Conformational movement of the C-terminal α7 helix in the integrin inserted (I) domain, a major ligand-binding domain that adopts an α/β Rossmann fold, has been proposed to allosterically regulate ligand-binding activity. Disulfide bonds were engineered here to reversibly lock the position of the α7 helix in one of two alternative conformations seen in crystal structures, termed open and closed. Our results show that pairs of residues with Cβ atoms farther apart than optimal for disulfide bond stereochemistry can be successfully replaced by cysteine, suggesting that backbone movement accommodates disulfide formation. We also find more success with substituting partially exposed than buried residues. Disulfides stabilizing the open conformation resulted in constitutively active αMβ2 heterodimers and isolated αM inserted domains, which were reverted to an inactive form by dithiothreitol reduction. By contrast, a disulfide stabilizing the closed conformation resulted in inactive αMβ2 that was resistant to activation but became activatable after dithiothreitol treatment.