Abstract
Phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) was studied with time-resolved Fourier transform infrared spectroscopy. ATP and ATP analogs (ITP, 2'- and 3'-dATP) were used to study the effect of the adenine ring and the ribose hydroxyl groups on ATPase phosphorylation. All modifications of ATP altered conformational changes and phosphorylation kinetics. The differences compared with ATP increased in the following order: 3'-dATP > ITP > 2'-dATP. Enzyme phosphorylation with ITP results in larger absorbance changes in the amide I region, indicating larger conformational changes of the Ca(2+)-ATPase. The respective absorbance changes obtained with 3'-dATP are significantly different from the others with different band positions and amplitudes in the amide I region, indicating different conformational changes of the protein backbone. ATPase phosphorylation with 3'-dATP is also much ( approximately 30 times) slower than with ATP. Our results indicate that modifications to functional groups of ATP (the ribose 2'- and 3'-OH and the amino group in the adenine ring) affect gamma-phosphate transfer to the phosphorylation site of the Ca(2+)-ATPase by changing the extent of conformational change and the phosphorylation rate. ADP binding to the ADP-sensitive phosphoenzyme (Ca(2)E1P) stabilizes the closed conformation of Ca(2)E1P.
Highlights
In skeletal muscle cells the Ca2ϩ-ATPase of sarcoplasmic reticulum (SR)1 pumps Ca2ϩ actively from the cytoplasm into the SR lumen [1,2,3,4,5,6]
The Conformational Change Observed by Infrared Spectroscopy—Previously we have estimated that the net change in secondary structure involves up to 10 amino acids for all partial reactions of the SR Ca2ϩ-ATPase [12]
Our results demonstrate that infrared spectroscopy can be used to probe ligand-protein interactions
Summary
In skeletal muscle cells the Ca2ϩ-ATPase of sarcoplasmic reticulum (SR) pumps Ca2ϩ actively from the cytoplasm into the SR lumen [1,2,3,4,5,6]. Infrared spectroscopy (14 –18) has several advantages for elucidating the molecular mechanism of proteins, such as high time resolution, universal applicability from small soluble proteins to large membrane proteins, and high molecular information content combined with a sensitivity high enough to detect a change in the environment around a single atom of a large protein. These properties make this method very useful to obtain information on enzyme-substrate recognition (18 –21). Photolabile derivatives, i.e. P3-1-(2-nitrophenyl)ethyl nucleotides (caged nucleotides) [30], were used to trigger the protein reaction directly in the infrared cuvette in order to detect the small infrared absorbance changes associated with phosphorylation of the Ca2ϩ-ATPase
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