Abstract
The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.
Highlights
Photochromic fluorescent proteins that are reversibly photoswitchable have been reported for constructs and mutants derived from different origins [1,2,3,4,5,6,7,8,9]
We present high resolution room temperature SFX structures of Skylan-NS in both ‘on’ and meta-stable ‘off’ state collected using two different X-ray free electron lasers (XFELs), the SPring-8 Angstrom Compact free electron Laser (SACLA), Japan and the Linear Coherent Light Source (LCLS), USA
The SPring-8 Angstrom Compact Free Electron Laser (SACLA) pipeline was used for hit selection and processing
Summary
Photochromic fluorescent proteins that are reversibly photoswitchable have been reported for constructs and mutants derived from different origins [1,2,3,4,5,6,7,8,9]. With few exceptions [10], this class of photochromic rsFPs have a dark/resting state with generally a high fluorescence quantum yield and a cis chromophore conformation which is anionic at neutral or alkaline pH [11]. Following green illumination this ‘on’ state converts to a meta-stable off-state which has a trans chromophore conformation and is neutral. The photochemical quantum yield of cis-trans (on→off) photoisomerisation differs for reported mutants and constructs, but are generally at the few percent level for the fastest switching cases [12,13,14]. In the dark at room temperature the off-state converts thermally to the resting ‘on’ state with typical time constant of minutes to hours depending on the details of the construct [12]
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