Molecular dynamics simulation of Escherichia coli dihydrofolate reductase and its protein fragments: Relative stabilities in experiment and simulations

Abstract

We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1-36, 37-88, and 89-159 to be unstable, with a C ␣ RMSD (C ␣ root mean squared deviation) >5 Å after 3.0 nsec of simulation. The unfolding of fragment 1-36 was immediate, whereas fragments 37-88 and 89-159 gradually unfolded because of the presence of the ␤-sheet core structure. In the absence of residues 1-36, the two distinct domains comprising fragment 39-159 associated with each other, resulting in a stable conformation. This conformation retained most of its native structural elements. We have further simulated fragments derived from computational protein cutting. These were also found to be unstable, with the exception of fragment 104-159. In the absence of ␣ 4 , the loose loop region of residues 120-127 exhibited a ␤-strand-like behavior, associating itself with the ␤-sheet core of the protein fragment. The current study suggests that the folding of dihydrofolate reductase involves cooperative folding of distinct domains which otherwise would have been unstable as independent folded units in solution. Finally, the critical role of residues 1-36 in allowing the two distinct domains of fragment 104-159 to fold into the final native conformation is discussed.

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

“…Current results are consistent with earlier experimental spectroscopic34 and MD simulation studies of EcDHFR fragments 30. Of the eight overlapping fragments studied by CD and fluorescence spectroscopy as a function of denaturation by urea, fragments consisting of the ABD, ND, and LD were observed to be disordered, whereas the fragment consisting of both the ABD and LD (residues 37–159) shows a significant extent of native secondary structural characteristics.…”

“…This fluctuation of the C α atoms relative to the reference structure is shown in Figure 5. Such a movement was also observed in another simulation of EcDHFR in explicit solvent but not to such a large extent 30. Such a movement of the loop region is a reflection of the high mobility, characteristic of this region as shown by the high β‐values obtained for this region from X‐ray studies 26.…”

“…Previous fragment studies34 showed that the LD does not fold spontaneously on its own. However, simulation studies have suggested that the domain is relatively stable in its native fold 30. This finding suggests that native LD, although thermodynamically stable, may consist of a large activation folding barrier.…”

Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity

“…Intramolecular chaperones are generally divided into two families: one family in which the chaperone region is proteolytically cleaved after the formation of the mature protein and the other family in which the chaperone region is not cleaved. Examples of the first group are the subtilisin from Bacillus subtilis and the ␣-lytic protease of Lysobacter enzymogenes (19), while an example of the second group is the DHFR of E. coli (29). It has been proposed that such fragments may exist in many proteins, though it is difficult to predict how many proteins have such sequences.…”

Folding with a protein's native shortcut network

“…DHFR (dihydrofolate reductase) is a much studied model protein with multiple PDB entries. We use the crystallized DHFR structure from E. coli 7DFR, which according to the SSE delineation 7 in [39] has four α-helices and eight β-strands. DHFR has two structural domains: ABD (adenine binding domain) between residues 38 and 88, and a discontinuous domain (DD) spanning residues 1...37 and 89...159 [78].…”