In cellular signaling cascades, protein activity can be controlled by molecular-switch directed ligand recognition. With differential ligand-binding arising from interconversion of two prolyl imide bond conformers, peptidyl-prolyl cis/trans isomerization has been identified as a noncovalent molecular switch mechanism. Involved in the T cell signaling pathway, Interleukin-2 tyrosine kinase (Itk) contains a Src homology 2 (SH2) regulatory domain that exists in solution as two conformers due to prolyl isomerization within the native state protein. To investigate the role that local structural factors play in governing and enabling prolyl-driven conformational heterogeneity within the Itk SH2 domain, variants from alanine-scanning mutagenesis and peptide models of the proline-containing loop were characterized using NMR spectroscopy. The data indicate that several factors play a role, including a stabilizing hydrophobic patch within the loop, the identity of the residues directly preceding the isomeric proline, and strain introduced at the base of the loop. Local factors alone did not account for the large degree of conformational heterogeneity observed, indicating that interactions within the folded protein also play an important role in enabling dual imide bond occupancy. As understanding of the molecular level determinants increases with continued structural characterizations, the mechanisms underlying molecular switches will be illuminated.