Sequence alignment of human TYR and human TYRP1.

<p>Conserved residues are highlighted in red. Putative TYR glycosylation sites are indicated with a green star. Two different TYR constructs were designed for recombinant expression, TYR456 (50 kDa, residues 19–456) and TYR469 (51.5 kDa, residues 19–469).</p

Enzymatic deglycosylation of human TYR.

<p><b>A.</b> Deglycosylation of TYR469 with PNGase. <b>B.</b> Deglycosylation of TYR469 with Endo H_f. M, molecular weight marker; C, non-treated protein as control; D, deglycosylated sample.</p

Characterization of TYR variants by gel filtration and enzyme activity assays.

<p>Overlap of the analytical gel filtration profiles of TYR456 (peak B, in red) and TYR469 (peak A, in blue) on a Superdex 200 10/300 GL column (GE Healthcare). Colorimetric activity assays with the corresponding eluted fractions using L-DOPA as substrate generated a pink or a dark pink pigment product (i.e. quinone-MBTH adduct), indicating that both variants are enzymatically active (10 μL of each elution fraction containing TYR was added in a 80 μL reaction well containing L-DOPA. 10 μL gel filtration buffer solution was used as a negative control). The apparent gradient of light pink to dark pink, and back to light pink in the reaction well indicates different protein concentrations of each elution fraction.</p

Determination of the thermal stability of the TYR variants by thermal shift assays.

<p>Fluorescence scan diagrams based on <i>Sypro Orange</i> (Thermo Fisher Scientific) binding upon thermal unfolding of TYR456 and TYR469 proteins, respectively. The apparent stability midpoint values (or melting temperatures) under the analysed buffer conditions are 72°C and 60°C for TYR456 and TYR469, respectively.</p

Crystallization of TYR469.

<p><b>A.</b> Picture of the initial crystallization hit of TYR469 in 100 mM (NH)₄SO₄, 10 mM MgCl₂, 50 mM MES, pH 5.9, and 18% PEG8000 (w/v). <b>B.</b> Picture of a Cryoloop containing multiple microcrystals suitable for an X-ray mesh scan. <b>C.</b> Heat map resulting from the cryoloop mesh scan. The cross spots indicate diffracting crystals positions. The quality of the diffraction is scored based on a colour gradient (from yellow—poor—to dark red—highest–). <b>D.</b> Picture of TYR469 crystals with 0.5% polyvinylpyrrolidone K15 (w/v) as an additive (HR2-138 condition E2, Hampton Research). <b>E.</b> Picture of TYR469 crystals with 10 mM spermine tetrahydrochloride as an additive (HR2-138 condition D3). <b>F.</b> Representative X-ray diffraction pattern of a crystal from E. Resolution circles are indicated.</p