Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini

Abstract

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

“…As slow exchange could be responsible for the broadening, spectra were acquired at higher temperatures (Figure S3); however, the W45 peak still remained broader than the W38 peak even at higher temperatures, which suggested broadening may arise from other reasons as well. The N‐termini of R67 DHFR are disordered, forming a collapsed structure, interacting with themselves and the monomer‐monomer interface of the tetramer core 2,18 . Since W45 is located at the monomer‐monomer interface, we hypothesized that the interaction of the disordered N‐termini with W45 could be responsible for the broad peaks.…”

“…Since W45 is located at the monomer‐monomer interface, we hypothesized that the interaction of the disordered N‐termini with W45 could be responsible for the broad peaks. The disordered N‐termini can be cleaved by α‐chymotrypsin from each of the protomers after Phe47 of R67 DHFR expressed from the pRSETb plasmid (Figure S4A), and the core of the enzyme remains intact and retains its activity 2,7,8,18–20 . Truncation of 5F R67 DHFR sharpened both peaks (Figure S4).…”

“…The chemical shift of W38 was unchanged by truncation. The N‐termini do not interact with the dimer‐dimer interface where W38 is located, 18 and the absence or presence of the N‐termini in the full‐length versus truncated proteins were not expected to alter the chemical shift of W38. W45 sharpened significantly into a single peak with similar line width to the W38 peak, which indicated the N‐termini induced the broadening of the W45 peaks.…”

“…Purified protein was dialyzed against deionized H 2 O, lyophilized, and stored at 4°C. α‐Chymotrypsin‐truncated R67 DHFR was prepared as described 18,19 . Protein concentrations were determined by measuring the absorbance at 280 nm using extinction coefficients (1.15, 1.38, 1.35, and 1.19 ml/mg for 4F, 5F, 6F, and 7F R67 DHFR, respectively) determined with a bicinchoninic acid assay (Pierce).…”

“…The N-termini of R67 DHFR are disordered, forming a collapsed structure, interacting with themselves and the monomer-monomer interface of the tetramer core. 2,18 Since W45 is located at the monomermonomer interface, we hypothesized that the interaction of the disordered N-termini with W45 could be responsible for the broad peaks. The disordered N-termini can be cleaved by α-chymotrypsin from each of the protomers after Phe47 of R67 DHFR expressed from the pRSETb plasmid ( Figure S4A), and the core of the enzyme remains intact and retains its activity.…”