The Escherichia coli biotin repressor, BirA, is an allosteric transcriptional regulatory protein that undergoes ligand-induced loop folding upon corepressor binding. The folding results in formation of a hydrophobic cluster composed of a tryptophan and two valine side chains. Functionally, corepressor binding results in enhanced repressor dimerization that is linked to assembly of the transcriptional repressor on DNA. Single alanine replacements in the hydrophobic cluster perturb both corepressor binding and homodimerization, indicating that loop folding is required for the allosteric response. In this work, further perturbation of the hydrophobic cluster was investigated by creating BirA variants with double and triple alanine replacements at these same positions. Isothermal titration calorimetry measurements of corepressor binding and sedimentation equilibrium measurements of homodimerization reveal non-additivity in the effects of the multiply substituted loop. The results further indicate that certain combinations of alanine substitutions lead to reversion of the allosteric response toward that observed for the wild type protein. The reversion may structurally reflect side chain repacking that yields a folded conformation of the loop that is competent for allosteric activation.