Effects of calcium binding on the internal dynamic properties of bovine brain calmodulin, studied by NMR and optical spectroscopy.
The dynamic properties of bovine brain calmodulin have been studied as a function of binding calcium ions, using a number of complementary spectroscopic methods. Rotational correlation times for proton-proton vectors within tyrosine and phenylalanine residues of calmodulin have been determined from time-dependent NOE measurements. In the presence of Ca2+, a range of rotational correlation times is observed. The longest value is consistent with Ca4-calmodulin having a markedly nonspherical shape in solution. In the absence of Ca2+, the rotational correlation times of all vectors are significantly shorter, indicating that several phenylalanine side chains in apocalmodulin have increased internal dynamics. Time-resolved tyrosine fluorescence anisotropy shows global correlation times broadly in agreement with the NMR results, but with an additional faster correlation time [approximately 600 ps]. Tyrosine residues in apocalmodulin have substantial segmental motion, which becomes significantly reduced, but not eliminated, when Ca2+ is bound. The correlation time for global rotation of Ca4-calmodulin increases from pH 7 to 4.5, indicating increased overall molecular asymmetry. This occurs without a significant change in total alpha-helix content as measured by circular dichroism. These results are consistent with the central region of Ca4-calmodulin being relatively flexible in solution at pH 7, but with the molecule adopting a more extended shape under more acidic conditions. The Ca(2+)-induced change in alpha-helix content can be mimicked by protonation. The alpha-helix content of Ca4-calmodulin in solution appears less than in the crystal structure; additional alpha-helix is induced in partially nonaqueous solutions, particularly at acidic pH, as used in crystallization conditions.
Török
K
K
National Institute for Medical Research, Mill Hill, London, U.K.
Lane
A N
AN
Martin
S R
SR
Janot
J M
JM
Bayley
P M
PM
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D013057:Spectrum Analysis
D002147:Calmodulin; D002118:Calcium