Substitution of four amino acid residues (L5V,F30V, Y33F,A34F) in the B1 domain of the immunoglobulin G binding protein (GB1) leads to the formation of a swapped dimer, shown to be in equilibrium with a native-like monomeric state of the protein (Byeon et al., J Mol Biol 2003;333:141-152). In this study, we employ protein design calculations and molecular dynamics simulations to investigate the role of these substitutions in fostering the swapping reaction. DESIGNER, a fully automatic procedure for computing the amino acid sequences likely to stabilize a given backbone structure is used to investigate the effect of the four substitutions on the stability of the wild type native monomeric conformation. Results indicate that at least three of these substitutions (L5V,F30V, A34F) have a destabilizing effect. The L5V forms destabilizing interactions with surrounding residues, whereas F30V causes local strain due to unfavorable interactions with its own backbone. A dual role in the swapping reaction is played by A34F. It destabilizes the monomer conformation while stabilizing the swapped dimer. Our calculations find an energetically favorable conformation for the 34F side chain in the core of the monomer, but only at the expense of forcing the wild type W43 side chain into a highly strained rotamer, and forming unfavorable interactions with both W43 and V54. Although detailed calculations could not be performed on the swapped dimer, due to the lower accuracy of the model, analysis revealed that the 34F side chain from both subunits are tightly packed against each other in the dimer core, suggesting that their replacement by the smaller Ala, as in the wild type, would be highly destabilizing through the creation of a large internal cavity possibly accompanied by a substantial conformational change. Analysis of room temperature molecular dynamics (MD) simulations of the wild type and the modeled quadruple mutant structures reveals that the latter structure fluctuates more than its wild type counterpart. In addition, its C-terminal beta-hairpin, which is exchanged in the swapping reaction, undergoes a conformation change, which pushes it further away from the remainder of the protein. Simulations at higher temperature (450 K) show that the quadruple mutant unfolds earlier and more completely than the wild type following a sequence of events that is compatible with the description of the highly fluctuating monomeric state of this mutant observed by NMR. Our findings thus support the notion that domain swapping in GB1 is fostered by three main factors: a decrease in stability and increased flexibility of the monomer conformation, concomitant with stabilization of the swapped dimer conformation through new interactions that have no counterparts in the monomer.