The temperature dependence of changes in the free energies for the dimeric association of βB1 and βA3 and the tetrameric association of βB1/βA3.

<p>Asssociation of βB1, βA3 and βB/βA3 are shown by blue open triangles, black open squares and red open circles, respectively. Panels A and B: van't Hoff plots where <i>ln(K_d/C_o)</i> is plotted as function of the reciprocal of absolute temperature (<i>1000/T</i>), <i>K_d's</i> are the dissociation constants obtained from analytical ultracentrifugation, and <i>C_o</i> is the µM concentration. Panel A: the difference in heat capacity (<i>ΔC_p</i>) is constrained to be 0, resulting in a linear function; Panel B: <i>ΔC_p</i> is not constrained and has a nonzero value. Panel C: temperature dependence of Gibbs free energy gained in formation of βB1/βA3. <i>ΔΔG_d (βB1/βA3)</i> is defined as a difference between Gibbs free energy changes of tetrameric βB1/βA3 and that of individual components (βB1 and βA3). Concentrations for βB1, βA3, and βB1/βA3 crystallins were each 0.5 mg/ml.</p

Thermodynamic profiles for the associations of homodimeric βB1 and βA3 and tetrameric βB1/βA3.

<p>Thermodynamics parameters, enthalpy <i>ΔH_a</i>, and entropy <i>ΔS_a</i> changes were determined using linear (<i>ΔC_p = 0</i>) and nonlinear (<i>ΔC_p≠0</i>) fitting functions into van't Hoff plots. The Gibbs free energy changes <i>ΔG_a</i> were calculated using formula <i>ΔG_a = ΔH_a−TΔS_a</i>, where <i>T</i> is temperature in K; e.u. = 1 cal/(deg mol).</p

Overview of the homo- and heteromolecular associations of β-crystallins.

<p>Top panel: βA3, βB1, and βB2 self-associate in a reversible manner to form dimers. The homo-associations of βA3 and βB2 exhibit endothermic enthalpy and are driven by entropy as a result of hydrophobic interactions between protein molecules. In contrast, the self-association of βB1 is driven by exothermic enthalpy due to van der Waals interactions and hydrogen bonds at the dimer interface. Bottom panel: The βB1/βA3 complex is likely formed by the association of hetero-dimers but we cannot rule out that it is formed from homodimers. Similar to that of βB1 alone, the formation of the tetramer is driven by exothermic enthalpy. Structures of βB1 and βB2 were obtained from the protein database RCSB (files: 1 oki and 1 blb, respectively). Closed and open structures of βA3 were modeled as described earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029227#pone.0029227-Sergeev1" target="_blank">[1]</a>. From our results we cannot say which monomer conformation exists within the hetero-tetramer. However, the majority of known crystal structures of β-crystallins (3 of 4) are of the closed monomer type suggesting this is the most stable conformation. Therefore, the structure of the hypothetical tetrameric βB1/βA3 complex was generated using the crystal packing of βB1 crystallin as a template (PDB file: 1 oki).</p

Gel filtration and enzymatic activities for the pure recombinant hTyrC_tr and two temperature sensitive mutant variants.

<p>Chromatographies were using Superdex 75 10/30 HR: hTyrC_tr (<b>B</b>) and of R422Q and R422W (<b>E</b>). The elution points of molecular mass standards are shown at the top for reference. Panels <b>A, C</b> and <b>D</b> show test tubes containing the L-DOPA colorimetric reactions for each protein fraction of hTyrC_tr, R422Q and R422W, respectively. Brown color (intensity proportional) in tube indicates diphenol oxidase activity. Corresponding Western blots bands were labeled by horizontal arrows.</p

Effect of Inhibitors on enzymatic activity of hTyrC_tr is shown by IC₅₀ values.

<p>Values for tyrosinase enzymes (EC 1.14.18.1) obtained from different sources and summarized in the BRENDA database, <a href="http://www.brenda-enzymes.info" target="_blank">http://www.brenda-enzymes.info</a>); ^**) complete inhibition.</p

Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

<div><p>Background</p><p>Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (<i>TYR)</i> is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.</p><p>Methodology/Principal Findings</p><p>The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in <i>T. ni</i> larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.</p><p>Conclusions/Significance</p><p>The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their <i>in vivo</i> function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.</p></div

Purification and characterization of hTyrC_tr.

<p><b>A</b>: IMAC using a HisTrap 5 ml column. The arrow indicates the start of the imidazole gradient. <b>B</b>: Gel filtration using Superdex 75 16/60 HR. Chromatography profile monitored at 260 nm (purple lines) and 280 nm (green lines). The inserts show the diphenol oxidase activity of hTyrC_tr measured in separate tube for each fraction after 30 min of incubation at 37°C with 3 mM L-DOPA in 50 mM sodium phosphate buffer, pH 7.5. <b>C</b>: SDS-PAGE (top panel) and Western blot (bottom panel) showing stepwise purification of hTyrC_tr. From left to right: L, protein ladder; 1, total lysate of larvae expressing hTyrC_tr; 2, flow through; 3, sample after 5 ml HisTrap; 4, sample after Superdex 75. <b>D</b>: Sedimentation equilibrium of hTyrC_tr. The protein concentration gradient (280 nm) versus radial distance is indicated. The red line shows calculated fit for an ideal monomer and blue circles the experimental values. The top panel shows the residuals of a fitted curve to the data points. Calculated fit of monomer was obtained assuming that the average partial specific volume of glycans is 0.63 cc/g and that the protein contains 10% carbohydrate.</p

Purification of recombinant hTyrC_tr and two temperature sensitive variants from 10 g of larval biomass^a.

<p>^a The total protein was estimated by the Warburg – Christian method using absorbance at 260/280 nm (Sigma-Aldrich); L-DOPA activity was determined as described in Materials and Methods section. Specific activity was obtained as the L-DOPA enzyme activity multiplied by the sample total volume and divided by total protein. The tyrosinase content of hTyrCtr, R422Q and R422W in protein extracts were estimated from SDS-PAGE gels as 100% X tyrosinase band intensity/total protein band intensity.</p

Far-UV CD Spectra of hTyrC_tr and temperature sensitive mutants R422Q/W.

<p>CD spectra for hTyrC_tr and two mutants, R422Q and R422W, are shown by blue, red, and green solid lines, respectively. Dashed lines show correspondent spectra measured in the presence of 0.5 mM tyrosine. Measurements were performed at 37°C (<b>A</b>) and 31°C (<b>B</b>). Scans (190–260 nm) were performed in 50 mM sodium phosphate buffer, pH 7.5 at protein concentrations of 0.2 mg/ml. <b>Inserts</b>: spectral differences at the range 200–230 nm are shown. Histograms C and D show ellipticity (Θ) ratios (%) in the absence (<b>C</b>) or the presence (<b>D</b>) of tyrosine with data from spectra shown in Panels <b>A</b>, <b>B</b>. The ratios were calculated as 100% × (Θ) 31°C/(Θ) 37°C determined at fixed wavelengths of 208, 222 nm (α-helix), and 215 nm (β-sheet). Dashes show a 100% level.</p

N-glycosylation of hTyrC_tr.

<p>N-glycosylation sites determined by MS are mapped to the human tyrosinase protein structure modeled as described in methods section. The protein backbone structure is shown by magenta ribbon. Two copper atoms, CuA and CuB, which coordinated by His residues are shown in orange. Fully and partially occupied N-glycosylation sites are represented by red and yellow spheres, respectively. Two potential N-glycosylation sites, not determined in in present study, are shown in grey. The location of mutant variants is indicated in green.</p