Unfolding mechanics of holo- and apocalmodulin studied by the atomic force microscope.
The structural stability of calmodulin (CaM) has been investigated previously by chemical and thermal methods. The calcium-loaded form of CaM has been found to be exceptionally stable, because it can be exposed to temperatures >90 degrees C or to a 9 M urea solution without a marked change in its tertiary structure, and is therefore not experimentally accessible for unfolding studies using conventional analytical methods. In this study, we have developed a system for measuring the force for mechanically unfolding CaM using an atomic force microscope (AFM) by stretching the protein from its N- and C-terminal residues; we have been successful in obtaining force versus extension (F-E) curves for both apo and holo forms of CaM. In our experiment, distinguishable F-E curves have been obtained upon stretching of apoCaM and holoCaM to their full extensions. A very low force observed upon stretching of apoCaM indicated a relatively high flexibility of the apo form. On the contrary, a relatively high unfolding force and the appearance of a characteristic force peak were noted during full stretching of holoCaM. The F-E curve of the latter form of CaM most likely reflects a more rigid and probably more organized conformation of holoCaM than that of apoCaM. These experiments confirmed that the AFM is able to clearly distinguish two functionally distinct forms of CaM in terms of their mechanical properties.
Hertadi
Rukman
R
Laboratory of Biodynamics, Tokyo Institute of Technology, Midoriku, Yokohama, Japan 226-8501.
Ikai
Atsushi
A
D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001696:Biomechanical Phenomena; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D018625:Microscopy, Atomic Force / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering
D001059:Apoproteins; D002147:Calmodulin; D002118:Calcium