman InstituteUNSPECIFIED
Arnold and Mabel Beckman FoundationUNSPECIFIED
NIHR21MH103824
Army Research Office (ARO)W911F-09-0001
NIH Predoctoral FellowshipF31MH102913
NIH Predoctoral FellowshipF32GM116319
Donna and Benjamin M. Rosen Bioengineering CenterUNSPECIFIED
Subject Keywords:membrane proteins | channelrhodopsin | structure-guided recombination | chimeragenesis
PubMed Central ID:PMC5380088
Record Number:CaltechAUTHORS:20170310-130542741
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20170310-130542741
Official Citation:Claire N. Bedbrook, Austin J. Rice, Kevin K. Yang, Xiaozhe Ding, Siyuan Chen, Emily M. LeProust, Viviana Gradinaru, and Frances H. Arnold Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins PNAS 2017 114 (13) E2624-E2633; published ahead of print March 10, 2017, doi:10.1073/pnas.1700269114
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:75032
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:10 Mar 2017 22:07
Last Modified:14 May 2019 20:01

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