Discovery of a microbial rhodopsin that is the most stable in extreme environments

Abstract

Microbial rhodopsin is a retinal protein that functions as an ion pump, channel, and sensory transducer, as well as a light sensor, as in biosensors and biochips. Tara76 rhodopsin is a typical proton-pumping rhodopsin that exhibits strong stability against extreme pH, detergent, temperature, salt stress, and dehydration stress and even under dual and triple conditions. Tara76 rhodopsin has a thermal stability approximately 20 times higher than that of thermal rhodopsin at 80°C and is even stable at 85°C. Tara76 rhodopsin is also stable at pH 0.02 to 13 and exhibits strong resistance in detergent, including Triton X-100 and SDS. We tested the current flow that electrical current flow across dried proteins on the paper at high temperatures using an electrode device, which was measured stably from 25°C up to 120°C. These properties suggest that this Tara76 rhodopsin is suitable for many applications in the fields of bioengineering and biotechnology.

Keywords: bioelectronics; biophysics; biotechnology; microbiology.

Graphical abstract

Figure 1

Phylogenetic tree and photochemical analysis of Tara76 rhodopsin (A) The evolutionary history was inferred using the maximum likelihood method based on the Jones-Taylor-Thornto matrix-based model. Evolutionary analyses were conducted in MEGA7 (Jones et al., 1992; Kumar et al., 2016). The red squares are Tara76 rhodopsin, and the PRs are shown in a group. (B) The absorption spectra of Tara76 rhodopsin at pH 0.02, 4, 7, 10, and 13, and orange color of Tara76 rhodopsin at neutral pH. (C) The proton outward pumping was measured. The black dotted line was measured in an unbuffered solution of 10 mM NaCl, and the red dotted line was the result of treatment with 10 mM NaCl and 10 uM CCCP (carbonyl cyanide m-chlorophenylhydrazone). The gray area is dark, and the white area is in the presence of light for 3 min at 25°C. (D–F) The absorption spectra were measured depending on pH changes. The red-shifted (red arrow) and blue-shifted (blue arrow) absorption spectra at each pH are indicated by arrows, and purified rhodopsins are exhibited at their representative pH. Panels D, E, and F represented at pH 0.02, 4, and 13. (G–I) The pH titration curve for the calculation of pKa. Equation ‘y = A/1+(pH 10-pKa)' was applied to fit the data using Origin Pro 7.0 where A is the maximal amplitude of relative absorbance changes (Zhu et al., 2013). Panels G, H, and I represented the measurements performed at pH range 0 to 4, 4 to 10, and 10 to 13.

Figure 2

pH resistance test and detergent resistance test (A) The stability was tested for 30 days at pH 0.02. Even under extreme acidic conditions, Tara76 rhodopsin retained a colored pigment for more than 1 month (B) pH-dependent absorption spectra showing how stable each rhodopsin was at each pH. The Y axis was calculated as the ratio between colored and denatured protein at 25°C. In comparison, GR (black square line), PR (red circle line), TR (blue triangle line), and Tara76 rhodopsin (pink reverse triangle line) were measured. (C) The resistance of GR, PR, TR, and Tara76 rhodopsin to Triton X-100 was measured. Each protein was solubilized by Triton X-100, and the maximum absorption spectra were measured over time. The temperature was maintained at room temperature and measured for 1,440 min (one day) starting at 0 min for 30 min after treatment for solubilization. The error bars are the standard deviations of three independent experiments (n = 3). GR, PR, TR, and Tara76 rhodopsin are designated as black, red, blue, and pink, respectively.

Figure 3

Thermal stability and denaturation curve (A–D) Protein denaturation curves were plotted with temperature changes. GR, PR, TR, and Tara76 rhodopsin were tested at 60°C, 70°C, 80°C, and 85°C, respectively. The black square line represents GR, the red circle line represents PR, the blue triangle line represents TR, and the pink reverse triangle line represents Tara76 rhodopsin. The starting point was measured at room temperature every 10 min for 1 hr and then every 60 min for 240 min at each temperature. The unit of the Y axis is the protein folding ratio (%), which is the rate that the retinal is bleached from the rhodopsin, rendering the pigments colorless. The unit of the X axis is reciprocal time [min⁻¹]. (E) The denaturation rate constant (k0.5) was determined from each sample data from temperature. The values are calculated at 80°C, and the values of 60°C and 70°C are shown in Table S1. The unit of the X axis is reciprocal temperature [K⁻¹].

