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. Study holds ProTherm entries: 17075, 17076, 17077, 17078 Extra Details: 10 mM EDTA was added in the experiment. porin; membrane protein stability; thermal unfolding; ir spectroscopy
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:49 p.m.
|Number of data points||7|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Derived Quantity: dTm|
|Libraries||Mutations for sequence MKKALIASTALLLTAGVAAADVKISGYGRTGVIYYENDSDVNESRVQSRLRMNIDASSSTDSGVDFGARFRLQWDQGEEGEAQTNAGKLWVSAQGFTVEVGNVDTAFASNGLIYETGLGSYDRSVGFSAVTGAFFAYDSNGYGIDEVDRVGIRALYTLGDLTLQASYVDPDQSGTVIGVEEEFGFSVDYTWNERLELSAAAAFNGAGIDGNDIYFLGARYAVMDNARIGLNFVDAGDEEDGYDGKTIALYGDYTLADGRTNLEAYIANNDRDSNETDNAFGIGVNYDLGGARLGASLQRDYAENVTADFGVRFEF|