The backbone dynamic properties of uniformly 15N-labeled calcium-saturated calmodulin (Ca 2+-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by 15N NMR relaxation methods. This particular solvent was chosen in order to mimic the conditions in which CaM was crystallized, which included the presence of alcohols. Special attention was paid to the central linker region of Ca 2+-CaM, which is a long, solvent-exposed α-helix in the crystal structure but is known to be partially unwound and flexible in solution. 15N T 1, T 2, and 15N–{ 1H} NOE values were determined for both Ca 2+-CaM in H 2O and Ca 2+-CaM in 35% TFE, and the results indicated that the presence of 35% TFE did indeed induce a more ordered conformation in the central linker, with order parameters for Asp78–Glu80 of 0.29, 0.17, and 0.27 in H 2O and 0.82, 0.66, and 0.64 in 35% TFE. However, 15N–{ 1H} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78–Glu80 15N–{ 1H} NOE=0.46, 0.46, and 0.51). Furthermore, there is still independent motion of the two lobes of Ca 2+-CaM in 35% TFE, with motional correlation times of ∼10 and ∼9 ns for the N- and C-lobes, respectively, indicating that 35% TFE was not sufficient to force Ca 2+-CaM into a rigid dumbbell-shaped molecule as seen in the crystal structure. Additional factors that could further stabilize the structure of CaM in the crystal include pH, temperature, and crystal packing.