The backbone dynamic properties of uniformly N-15-labeled calcium-saturated calmodulin (Ca2+-CaM) in 35% 2,2,2-trifluoroethanol (TEE) have been examined by N-15 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 Ca2+-CaM, which is a long, solvent-exposed a-helix in the crystal structure but is known to be partially unwound and flexible in solution. N-15 T-1, T-2. and N-15-{H-1} NOE values were determined for both Ca2+-CaM in H2O and Ca2+-CaM in 3% TFE, and the results indicated that the presence of 35% TEE 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 H2O and 0.82, 0.66, and 0.64 in 35% TEE. However, N-15-{H-1} NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78-Glu80 N-15-{H-1} NOE = 0.46 0.46. and 0.51). Furthermore, there is still independent motion of the two lobes of Ca2+-CaM in 35% TFE, with motional correlation times of similar to10 and similar to9ns for the N- and Globes, respectively, indicating that 35% TFE was not sufficient to force Ca2+-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. (C) 2003 Elsevier Inc. All rights reserved.