Probing conformational changes within LC2 domains of cardiac myosin.
Nine monoclonal antibodies were used to test calcium and EDTA effects on the molecular conformation of ventricular VLC2 within myosin. Antibody epitopes were located in six domains of VLC2 using recombinant proteins. The apparent association constants of these antibodies were measured in solution in the presence of calcium or EDTA. An immunofluorescence study was performed to establish whether the observed effects would occur in more integrated systems, as compared to isolated proteins in solution. Our results showed (1) a slight effect of calcium on isolated VLC2, located in the aa 29-45 domain, (2) a clear-cut effect of calcium on VLC2 within myosin, only in the aa 45-59 domain, and (3) in the presence of EDTA, antibody affinities for VLC2 within myosin similar to the affinities for isolated VLC2. These results are discussed in terms of spatial arrangements and binding mechanisms between HC and VLC2. They suggest that there are two processes for stabilizing HC/VLC2 complex formation: one binding via calcium chelation and another involving hydrophobic interactions.
Eldin
P
P
Institut National de la Santé et de la Recherche Médicale, INSERM U300, Faculté de Pharmacie, Montpellier, France.
Cathiard
A M
AM
Anoal
M
M
Léger
J
J
Mornet
D
D
Léger
J J
JJ
Cornillon
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D004492:Edetic Acid / Q000737:chemistry; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry*; D009210:Myofibrils / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D004492:Edetic Acid; D009218:Myosins; D002118:Calcium