In proteins, amino acid changes at "rheostat" positions exhibit functional changes that vary with the substitution chosen: some substitutions enhance function, some are like wild-type, some are partially detrimental, while others abolish function. One way that substitutions might exert their complex effects is by altering protein conformational landscapes. To test this, we studied five substitutions of V52 in E. coli LacI, an experimentally-known rheostat position. For each variant, we mapped the accessible conformational landscapes by performing molecular dynamics simulations at ambient conditions and under three perturbations: increased pressure, binding to allosteric ligand "ONPF", and ONPF plus pressure. The simulated DNA binding domain landscapes were compared to published experimentally-measured parameters, and the results suggest that complex combinations of dynamic parameters and/or additional simulations in the presence of DNA are needed to predict DNA binding specificity. For the variants regulatory domains all landscapes displayed boundaries similar to wild-type, but changes within the boundaries were unique. Of these, V52A/ONPF was striking: The regulatory domains for ONPF-bound, wild-type LacI are in an "Open" conformation and, experimentally, ONPF enhances DNA binding. Four variants responded to ONPF like wild-type, but ONPF binding to V52A shifted these domains to a "Closed" conformation that is associated with diminished DNA binding for wild-type LacI. This finding predicted that ONPF's allosteric regulation of V52A would change from "anti-inducer" to "inducer", which we experimentally validated in vivo and in vitro. This supports the hypothesis that substituting rheostat positions can alter function by altering the relative populations on protein conformational landscapes.