The Secondary Structure of a Major Wine Protein is Modified upon Interaction with Polyphenols

Abstract

Polyphenols are an important constituent of wines and they are largely studied due to their antioxidant properties and for their effects on wine quality and stability, which is also related to their capacity to bind to proteins. The effects of some selected polyphenols, including procyanidins B1 and B2, tannic acid, quercetin, and rutin, as well as those of a total white wine procyanidin extract on the conformational properties of the major wine protein VVTL1 (Vitis vinifera Thaumatin-Like-1) were investigated by Synchrotron Radiation Circular Dichroism (SRCD). Results showed that VVTL1 interacts with polyphenols as demonstrated by the changes in the secondary (far-UV) and tertiary (near-UV) structures, which were differently affected by different polyphenols. Additionally, polyphenols modified the two melting temperatures (T_M) that were found for VVTL1 (32.2 °C and 53.9 °C for the protein alone). The circular dichroism (CD) spectra in the near-UV region revealed an involvement of the aromatic side-chains of the protein in the interaction with phenolics. The data demonstrate the existence of an interaction between polyphenols and VVTL1, which results in modification of its thermal and UV denaturation pattern. This information can be useful in understanding the behavior of wine proteins in presence of polyphenols, thus giving new insights on the phenomena that are involved in wine stability.

Keywords: Synchrotron Radiation Circular Dichroism (SRCD); VVTL1; polyphenols; protein interaction; protein structure; wine.

Figure 1

(A) Mapping of the electrostatic potentials on the molecular surface of the grape VVTL1 4L5H. The negative and positive potentials are colored in red and blue, respectively, while neutral areas are in white. The arrow indicates the acidic cleft located between domains I and II. (B, C) Mapping of the surface hydrophobicity of the VVTL1 4L5H: (B) front view; and, (C) back view (rotated approximately 180˚). Hydrophobicity continuum starts from dodger blue for the most hydrophilic areas, to white, to orange and to red for the most hydrophobic areas. The images were generated using the Chimera 1.14 software [27].

Figure 2

Chemical structures of proanthocyanidins B1 (PB1) and B2 (PB2), quercetin, rutin, and tannic acid.

Figure 3

Far-UV Synchrotron Radiation Circular Dichroism (SRCD) spectra of VVTL1 (0.400 mg/mL) alone (black line in both pictures) or in presence of 2 eq. of polyphenols (A: PB1 in red, PB2 in blue, wine total extract (WTE) in green; B: Q in magenta, R in cyan, TA in orange) in model wine solution (MWS). Spectra were recorded at 20 °C using a Suprasil 0.02 cm cell (Hellma) filled with 60 µL of solution, integration time 1 s, 1 nm digital resolution, 39 nm/min. scan speed, and monochromator slit widths 1.2 nm bandwidth.

Figure 4

Near-UV CD spectra in MWS of polyphenols alone (blue lines), VVTL1 alone (black lines) and VVTL1 in presence of 2 eq. of selected polyphenols, measured (red lines) or calculated (green lines). Polyphenols were Procyanidin B2 (PB2) (A), Wine Tannins Extract (WTE) (B), Rutin (R) (C), and tannic acid (TA) (D). PB1 and Quercetin are not shown as the differences between calculated and measured spectra were equivalent to those shown in (A) and (B), respectively. VVTL1 concentration was 0.500 mg/mL. The CD spectra were measured using nitrogen flushed Jasco J-715 spectropolarimeter, scanning speed 50 nm/min., data pitch 0.5 nm, response time 4 s, bandwidth 2 nm using a Suprasil 1.0 cm cell (Hellma) that was filled with 900 µL of solution.

Figure 5

Thermal denaturation experiments: far-UV SRCD spectra of VVTL1 (0.400 mg/mL in MWS) alone (A) and in presence of Procyanidin B1 (PB1) (B) and B2 (PB2) (C), tannic acid (TA) (D), Wine Tannins Extract (WTE) (E), Rutin (R) (F), and Quercetin (Q) (G) were recorded at different temperatures (indicated) using a Suprasil 0.02 cm cell (Hellma) filled with 60 µL of solution, integration time 1 s, 1 nm digital resolution, 39 nm/min scan speed and monochromator slit widths to 1.2 nm bandwidth. Grey lines indicate intermediate temperatures with steps of 5 °C.