Abstract
Here, we bind the sodium dependent amino acid transporter on nitrilotriacetic acid/polyethylene glycol functionalized gold sensors in detergents and perform a detergent-lipid exchange with phosphatidylcholine. We characterize the LeuT structure in the adsorbed film by magnetic contrast neutron reflection using the predicted model from molecular dynamic simulations.
Highlights
We bind the sodium dependent amino acid transporter on nitrilotriacetic acid/polyethylene glycol functionalized gold sensors in detergents and perform a detergent–lipid exchange with phosphatidylcholine
This is mainly due to the highly amphipathic character of membrane proteins that turns them into biomolecules that are extremely difficult to study. This is demonstrated by the fact that only B1% of the known total protein structures (101, 218 protein crystal structure appeared on the protein data bank on May 20th, 2015) belong to the membrane protein category, yet they are among the most important drug targets and potential biomarkers. In this communication we outline how we control membrane protein tethering on surfaces by using self-assembled monolayers (SAMs) made from a nitrilotriacetic acid/polyethylene glycol (NTA–PEG) anchor and a polyethylene glycol (PEG) spacer (Fig. 1A)
The high affinity of the metal–NTA technology together with the protein-repelling properties of PEG10 constitutes a valid approach to minimize non-specific protein adsorption to surfaces.[11,12]. In this communication we show that NTA–PEG coated Au surfaces can be used to promote the specific immobilization of LeuT in detergents
Summary
Cite this: Soft Matter, 2015, 11, 7707 Received 16th June 2015, Accepted 19th August 2015. This method was proposed earlier for biomembrane reconstitution of a membrane protein immobilized via the Ni-NTA technology on non-PEGylated linkers.[15] Initially, two different detergents, n-dodecyl b-D-maltopyranoside (DDM) or maltose-neopentyl glycol (MNG-3), were used for the preparation of LeuT Both detergents are known to preserve the stability and activity of LeuT.[16] QCM-D was used to follow the changes in frequency and dissipation as a function of time and in this way follow the LeuT immobilization process and the reconstitution of a lipid environment in situ (Fig. 2). Density profiles from MD simulations of lipid monolayers were compared to those fitted to NR data.[24]
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