We have investigated outer membrane protein porin from Paracoccus denitrificans for its stability against heat and pH. Pathways of unfolding and refolding have been analyzed. Porin incubated at pH 12.5 and above undergoes a slow unfolding into an unordered structure. The unfolded protein could be refolded into a nativelike structure that is functionally active but with distinct deviation from the native protein. This nativelike structure exhibited an entirely different thermal stability. Although aggregation is normally considered a structural "dead-end", the possibility of opening an aggregated porin and forming a functionally active structure was analyzed here. Porin aggregates on heating above 86.2 degrees C. Incubating the heat-aggregated protein at high pH (> or = 12.5) leads to a slow opening of the protein into an unordered structure. It was possible to refold this unordered protein into a trimeric nativelike structure which was capable of forming active pores. However, the thermal stability of the refolded porin was unlike that of the native porin. To understand the basic mechanism behind the unfolding processes, the protein was subjected to heating at various pH values. It was observed that at pH > or = 12.5 the protein does not aggregate upon heating; instead, it opens into an unordered structure. We conclude that at high pH values, the electrostatic interactions of various amino acid residues are perturbed which leads to unfolding into an unordered structure. This study shows for the first time an entirely new unfolding and refolding pathway for porin. Study holds ProTherm entries: 19915, 19916, 19917, 19918 Extra Details: outer membrane protein; nativelike structure; refolding pathway
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||4|
|Assays/Quantities/Protocols||Experimental Assay: Tm pH:10.0 ; Experimental Assay: Tm pH:8.0 ; Experimental Assay: Tm pH:4.0 ; Experimental Assay: Tm pH:1.5|
|Libraries||Mutations for sequence MKKALIASTALLLTAGVAAADVKISGYGRTGVIYYENDSDVNESRVQSRLRMNIDASSSTDSGVDFGARFRLQWDQGEEGEAQTNAGKLWVSAQGFTVEVGNVDTAFASNGLIYETGLGSYDRSVGFSAVTGAFFAYDSNGYGIDEVDRVGIRALYTLGDLTLQASYVDPDQSGTVIGVEEEFGFSVDYTWNERLELSAAAAFNGAGIDGNDIYFLGARYAVMDNARIGLNFVDAGDEEDGYDGKTIALYGDYTLADGRTNLEAYIANNDRDSNETDNAFGIGVNYDLGGARLGASLQRDYAENVTADFGVRFEF|