Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II

Abstract

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related.

A comparative infrared spectroscopic study of glycoside hydrolases from extremophilic archaea revealed different molecular mechanisms of adaptation to high temperatures

“…These data are in agreement with the experiments of differential scanning calorimetry showing that the temperature of melting ( T m ) of this enzyme was 108°C 28. Thermal denaturation curves for Ssβ‐gly and Taβ‐gly were obtained by plotting the changes of the amide I′ bandwidth as a function of the temperature29, 30 (data not shown). The temperature of melting for Ssβ‐gly and Taβ‐gly resulted 98.1 and 98.4°C, respectively and was calculated as described 18…”

Structure/function of KRAB repression domains: Structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses

“…The amide II band intensity (absorption close to 1550 cm −1 ) is very sensitive to 1 H/ 2 H exchange. A large decrease in amide I band intensity reflects a high protein flexibility and/or a low compact structure 35–38. The infrared spectra of KRAB‐A and KRAB‐AB analyzed in 2 H 2 O medium at 20°C showed a very low absorption close to 1550 cm −1 (data not shown) that did not decrease significantly at high temperatures (data not shown), indicating that at 20°C the 1 H/ 2 H exchange was complete, and suggesting, in turn, a highly flexible protein structure 35–38…”

“…These spectra (Fig. 5) show that, with the increase in temperature, the intensity of the amide I′ band decreases while the intensity of the bands close to 1615 and 1682 cm −1 (aggregation bands) increases, reflecting denaturation and aggregation, respectively 37. Hence, the denaturation and aggregation process may be followed by plotting the ratio between the intensity of the amide I′ band and the intensity of the 1615 cm −1 aggregation band.…”

“…At 70°C the spectrum is characterized by a broad band at 1642 cm −1 (unordered structures) and by the two aggregation bands, indicating that the protein is denatured and aggregated. It must be pointed out that the aggregation process is caused by denaturation of the protein, and that the aggregation bands reveal that some unfolded elements undergo intermolecular interactions (aggregation) and form antiparallel β‐sheet‐like structures 36–38. If the denaturation and aggregation processes occur concomitantly, the onset of the aggregation bands corresponds to the onset of protein denaturation.…”

Fabrication of an electrochemical biosensor with ZnO nanoflakes interface for methylglyoxal quantification in food samples

“…An increase in peak intensity corresponding to (101) crystal plane indicated that the growth of ZnO flakes was preferentially oriented towards (101) crystal plane. [41] and b sheets of GLO 1 [42,43] respectively. The last peak confirmed the immobilization of GLO 1 with ZnO flakes.…”