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Investigation of the thermal stability of porin from Paracoccus denitrificans by site‐directed mutagenesis and Fourier transform infrared spectroscopy
Author(s) -
Sukumaran S.,
Zscherp C.,
Mäntele W.
Publication year - 2004
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.20049
Subject(s) - paracoccus denitrificans , chemistry , protein secondary structure , porin , micelle , trimer , circular dichroism , sodium dodecyl sulfate , gel electrophoresis , thermal stability , protein quaternary structure , bacterial outer membrane , crystallography , biochemistry , organic chemistry , escherichia coli , dimer , protein subunit , aqueous solution , gene , enzyme
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 β‐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°C. In native‐like conditions (provided by liposomes), no change was observed in the secondary structure of the protein until 95°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. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004