Published August 3, 2020
| Version v1
Journal article
Open
Incorporation of Sensing Modalities Into De Novo Designed Fluorescence-activating Proteins
Authors/Creators
-
Jason C. Klima1
- Lindsey A. Doyle2
- Justin Daho Lee3
- Michael Rappleye4
- Lauren A. Gagnon5
- Min Yen Lee5
- Emilia P. Barros6
- Anastassia A. Vorobieva1
- Jiayi Dou1
- Samantha Bremner4
- Jacob S. Quon7
- Cameron M. Chow1
- Lauren Carter1
- David L. Mack4
- Rommie E. Amaro6
- Joshua C. Vaughan5
- Andre Berndt3
- Barry L. Stoddard2
- David Baker1
- 1. Department of Biochemistry, University of Washington, Seattle, WA, USA.
- 2. Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
- 3. Molecular Engineering Ph.D. Program, University of Washington, Seattle, WA, USA.
- 4. Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA.
- 5. Department of Chemistry, University of Washington, Seattle, WA, USA.
- 6. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
- 7. Institute for Protein Design, University of Washington, Seattle, WA, USA.
Description
Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.
Files
Figure_5d_after_photobleach_1_raw.tif
Files
(20.2 GB)
| Name | Size | |
|---|---|---|
|
md5:2992a11f3705e03b2961d2e34393ed41
|
1.2 MB | Download |
|
md5:83b676687347c930a592664d73c407c7
|
1.2 MB | Download |
|
md5:1ac3299dd682201aaae5431b650b6013
|
1.2 MB | Download |
|
md5:a0e243122311add13b32158486f7bccb
|
1.2 MB | Download |
|
md5:0edaab1f3e1493d91521f504fa4cbe0e
|
1.2 MB | Download |
|
md5:7b6ac0f1021309e0f2ce3e8499c4153f
|
1.2 MB | Download |
|
md5:82016d238126a7d6d2ec3fca98f40812
|
1.2 MB | Download |
|
md5:1a3682624b462848841dcdbc392bc11f
|
1.2 MB | Download |
|
md5:44081317327a37d603aeff7525e33f65
|
1.2 MB | Download |
|
md5:8c9bc2303c9d58f150ec0d7764c42c52
|
1.2 MB | Download |
|
md5:bfd5388548062d63cea08cee8e485b7e
|
1.2 MB | Download |
|
md5:313412147ea326cd50c82e1885825fc5
|
1.2 MB | Download |
|
md5:334775372175829adb79fdbb0dc003e1
|
1.2 MB | Download |
|
md5:f28ef502ecb1463bbcae10d5add39e75
|
1.2 MB | Download |
|
md5:addd1f0669703578a1db8186a8005f38
|
1.2 MB | Download |
|
md5:71ceaf57329daf870a00253f78175fb2
|
1.2 MB | Download |
|
md5:9ae235bddd33ae1146aaa94293e1c98c
|
1.2 MB | Download |
|
md5:69dcca5beecf7eaf634bbd92305d438c
|
1.2 MB | Download |
|
md5:4987ecd01fa293c797fa260df01baa6b
|
1.2 MB | Download |
|
md5:9baa8e5ac5fe059bc0c5f77ff86397e0
|
1.2 MB | Download |
|
md5:6eaf683c596d024c89b38c3aaf8a201e
|
1.2 MB | Download |
|
md5:b45cd5b3436889e4a9da9f939e566798
|
1.2 MB | Download |
|
md5:a70186ae5eb7912a36c966e495eef8cf
|
1.2 MB | Download |
|
md5:8e92c05176e1d880866f79c5071044df
|
1.2 MB | Download |
|
md5:12aa8b7499522f9945903daf6fb72ae3
|
1.2 MB | Download |
|
md5:5d127b83bd3ea9ce49dc4ace81d23b26
