Identifying lipids tightly bound to an integral membrane protein

Abstract

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.

Keywords: Enterobacterial common antigen; Membrane proteins; Non-annular lipids; Phosphatidylethanolamine; Protein-lipid interactions; Solid state NMR.