Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Calmodulin (CaM) is a crucial calcium-binding protein involved in numerous cellular processes.
• Understanding CaM's conformational dynamics is key to elucidating its diverse functions.
• CaM exists as a two-domain protein linked by a flexible interdomain helix.

Purpose of the Study:

• To explore the conformational landscape of the two-domain protein calmodulin.
• To investigate the preferred conformations adopted by calmodulin in solution.
• To develop and apply a novel NMR-based approach for studying multidomain protein dynamics.

Main Methods:

• Utilized Nuclear Magnetic Resonance (NMR) spectroscopy.
• Employed lanthanide-substituted calmodulin (using Tb(3+) and Tm(3+)).
• Measured pseudocontact shifts and residual dipolar couplings of the C-terminal domain relative to the N-terminal domain.

Main Results:

• Identified specific, non-uniform population distributions across sterically allowed conformations.
• The C-terminal domain preferentially occupies a region defined by a wide elliptical cone relative to the N-terminal domain.
• The preferred conformations differ significantly from the extended helix or closed states observed in other CaM forms.

Conclusions:

• Calmodulin samples a restricted conformational space, favoring specific orientations and rotations of its C-terminal domain.
• The developed NMR approach provides unique structural insights into multidomain proteins with dynamic conformational ensembles.
• This methodology is applicable to other multidomain proteins and macromolecular interactions.