Tolerance of Arc repressor to multiple-alanine substitutions

Abstract

Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.

Figure 1

(A) Orthogonal views of the Arc repressor dimer prepared by using molscript (41). The C_α atoms at each of the 22 positions (44 in dimer) substituted by alanines are marked by spheres. (B) Arc sequence, secondary structure, and positions of multiple-alanine substitutions in the 3A, 7A, 11A, 12A, and 15A mutants.

Figure 2

NMR spectra in the amide-aromatic and methyl regions of wild-type Arc-st11 and the 15A mutant.

Figure 3

Copper-phenanthroline footprinting of the arc operator at 22°C by wild-type Arc-st11 and the multiple-alanine mutants at protein concentrations of 1 μM in Tris⋅HCl (pH 7.5), 3 mM MgCl₂, 100 mM KCl, 0.1 mM EDTA, 100 μg/ml BSA, and 0.02% Nonidet P-40.

Figure 4

Thermal denaturation of the multiple-alanine mutants (10 μM) in buffer S. Normalized ellipticity is calculated as (ɛ − ɛ₂₅)/(ɛ₉₅ − ɛ₂₅) where ɛ, ɛ₂₅, and ɛ₉₅ are the CD ellipticities at 222 nm at any temperature, 25°C, and 95°C, respectively.

Figure 5

(A) Unfolding and dissociation rate constants determined after jumps into different concentrations of urea or GuHCl (25°C, buffer S). (B) Refolding and association rate constants determined after dilution from 6 M GuHCl. (C) Reaction-coordinate diagrams for wild-type Arc and the 15A mutant.