A Single Approach Reveals the Composite Conformational Changes, Order of Binding, and Affinities for Calcium Binding to Calmodulin

High Resolution Image Download MS PowerPoint Slide We found that a newly developed method named LITPOMS ( li gand t itration, fast p hotochemical o xidation of proteins and m ass s pectrometry) can characterize section-by-section of a protein the conformational changes induced by metal-ion binding. Peptide-level LITPOMS applied to Ca 2+ binding to calmodulin reveals binding order and site-specific affinity, providing new insights on the behavior of proteins upon binding Ca 2+ . We established that EF hand-4 (EF-4) binds calcium first, followed by EF-3, EF-2, and EF-1 and determined the four affinity constants by modeling the extent-of-modification curves. We also found positive cooperativity between EF-4, EF-3 and EF-2, EF-1 and allostery involving the four EF-hands. LITPOMS recapitulates via one approach the calcium-calmodulin binding that required decades of sophisticated development to afford versatility, comprehensiveness, and outstanding spatial resolution.