Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli

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Abstract
We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded .beta. sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner. Thus, for the most part, these results show that it is the role of position 75 in the folding of hydrofolate reductase that has been examined by mutagenesis. However, the tyrosine substitution produced striking effects in both equilibrium and kinetic studies that were not detected in the other mutant proteins. Therefore, a specific amino acid replacement can result in unique effects when compared with other replacements at the same site.