Figure 4

Dual conditions for the stability of Tara76 rhodopsin (A) Thermal stability at various pH values. 3D color map surface that measured the stability of Tara76 rhodopsin with the pH, from pH 0.14 to pH 13 at each temperature from 0 to 240 min. The Y axis calculated the protein folding ratio (%) that the sample color was maintained at each temperature condition: it is 0% in purple color and 100% in red color. (B) Detergent stability under acidic and alkaline pH conditions. Combined result of pH resistance to strong detergent. Protein folding ratio of each sample solubilized with 1% Triton X-100 (final concentration) was plotted according to pH at 25°C. GR is a black square, PR is a red circle, TR is a blue triangle, and Tara76 rhodopsin is a pink reverse triangle. In the case of TR, the detergent resistance was low, and low resistance was observed according to pH. The error bars are the standard deviations of three independent experiments (n = 3). (C) Thermal stability with detergent resistance. The bar graph confirmed the stability of each temperature in the detergent condition. The temperature stability of GR, PR, TR, and Tara76 rhodopsin was observed after solubilization using strong detergent Triton X-100 (1% final con.). The low values of the figure were enlarged as black line boxes. The protein folding ratios were obtained by maximum absorption at 60°C, 70°C, and 80°C, respectively. Dark gray is at 60°C, medium gray is at 70°C, and light gray is at 80°C.

Figure 5

Five primal radar charts The results of GR, PR, TR, and Tara76 rhodopsin are summarized. Each rhodopsin is shown radially, including pH resistance, detergent resistance, thermal stability, and dual condition. These values were obtained through the respective denaturation rate constants. The green color is GR, the red color is PR, the pink color is TR, and the yellow color is Tara76 rhodopsin.

Figure 6

Comparison of stability under high salt conditions and recovery after heat drying and electrical conductivity of proteins (Tara76 rhodopsin, TR, PR, and GR) (A) Protein stability of GR, PR, TR, and Tara76 rhodopsin is confirmed at high concentrations of 4 M sodium chloride. The measurements were made after 24 hr of treatment. The dark bar graph is under the normal condition, and the light gray bar is at the high salt concentration. These are shown as a ratio (B–D) (B) Each sample was dried for 16 hr at 55°C. The dark bar graph shows the state before drying, and the light gray bar shows that the color remained after recovery in 0.02% DDM solution. The error bars are the standard deviations of three independent experiments (n = 3). Electrical current flow through the proteins in the paper matrix during (C) various voltages and (D) various temperatures and 4-volt were applied. The inset of (C) is a setup for current measurement.

Figure 7

Hydrophobicity analysis of helix E The hydrophobicity of Tara76 rhodopsin was compared with that of GR, PR, and TR. (A) The hydrophobicity score of all amino acids is shown in helix E. The numerical value of the amino acid was determined through ProtScale from ExPAsy, and the helix E portion of each protein was indicated by a red line. In the case of Tara76 rhodopsin, from the 129^th residue to the 152^nd residue, PR indicates 147^th to 170^th residue, GR indicates 174^th to 197^th, and TR indicates 148^th to 17. (B) The hydrophobicity score of the helix E region was added and compared with the bar graph. (C) The urea gradient gel analysis, which is the result of hydrophobicity depending on how far the protein migrates through transverse urea gradient gel electrophoresis (TUG-GE). The concentration is 0 M at the top and 8 M at the bottom. Since it forms a continuous concentration, the stability of the protein depending on the concentration can be seen. This is expressed as a ratio according to the distance moved away. The error bars are the standard deviations of three independent experiments (n = 3).