Tolerance of Arc repressor to multiple-alanine substitutions

Arc repressor mutants containing from three to 15 multiple-alanine substitu tions have spectral properties expected for native Arc proteins, form heter odimers with wild-type Arc, denature cooperatively with T(m)s equal to or g reater than wild type, and, in some cases, fold as much as 30-fold faster a nd unfold as much as 50-fold slower than wild type. Two of the mutants, con taining a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutat ions is significantly greater than that predicted from the sum of the singl e substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that al most half of the residues in Arc can be replaced by alanine en masse withou t compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.