Optically modulatable blue fluorescent proteins

Abstract

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Figure 1

Absorption, excitation, and fluorescence spectra of modBFP.

Figure 2

A) Enhancement as a function of modulation frequency fitted to phase-resolved lifetime equations. B) Characteristic frequencies are plotted as a function of 405 nm primary excitation for modBFP and modBFP/H148K. The y-intercept yields the natural dark state decay rate k_decay of each protein.

Figure 3

Enhancement factor versus primary intensity of pulsed 372 nm (solid) and cw 405 nm (dashed) excitation of modBFP and modBFP/H148K. Enhancement factor of 1.0 indicates no fluorescent increase over primary alone.

Figure 4

Live cell demodulation of mitochondria-targeted modBFP/H148K. Upon 405 nm illumination, blue fluorescence is collected from modBFP/H148K-mito and autofluorescence. Co-illumination at 514.5nm, modulated at 2 Hz (secondary illumination only within the white circle) recovers only the modBFP/H148K-mito signal on a greatly reduced background (lower circle). Scale bar is 20 μm.

Figure 5

A possible schematic of energy levels involved in BFP modulation. Excitation of cis neutral state at 405 nm forms an excited state that can interconvert into what we hypothesize as being the anionic trans state. The natural decay out of that state is k_decay and can be related to the activation energy for back reversion, E_rev. If a molecule exists in its dark state, 514 nm excitation can regenerate the blue-absorbing, fluorescent manifold.

Figure 6

Speculative structures of the tyrosine-based chromophore in A) cis and B) trans position in the chromophore pocket based on the very similar PDB: 2QLE.