Investigation of the thermal stability of porin from Paracoccus denitrificans by site‐directed mutagenesis and Fourier transform infrared spectroscopy

Abstract

The folding of membrane proteins was addressed using outer membrane protein porin from the soil bacterium Paracoccus denitrificans (P. den.). IR spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis were used to probe the effect of mutagenesis on the thermal stability of the protein. Secondary structure analysis by amide I ir spectroscopy showed that the wild-type protein was predominantly composed of beta-sheet, which supports the x-ray crystal structure information (A. Hirsch, J. Breed, K. Saxena, O.-M. H. Richter, B. Ludwig, K. Diederichs, and W. Welte, FEBS Letters, 1997, Vol. 404, pp. 208-210). The mutants E81Q, W74C, and E81Q/D148N were shown to have similar secondary structure composition as the wild type. Wild-type protein and the mutants in detergent micelles underwent irreversible denaturation as a result of heating. Transition temperature calculated from the amide I analysis revealed that mutant porins were slightly less stable compared to the wild type. The protein in micelles showed complete monomerization of the trimer above 85 degrees C. In native-like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95 degrees C. This is supported by SDS-PAGE as no change in quaternary structure was observed, proving that the proteins are structurally thermostable in liposomes as compared to micelles. Our studies demonstrated that porins resistant to detergents and proteases are highly thermostable as well.

Correlation between the OmpG Secondary Structure and Its pH-Dependent Alterations Monitored by FTIR

“…3c and d). Similar to other β-barrel outer-membrane proteins, which exhibit remarkable thermal stability, 15,23,24 all investigated forms of OmpG were stable in detergent solution and did not show a transition in their secondary structure at either p 2 H, up to 60°C (Fig. 3c).…”

“…1a), corresponding most likely to the low-and high-frequency β-sheet signals. [15][16][17] The FTIR spectrum of OmpG WT incubated at p 2 H 5.4 (Fig. 1a, black line) shows slight differences in the β-sheet signal positions compared to the spectrum of OmpG WT at p 2 H 8.0 (red line).…”

Thermal stability of outer membrane protein porin from Paracoccus denitrificans: FT‐IR as a spectroscopic tool to study lipid–protein interaction

“…3 ). The plot clearly indicates that the protein in detergent micelles undergoes aggregation with a transition temperature of 86.2 °C, whereas for the protein reconstituted into lecithin, there is no change in their global secondary structure [5]. Fig.…”

“…We have earlier reported the details on the thermal stability of porin from Paracoccus denitrificans [5]. It was found that porin undergoes temperature induced aggregation above the transition temperature of 86.2 °C if the protein is in detergent micelles, whereas no change in its secondary structure is observed up to 95 °C if the protein is reconstituted into liposomes.…”

Into the Lipid Realm: Stability and Thermodynamics of Membrane Proteins

“…These transitions involve partial disruption of the secondary structure [107], and they are reversible under certain conditions.…”