|
1.2 MB | Download |
|
md5:5f4c97f8b3c94fe2dc0bf8782baac0de
|
1.2 MB | Download |
|
md5:efc98a2606244ab30f8ccd84ee11d913
|
1.2 MB | Download |
|
md5:71697bdb54323a1b1fe576da385d1996
|
1.2 MB | Download |
|
md5:93772fc9cd9813434c384c42d519938f
|
1.2 MB | Download |
|
md5:f67a595b18a77b153bc3958faf903d68
|
1.2 MB | Download |
|
md5:a939a8c241aeb60d77d4d856a2eba30c
|
1.2 MB | Download |
|
md5:845b772084c879917c97945f81e62c55
|
1.2 MB | Download |
|
md5:389680dad59b20d9e4482149d290b6f2
|
1.2 MB | Download |
|
md5:0fd246b5f9d3c7e5229a30a6f46ccc5c
|
1.2 MB | Download |
|
md5:6342dac0c5a1ef9b3257d5c0e8c61917
|
1.2 MB | Download |
|
md5:03364811d48085fd0f1d3c4059e0ff92
|
1.2 MB | Download |
|
md5:542162a7c85a87ed3bf7efde7d83e22b
|
1.2 MB | Download |
|
md5:92d8cb8206732456d3aeb63bc7eaa955
|
1.2 MB | Download |
|
md5:e4d063dc1de6663c3f38545bacd6d741
|
1.2 MB | Download |
|
md5:0ba45cdd4a3cd191c5477ccafaf8db6c
|
229.0 MB | Download |
|
md5:aceb232ac8106ed66f10fbaaaa5df9e4
|
229.0 MB | Download |
|
md5:9f882d5952c6568e6d70e4b5d8226813
|
229.0 MB | Download |
|
md5:f97a45a1335e9ece08a0b160ebb7f7f0
|
229.0 MB | Download |
|
md5:9915086ae491662cf3eb70dabd8d3090
|
229.0 MB | Download |
|
md5:ed47a6f21a3d5d12834213db2b4d0019
|
229.0 MB | Download |
|
md5:6e79852b64ed793071f6d3453d99023c
|
229.0 MB | Download |
|
md5:b751eaef0375cbc9a4e0536156f306c6
|
229.0 MB | Download |
|
md5:7f2ad983e0d331cbd9140bd2c78d02df
|
229.0 MB | Download |
|
md5:1a04c153ad6a3956c8b4cc50f9256812
|
229.0 MB | Download |
|
md5:cb5750fff574ca0e3943e5a02b34a21d
|
229.0 MB | Download |
|
md5:b51f73a2baf74cebeec257128e625097
|
229.0 MB | Download |
|
md5:8c9574181cc99d1efdc698320aee148f
|
229.0 MB | Download |
|
md5:308957937b125cf3b01a8f9886a29dae
|
229.0 MB | Download |
|
md5:60d3734c14f403f4cc4685ac77c116fb
|
229.0 MB | Download |
|
md5:16626dae5eec0ba7305d6c685e5bf32b
|
229.0 MB | Download |
|
md5:4a54afff09833ca42d1aa366c4fc8d52
|
229.0 MB | Download |
|
md5:2ab0d0f2a5551bce661be4a644620ebb
|
229.0 MB | Download |
|
md5:9ae1837ef586860181f6ff61e1f27b7d
|
229.0 MB | Download |
|
md5:24c95a91b546116ba85fd793f60e43f3
|
229.0 MB | Download |
|
md5:ad02a68d6bc7936b2902f4a8318f6510
|
229.0 MB | Download |
|
md5:f0483a13b7e030545873ff9642d26482
|
229.0 MB | Download |
|
md5:a023279be71d39e8bc88dfec4481395d
|
229.0 MB | Download |
|
md5:15b163ca8fd1a0d71f690f5e5715242d
|
229.0 MB | Download |
|
md5:82d3ac7d56830611494e0e2639aeac8e
|
229.0 MB | Download |
|
md5:32fe6f364e89ff186c1404077d004784
|
229.0 MB | Download |
|
md5:0a2c824899368962d533aca73bcefebe
|
229.0 MB | Download |
|
md5:769accb8fc47e2e3cf69341a416881b2
|
229.0 MB | Download |
|
md5:b4fb823f372e8d8809f351e32a87b513
|
702.5 MB | Preview Download |
