title: 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.
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Claire N
authors.initials: CN
authors.lastname: Bedbrook
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Austin J
authors.initials: AJ
authors.lastname: Rice
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Kevin K
authors.initials: KK
authors.lastname: Yang
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Xiaozhe
authors.initials: X
authors.lastname: Ding
authors.affiliation: Twist Bioscience, San Francisco, CA 94158.
authors.forename: Siyuan
authors.initials: S
authors.lastname: Chen
authors.affiliation: Twist Bioscience, San Francisco, CA 94158.
authors.forename: Emily M
authors.initials: EM
authors.lastname: LeProust
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Viviana
authors.initials: V
authors.lastname: Gradinaru
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125; fha@cheme.caltech.edu.|Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125.
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000075402:Channelrhodopsins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000075402:Channelrhodopsins; D011993:Recombinant Fusion Proteins; D012243:Rhodopsin
keywords: channelrhodopsin; chimeragenesis; membrane proteins; structure-guided recombination