BCH 4122 Lecture 9: Lecture 9 Notes for Engineering an improved light-2

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rubyswan138

29 Mar 2018

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Notes for lecture 9 engineering an improved light-induced dimer (ilid) for controlling the localization and activity of signaling proteins. Significance (cid:862)photoa(cid:272)ti(cid:448)ata(cid:271)le p(cid:396)otei(cid:374)s a(cid:396)e po(cid:449)e(cid:396)ful tools fo(cid:396) stud(cid:455)i(cid:374)g (cid:271)iologi(cid:272)al processes. Control of protein-protein interactions with light using cry2/cib. Cry2-mcherry fusion protein to a membrane localized cib fragment: reversible light-triggered recruitment of proteins to the plasma membrane occurs within seconds http://pharmacology. ucdenver. edu/tucker/research. htm. Purpose (what they say they did) (cid:862)develop and apply methods for identifying mutations that improve the effectiveness of a light-i(cid:374)du(cid:272)ed di(cid:373)e(cid:396). (cid:863) Associate quickly with signal /dissociate quickly when no signal. (cid:862)full(cid:455) orthogonal to the o(cid:396)ga(cid:374)is(cid:373)s(cid:863) (cid:449)he(cid:396)e used (cid:894)o(cid:396)thogo(cid:374)al = statisti(cid:272)all(cid:455) i(cid:374)depe(cid:374)de(cid:374)t(cid:895) Show a large change in binding affinity upon light stimulation. Not cross-react with other molecules in the cell. Be easily used in a variety of organisms to recruit proteins of interest to each other. Their system: ssra-sspb in the lov2 domain of phototropin 1 from avena sativa (as) Senses blue light and activates a c-terminal kinase domain.

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What are the claims (conclusions) being made in the abstract below? Thank you!!

The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

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BCH 4122 Lecture 9: Lecture 9 Notes for Engineering an improved light-2

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