To determine how much information can be transferred from folding and unfolding studies of one protein to another member of the same family or between the mesophilic and thermophilic homologues of a protein, we have characterized the equilibrium unfolding process of the dimeric enzyme serine hydroxymethyltransferase (SHMT) from two sources, Bacillus subtilis (bsSHMT) and Bacillus stearothermophilus (bstSHMT). Although the sequences of the two enzymes are highly identical ( approximately 77%) and homologous (89%), bstSHMT shows a significantly higher stability against both thermal and urea denaturation than bsSHMT. The GdmCl-induced unfolding of bsSHMT was found to be a two-step process with dissociation of the native dimer, resulting in stabilization of a monomeric species, followed by the unfolding of the monomeric species. A similar unfolding pathway has been reported for Escherichia coli aspartate aminotransferase, a member of the type I fold family of PLP binding enzymes such as SHMT, the sequence of which is only slightly identical ( approximately 14%) with that of SHMT. In contrast, for bstSHMT, a highly cooperative unfolding without stabilization of any monomeric intermediate was observed. These studies suggest that mesophilic proteins of the same structural family even sharing a low level of sequence identity may follow a common unfolding mechanism, whereas the mesophilic and thermophilic homologues of the same protein despite having a high degree of sequence identity may follow significantly different unfolding mechanisms. Study holds ProTherm entries: 15604, 15605, 15606, 15607 Extra Details: Transition 1 dimeric enzyme; two-step process; monomeric intermediate; thermophilic
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:46 p.m.
|Number of data points||4|
|Proteins||Serine hydroxymethyltransferase ; Serine hydroxymethyltransferase ; Serine hydroxymethyltransferase|
|Assays/Quantities/Protocols||Experimental Assay: Tm|
|Libraries||Mutations for sequence MKYLPQQDPQVFAAIEQERKRQHAKIELIASENFVSRAVMEAQGSVLTNKYAEGYPGRRYYGGCEYVDIVEELARERAKQLFGAEHANVQPHSGAQANMAVYFTVLEHGDTVLGMNLSHGGHLTHGSPVNFSGVQYNFVAYGVDPETHVIDYDDVREKARLHRPKLIVAAASAYPRIIDFAKFREIADEVGAYLMVDMAHIAGLVAAGLHPNPVPYAHFVTTTTHKTLRGPRGGMILCQEQFAKQIDKAIFPGIQGGPLMHVIAAKAVAFGEALQDDFKAYAKRVVDNAKRLASALQNEGFTLVSGGTDNHLLLVDLRPQQLTGKTAEKVLDEVGITVNKNTIPYDPESPFVTSGIRIGTAAVTTRGFGLEEMDEIAAIIGLVLKNVGSEQALEEARQRVAALTDPTSRSAAGTMEFEA ; Mutations for sequence MKHLPAQDEQVFNAIKNERERQQTKIELIASENFVSEAVMEAQGSVLTNKYAEGYPGKRYYGGCEHVDVVEDIARDRAKEIFGAEHVNVQPHSGAQANMAVYFTILEQGDTVLGMNLSHGGHLTHGSPVNFSGVQYNFVEYGVDKETQYIDYDDVREKALAHKPKLIVAGASAYPRTIDFKKFREIADEVGAYFMVDMAHIAGLVAAGLHPNPVPYADFVTTTTHKTLRGPRGGMILCREEFGKKIDKSIFPGIQGGPLMHVIAAKAVSFGEVLQDDFKTYAQNVISNAKRLAEALTKEGIQLVSGGTDNHLILVDLRSLGLTGKVAEHVLDEIGITSNKNAIPYDPEKPFVTSGIRLGTAAVTSRGFDGDALEEVGAIIALALKNHEDEGKLEEARQRVAALTDKFPLYKELDY|