Minimal domain of bacterial phytochrome required for chromophore binding and fluorescence

Abstract

Fluorescent proteins (FP) are used to study various biological processes. Recently, a series of near-infrared (NIR) FPs based on bacterial phytochromes was developed. Finding ways to improve NIR FPs is becoming progressively important. By applying rational design and molecular evolution we have engineered R. palustris bacterial phytochrome into a single-domain NIR FP of 19.6 kDa, termed GAF-FP, which is 2-fold and 1.4-fold smaller than bacterial phytochrome-based NIR FPs and GFP-like proteins, respectively. Engineering of GAF-FP involved a substitution of 15% of its amino acids and a deletion of the knot structure. GAF-FP covalently binds two tetrapyrrole chromophores, biliverdin (BV) and phycocyanobilin (PCB). With the BV chromophore GAF-FP absorbs at 635 nm and fluoresces at 670 nm. With the PCB chromophore GAF-FP becomes blue-shifted and absorbs at 625 nm and fluoresces at 657 nm. The GAF-FP structure has a high tolerance to small peptide insertions. The small size of GAF-FP and its additional absorbance band in the violet range has allowed for designing a chimeric protein with Renilla luciferase. The chimera exhibits efficient non-radiative energy transfer from luciferase to GAF-FP, resulting in NIR bioluminescence. This study opens the way for engineering of small NIR FPs and NIR luciferases from bacterial phytochromes.

Obatoclax kills anaplastic thyroid cancer cells by inducing lysosome neutralization and necrosis

“…Obatoclax fluorescence excitation and emission dependency on pH was measured as described in [56]. In brief, pH titrations of 4 μM Obatoclax solutions were performed using a series of Hydrion buffers with a range pH values from 1.97 to 11.55 (Micro Essential Laboratory).…”

Rational construction of compact de novo-designed biliverdin-binding proteins

“…The resultant 15-kD monomer fluoresced modestly in the far-red spectrum (ex = 648 nm, em = 662 nm, F = 1.58%). This quantum yield is similar to sandercyanin, a naturally occurring fluorescent BV-binding fish pigment (F = 1.6%) 41 , and less than those of BV-binding directed evolution products derived from Bph-and AP (F ~ 7-18%) [17][18][19][20][21][22][23]31 . For simplicity, we hereon call this de novo-designed fluorescence-activating protein, "dFP."…”

“…Structure-function insights on cofactor stabilization in reported engineered bili-proteins can inform rational approaches for constructing de novo-designed ones. To date, fluorescent proteins (FPs) have been evolved from natural bacteriophytochromes (Bph) [17][18][19][20][21][22][23] , phytochromes (Phy) [28][29][30] , allophycocyanin light-harvesting complexes [31][32][33] (AP), and fatty acid-binding muscle proteins [34][35] (FABP). These engineered proteins follow the general principle of rigidifying the protein (i) to stabilize the floppy cofactor in a fluorescent conformation, (ii) to limit solvent and oxygen access to the cofactor, and (iii) to prevent the intrinsic protein structural re-arrangements associated with their natural signaling roles.…”

“…For reference, the mini is approximately one-third the size of canonical GFP (27 kD) and monomeric Bph-FPs (30-35 kD) 18,21 , and half the size of a minimal fluorescent domain engineered from a Bph GAF domain (18 kD) 20 . The facile truncation and overall reduction in size compared to Bph-derived fluorochromes reflects the structural simplicity of the de novo-designed scaffold.…”

“…Here, we report the rational construction of compact de novo-designed proteins that bind biliverdin (BV), the optically active cofactor in natural photosensory bacteriophytochromes (Bph) and Bph-derived protein tools [17][18][19][20][21][22][23][24] . Energy minimization modeling in Rosetta [13][14][25][26][27] informed the strategic placement of residues that stabilized BV within the core and consequently increased its far-red fluorescence, despite lacking sequence or structural homology to natural biological fluorochromes.…”