|
md5:12747443f206e77581d188b7fa629cb5
|
702.5 MB | Preview Download |
|
md5:8fc59b1d741d7f3f14072e0756a27125
|
702.5 MB | Preview Download |
|
md5:b6389747250f9bab6d75052e8c1b9795
|
196.3 kB | Download |
|
md5:a5835f3b9b2cc4ebce46afa77eb99af1
|
702.5 MB | Preview Download |
|
md5:046008286a2560d580b7804245bae943
|
702.5 MB | Preview Download |
|
md5:33935cccad4406fec9c50304947bcb0a
|
702.5 MB | Preview Download |
|
md5:b81308c3b8cfcd87a7857aeabe388e11
|
241.1 kB | Download |
|
md5:d20a1e0dc82758925aae1df970871ce5
|
702.5 MB | Preview Download |
|
md5:ac26eff461c6a7f8e63cff6b8e4845b5
|
702.5 MB | Preview Download |
|
md5:0cc47f3725792018a25bfe7284d18d7a
|
702.5 MB | Preview Download |
|
md5:e8892cecede8bf75182fd899614a2d0f
|
702.5 MB | Preview Download |
|
md5:9c5b305687b504644c3b95f421021f7f
|
702.5 MB | Preview Download |
|
md5:26711bd3ed7a1821b647971ba2b69471
|
702.5 MB | Preview Download |
|
md5:8ac0d6a36c7c63c779d0d40949860f10
|
702.5 MB | Preview Download |
|
md5:6af184aab4b8c140738caa974f61ed27
|
702.5 MB | Preview Download |
|
md5:7396cf2a319c21a4430ce32144c800ea
|
702.5 MB | Preview Download |
|
md5:4bd8cb53dfaa1175637c7c537177c028
|
702.5 MB | Preview Download |
|
md5:30250c1231e06ed8f089d2fcb3035e6e
|
702.5 MB | Preview Download |
|
md5:6ce9090c05fda16b4f8a79d433c57877
|
702.5 MB | Preview Download |
|
md5:1c19eba11b196f9d42eb655d81aa086c
|
936.6 MB | Preview Download |
|
md5:a582ec53ff64009c38e4b045e993f474
|
8.8 kB | Download |
|
md5:1b3c980d31fd97a2140e3d3f145e2eb7
|
14.2 kB | Download |
|
md5:7963b35166802a7efb8ebe448e5dcd8f
|
21.9 kB | Download |
|
md5:872ba07ca6c6b5de95b77e8b09e07791
|
60.9 kB | Download |
|
md5:809cb75b2c50e82867440f98a6602209
|
72.1 kB | Download |
|
md5:e05bff432784c75747b73c77978772c4
|
8.5 kB | Download |
|
md5:f65424c1aed07e57ba8bce30a9f822e1
|
8.5 kB | Download |
|
md5:2ecb8d29372a6a1e2f8f18dd63d1346d
|
8.4 kB | Download |
|
md5:afbe82e9e1388f5f24591165c4e90ce5
|
8.4 kB | Download |
|
md5:fedf3c9a7a545c3232547c03ce5bb095
|
8.4 kB | Download |
|
md5:ae91b4363d3c08eb05a75746141c030a
|
8.4 kB | Download |
|
md5:b9d60b45a75140f33177245f9b0df9c7
|
8.3 kB | Download |
|
md5:12b92b80a5b729c02dc054ca7501985c
|
8.4 kB | Download |
|
md5:1b90acf6d82d5a4c2478fa2f1c5922fb
|
8.4 kB | Download |
|
md5:dcb0085a40aa3fd6f2909837cabfcabb
|
2.6 MB | Preview Download |
|
md5:62ca34de58829aec9c293c0a5f462197
|
2.6 MB | Preview Download |
|
md5:6f39631cc0f5adb58e62564307838680
|
2.6 MB | Preview Download |
|
md5:8a529ce412805125f3ecc71ef5efe9f2
|
2.5 MB | Preview Download |
|
md5:bf998f9dbbbeaffbbcd35b2d348760f6
|
2.7 MB | Preview Download |
|
md5:7ae2387bedc6ac6e01dfa095528dd5c6
|
2.6 MB | Preview Download |
|
md5:9fe27925cc7c401d9882054645725be7
|
