Effect of urea on Ca²⁺-ATPase conformation and activity studied by infrared spectroscopy

Abstract

Sarcoplasmic reticulum Ca²⁺-ATPase is an integral protein that pumps calcium out of the cytoplasm during striated muscle relaxation [1]. This is a P-type ATPase whose structure has been solved at 8 Å resolution by electron cryocrystallography [2].The P-type ATPases are a family of transmembrane ion pumps primarily involved in the ATP hydrolisis-dependent transmembrane movement of a variety of different inorganic cations against a concentration gradient having in common ten predicted transmembrane helical segments [3 ]. The protein appears to consist of an extensive beak-shaped cytoplasmic domain containing interconnected α-helical and β-strand segments, a stalk connecting the beak with the membrane, and ten transmembrane segments characteristic of P-type ion pumps [4], The cytoplasmic domain contains the active sites of ATP hydrolysis and phosphorylation while the Ca²⁺ channel is expected to be associated to the transmembrane domain. Some Ca²⁺-binding sites, probably related to Ca²⁺ translocation, which is in turn coupled to ATP hydrolysis, can also be found in the cytoplasmic and lumenal regions of the membrane close to the transmembrane helices. Different experimental approaches, such as susceptibility of protein domains to protease digestion or changes in the interaction between protomers to form oligomeric structures, have been undertaken in order to understand the structural changes that link protein phosphorylation with cation translocation.