Raman Spectroscopy of Conformational Changes in Membrane-Bound Sodium Potassium ATPase

Claus Helix Nielsen, Salim Abdali, Jens August Lundbæk, Flemming Cornelius

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In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins — in this case, the sodium potassium adenosine triphosphatase (Na+,K+-ATPase). Spectral analysis of protein-lipid complexes is complicated by the presence of a lipidic environment, rendering the amide I spectral region difficult to analyze. We
have therefore focused on spectral changes in the disulfide region arising from cysteine (Cys) cross-linking and the tyrosine (Tyr) Fermi doublet region arising from changes in Tyr hydrogen bonding environment. Specifically, we have studied the conformational changes in the Na+,K+- ATPase undergoing the E2→E1 transition as controlled by Na+. These conformational changes
were compared to the E1 conformation stabilized by controlled proteolytic digestion. Our results show that controlled digestion promoting the E1 conformation leads to changes in cross-linking between Cys residues as well as changes in the Tyr hydrogen bonding environment. In contrast, Na+ binding, which also promotes the E1 conformation, does not lead to any changes in these
spectral regions. This demonstrates that the functional E1 state of the Na+,K+-ATPase stabilized by N-terminal truncation differs from that induced by Na+ binding, and that the N-terminal truncation leads to changes in protein structure that affect the average hydrophobic environment of protein Tyr, possibly reflecting changes in the hydrophobic coupling between protein and membrane. This illustrates the stabilizing role of the N-terminal domain under physiological conditions. More generally, it shows that Raman spectroscopy might be a useful tool in understanding the relationship between functional states and structural changes in membrane-bound proteins.

Original languageEnglish
Issue number2
Pages (from-to)52-63
Publication statusPublished - 2007


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