| Abstract
| - It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-handproteins such as calmodulin or troponin C. In this work we investigate the structure and stability of thefour EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presenceand absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structureand flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimentedwith steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II(F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structuresimilar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain.The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partiallyunfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stablestructure. The data reveal significant communication between the N- and C-lobes of CIB1, and show thattransient intermediate conformations are formed along the unfolding pathway for each form of the protein.Collectively the data demonstrate that the communication between the paired EF-hand domains as wellas between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C,which might be important for the unique function of CIB1 in numerous biological processes.
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