Backbone dynamic properties of the central linker region of calcium-calmodulin in 35% trifluoroethanol.
The backbone dynamic properties of uniformly (15)N-labeled calcium-saturated calmodulin (Ca(2+)-CaM) in 35% 2,2,2-trifluoroethanol (TFE) have been examined by (15)N 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 alpha-helix in the crystal structure but is known to be partially unwound and flexible in solution. (15)N T(1), T(2), and (15)N-[(1)H] NOE values were determined for both Ca(2+)-CaM in H(2)O 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(2)O and 0.82, 0.66, and 0.64 in 35% TFE. However, (15)N-[(1)H] NOE values showed that these residues were still slightly more flexible than the rest of the molecule in 35% TFE (Asp78-Glu80 (15)N-[(1)H] 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 approximately 10 and approximately 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.
Brokx
Richard D
RD
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Dr. N.W., Calgary, Alta., Canada T2N 1N4.
Scheek
Ruud M
RM
Weljie
Aalim M
AM
Vogel
Hans J
HJ
D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry*; D007700:Kinetics; D009038:Motion; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D011994:Recombinant Proteins; D014270:Trifluoroethanol / Q000494:pharmacology; D014867:Water
D002147:Calmodulin; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014867:Water; D014270:Trifluoroethanol; D002118:Calcium