AbstractThe SPRY domain was identified originally as a sequence repeat in the dual‐specificity kinase splA and ryanodine receptors and subsequently found in many other distinct proteins, including more than 70 encoded in the human genome. It is a subdomain of the B30.2/SPRY domain and is believed to function as a protein–protein interaction module. Three‐dimensional structures of several B30.2/SPRY domain–containing proteins have been reported recently: murine SSB‐2 in solution by NMR spectroscopy, a Drosophila SSB (GUSTAVUS), and human PRYSPRY protein by X‐ray crystallography. The three structures share a core of two antiparallel β‐sheets for the B30.2/SPRY domain but show differences located mainly at one end of the β‐sandwich. Analysis of SSB‐2 residues required for interactions with its intracellular ligands has provided insights into B30.2/SPRY binding specificity and identified loop residues critical for the function of this domain. We have investigated the backbone dynamics of SSB‐2 by means of Modelfree analysis of its backbone 15N relaxation parameters and carried out coarse‐grained dynamics simulation of B30.2/SPRY domain–containing proteins using normal mode analysis. Translational self‐diffusion coefficients of SSB‐2 measured using pulsed field gradient NMR were used to confirm the monomeric state of SSB‐2 in solution. These results, together with previously reported amide exchange data, highlight the underlying flexibility of the loop regions of B30.2/SPRY domain–containing proteins that have been shown to be important for protein–protein interactions. The underlying flexibility of certain regions of the B30.2/SPRY domain–containing proteins may also contribute to some apparent structural differences observed between GUSTAVUS or PRYSPRY and SSB‐2.