Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein

Abstract

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

“…Protein Purification-High yields of R67 DHFR were obtained as described previously (17). Briefly, ammonium sulfate precipitation and ion-exchange column chromatography were used to purify the protein to homogeneity.…”

Crystal Structure of a Type II Dihydrofolate Reductase Catalytic Ternary Complex

“…Recombinant R67 DHFR was purified as previously described (17). The 16 N-terminal residues were removed using chymotrypsin, yielding a fully active protein composed of 62-residue peptides (17).…”

Conformational stability of dimeric proteins: Quantitative studies by equilibrium denaturation

“…Tests for the 2-state nature of the dimer dissociation are critical and should be made extensively with different protein concentrations and conditions. Tryptophan fluorescence (static, dynamic, and/or anisotropic) to detect tertiary structural changes is frequently used in conjunction with CD to assess secondary structure (Bowie & Sauer, 1989a;Reece et al, 1991;Timm & Neet, 1992;Mann et al, 1993). NMR is powerful but less often used (Raleigh & DeGrado, structure that are occasionally utilized (Dirr & Reinemer, 1991).…”

“…NMR is powerful but less often used (Raleigh & DeGrado, structure that are occasionally utilized (Dirr & Reinemer, 1991). The dependence of these techniques on protein concentration is a unique characteristic of the coupled denaturation and dissociation of oligomeric protein systems (2-or 3-state) and should be used as a definitive criterion for assigning the type of system as well as for evaluating the thermodynamic parameters (Bowie & Sauer, 1989a;Gittelman & Matthews, 1990;De Francesco et al, 1991;Liang & Terwilliger, 1991;Reece et al, 1991;Grant et al, 1992;Monera et al, 1992;Timm & Neet, 1992;Thompson et al, 1993;Timm et al, 1994). Alternatively, methods that directly determine the association state, such as ultracentrifugation or gel filtration, can directly detect the dissociation step.…”

“…Two-residue substitutions in either the N-terminal or the C-terminal region of a 34-residue peptide from troponin C decrease the conformational stability of the dimeric peptide by 4 kcal/mol (Monera et al, 1992). Deletion of 16 residues from the N-terminus of the 78-residue R67 DHFR peptide decreases the stability by 2.6 kcal/mol (Reece et al, 1991). Human and mouse NGF differ in 12 of 118 residues but differ by 4 kcal/mol (Timm et al, 1994); key residues have been suggested to account for this difference.…”