Thermal unfolding molecular dynamics simulation of Escherichia coli dihydrofolate reductase: Thermal stability of protein domains and unfolding pathway

Abstract

Temperature induced unfolding of Escherichia coli dihydrofolate reductase was carried out by using molecular dynamic simulations. The simulations show that the unfolding generally involves an initial end-to-end collapse of the adenine binding domain into partially extended loops, followed by a gradual breakdown of the remaining beta sheet core structure. The core, which consists of beta strands 5-7, was observed to be the most resistant to thermal unfolding. This region, which is made up of part of the N terminus domain and part of the large domain of the E. coli dihydrofolate reductase, may constitute the nucleation site for protein folding and may be important for the eventual formation of both domains. The unfolding of different domains at different stages of the unfolding process suggests that protein domains vary in stability and that the rate at which they unfold can affect the overall outcome of the unfolding pathway. This observation is compared with the recently proposed hierarchical folding model. Finally, the results of the simulation were found to be consistent with a previous experimental study (Frieden, Proc Natl Acad Sci USA 1990;87:4413-4416) which showed that the folding process of E. coli dihydrofolate reductase involves sequential formation of the substrate binding sites.

The Role of Large-Scale Motions in Catalysis by Dihydrofolate Reductase

“…Faster H/D exchange in EcDHFR than in MpDHFR is also seen in helix αE and nearby residues (Figure 6), a region of EcDHFR thought to lose structure early in the thermal unfolding process, 60 also suggesting high flexibility, although it should be noted that the residues that show no exchange after 24 h in apo-EcDHFR are also in this region. Apo-DHFR from the thermophile Geobacillus stearothermophilus (BsDHFR) has been found to have greater flexibility than EcDHFR at comparable temperatures 61 demonstrating that the general tenet that thermophilic enzymes have reduced and psychrophilic enzymes greater flexibility does not necessarily always apply.…”

Highly site‐selective stability increases by glycosylation of dihydrofolate reductase

“…N18, E120 and D132 are all found in the loop domain, which retains structure until relatively late in the thermal unfolding process [40,41], whereas R52 is formally located in the adenosine‐binding domain but forms part of the substrate‐binding pocket. Sugars bound at position 52 are therefore more likely to interact with the relatively stable [40–42] substrate‐binding domain (Fig. 1).…”

A detailed unfolding pathway of a (β/α)8‐barrel protein as studied by molecular dynamics simulations

“…A native tertiary contact was defined as one where the distance between any two non-neighboring C ␣ atoms (contacts i ϩ 1 and i ϩ 2 were excluded) was within 6.0 Å and was present, in the 300 K simulation, more than 70% of the time. 38…”