Dihydrofolate reductase (DHFR) has been a well-established model system for protein folding. The enzyme DHFR from the hyperthermophilic bacterium Thermotoga maritima (TmDHFR) displays distinct adaptations toward high temperatures at the level of both structure and stability. The enzyme represents an extremely stable dimer; no isolated structured monomers could be detected in equilibrium or during unfolding. The equilibrium unfolding strictly follows the two-state model for a dimer (N(2) right harpoon over left harpoon 2U), with a free energy of stabilization of DeltaG = -142 +/- 10 kJ/mol at 15 degrees C. The two-state model is applicable over the whole temperature range (5-70 degrees C), yielding a DeltaG vs T profile with maximum stability at around 35 degrees C. There is no flattening of the stability profile. Instead, the enhanced thermostability is characterized by shifts toward higher overall stability and higher temperature of maximum stability. TmDHFR unfolds in a highly cooperative manner via a nativelike transition state without intermediates. The unfolding reaction is much slower (ca. 10(8) times) compared to DHFR from Escherichia coli (EcDHFR). In contrast to EcDHFR, no evidence for heterogeneity of the native state is detectable. Refolding proceeds via at least two intermediates and a burst-phase of rather low amplitude. Reassociation of monomeric intermediates is not rate-limiting on the folding pathway due to the high association constant of the dimer. Study holds ProTherm entries: 5415, 5416, 5417, 5418, 5419, 5420, 5421 Extra Details: additive : EDTA(0.2 mM), hyperthermophilic bacterium; cooperative manner;,burst-phase; folding pathway; association constant
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:30 p.m.
|Number of data points||14|
|Proteins||Dihydrofolate reductase ; Dihydrofolate reductase|
|Assays/Quantities/Protocols||Experimental Assay: m temp:70.0 C ; Experimental Assay: dG_H2O temp:70.0 C ; Experimental Assay: m temp:55.0 C ; Experimental Assay: dG_H2O temp:55.0 C ; Experimental Assay: m temp:37.0 C ; Experimental Assay: dG_H2O temp:37.0 C ; Experimental Assay: m temp:25.0 C ; Experimental Assay: dG_H2O temp:25.0 C ; Experimental Assay: m temp:15.0 C ; Experimental Assay: dG_H2O temp:15.0 C ; Experimental Assay: m temp:15.0 C ; Experimental Assay: dG_H2O temp:15.0 C ; Experimental Assay: m temp:5.0 C ; Experimental Assay: dG_H2O temp:5.0 C|
|Libraries||Mutations for sequence AKVIFVLAMDVSGKIASSVESWSSFEDRKNFRKITTEIGNVVMGRITFEEIGRPLPERLNVVLTRRPKTSNNPSLVFFNGSPADVVKFLEGKGYERVAVIGGKTVFTEFLREKLVDELFVTVEPYVFGKGIPFFDEFEGYFPLKLLEMRRLNERGTLFLKYSVEKSHR|