2.6 MB | Preview Download |
|
md5:31d7a26d47abae1b9f512579b7dab2b7
|
2.5 MB | Preview Download |
|
md5:9117819e1dc67239495d5dc3ad01bfb0
|
2.5 MB | Preview Download |
|
md5:b8b2f9a8c0f1ccd6c0d1f52578681a91
|
2.6 MB | Preview Download |
|
md5:1c5da147bba6974f952baddab25d732b
|
1.3 MB | Preview Download |
|
md5:0acdc0e9ea341d141eba9c79fc638a64
|
4.5 MB | Download |
|
md5:a70cd1c6dd2e050d22938536f6314a60
|
4.5 MB | Download |
|
md5:85604e2c2506fef14436e1ee5817d423
|
4.5 MB | Download |
|
md5:72bba4c860cd7178b32d7c381f2433a1
|
4.5 MB | Download |
|
md5:516386a579f705ceb996202d2c543d78
|
4.5 MB | Download |
|
md5:1bbe6c22b8ad72e416bd8fa70d8c38b0
|
4.5 MB | Download |
|
md5:ef3a4bf5910a5fcd8c9bc69f71cfc605
|
4.5 MB | Download |
|
md5:cf4a4652ef2c84bd1de401eb80bdca6a
|
4.5 MB | Download |
|
md5:56be5d6790354dca7fca7211e8037851
|
4.5 MB | Download |
|
md5:450b25868040a3209e52a91920800983
|
4.5 MB | Download |
|
md5:7c3ddd9c240832986357463f5811c93a
|
4.5 MB | Download |
|
md5:71c0d424af36b2a8b8552edce80c23be
|
4.5 MB | Download |
|
md5:a409d8c2a7903bcbe273bfca716eb449
|
4.5 MB | Download |
|
md5:ae77ea9eeb3b0f2586e0569f2e7f0ed7
|
4.5 MB | Download |
|
md5:9ddf6815476f08b269f9d8c0f2482821
|
4.5 MB | Download |
|
md5:ce951076456aeb4dfef8a3f297c02fe9
|
4.5 MB | Download |
|
md5:04d5c42c0ddbc7c0723cf95ba6a7130b
|
4.5 MB | Download |
|
md5:bd72a19b35d5dde6a6593370d845a8cf
|
4.5 MB | Download |
|
md5:0ab2ff0f2335ca98d70cd7ae36e9521f
|
4.5 MB | Download |
|
md5:0d618648e6921baf841ad30808d9c5d4
|
4.5 MB | Download |
|
md5:812df04f828256eef7728053b136c016
|
191.5 kB | Preview Download |
|
md5:f36f777b8b68c64822973a5b0943ecba
|
173.0 kB | Preview Download |
|
md5:fae1d0d1d2f7d5476c2c4b033241fe01
|
189.6 kB | Preview Download |
|
md5:68685c468a3d24900128eba62ac913e5
|
247.9 kB | Preview Download |
|
md5:0baead2c6de3257c29a234dab1b74acb
|
237.0 kB | Preview Download |
|
md5:c2a74001bf262064ba605eeac3485613
|
186.9 kB | Preview Download |
|
md5:96124043679f2f238476100053a0423a
|
238.5 kB | Preview Download |
|
md5:989ee7a9fce9c888fb4029aff4a6cc5b
|
238.3 kB | Preview Download |
|
md5:ab45df478b714a64a38fb070c2154dcb
|
826 Bytes | Preview Download |
|
md5:a12fcd7d06013d3775e74415086720fd
|
248.2 kB | Preview Download |
|
md5:4e7f0ae9bf221478b753e46bc1f053a8
|
8.1 kB | Download |
|
md5:1c18577367ef23d9ba0bfaaec3f7b70c
|
8.1 kB | Download |
|
md5:66a0c15f628e23b4fa0ce30f10faaaa3
|
8.3 kB | Download |
|
md5:f438b5cf046dd6271d264ed6b1a12872
|
8.2 kB | Download |
|
md5:4b6a21d66dcfe95059fa9107b9a7321f
|
6.4 kB | Download |
|
md5:b736125bf70b4328970fb6cf77183334
|
6.3 kB | Download |
|
md5:f3ed92003bf9acbeb2eabf289969883f
|
6.5 kB | Download |
|
md5:c7e3dbe28b8e8e4afc8b4524b02134a5
|
6.5 kB | Download |
