Abstract
Purified integral membrane proteins require amphipathic molecules to maintain their solubility in aqueous solutions. These complexes, in turn, are used in studies to characterise the protein structures by a variety of biophysical and structural techniques, including spectroscopy, crystallography, and cryo‐electron microscopy. Typically the amphilphiles used have been detergent molecules, but more recently they have included amphipols, which are polymers of different sizes and compositions designed to create smaller, more well‐defined solubilised forms of the membrane proteins. In this study we used circular dichroism spectroscopy to compare the secondary structures and thermal stabilities of the NavMs voltage‐gated sodium channel in different amphipols and detergents as a means of identifying amphipathic environments that maximally maintain the protein structure whilst providing a stabilising environment. These types of characterisations also have potential as means of screening for sample types that may be more suitable for crystallisation and/or cryo‐electron microscopy structure determinations.
Highlights
Considerations in the choice of amphipathic molecules are the ability to maintain the structural and functional integrity of the protein in the Membrane proteins require amphipathic molecules for solubility and amphipathic complex, as well as to match the sizes and shapes of the stability in aqueous solutions.[1]
Whilst detergents are generally used for crystallography (and NavMs crystals diffracted to high resolution (2.45 Å) in HEGA10), amphipols have been shown to be more suitable for cryoEM structure determinations of a number channel of proteins.[17]
Circular dichroism spectroscopy was utilised to monitor the thermal stability of a membrane protein
Summary
Considerations in the choice of amphipathic molecules are the ability to maintain the structural and functional integrity of the protein in the Membrane proteins require amphipathic molecules for solubility and amphipathic complex, as well as to match the sizes and shapes of the stability in aqueous solutions.[1]. Circular dichroism (CD) spectroscopy is an important method for characterising membrane protein structure and stability.[5] It can probeen shown to be effective alternative environments in which mem- vide information on the secondary structure of proteins in brane proteins can retain their structure and function. They have pro- amphiphiles, and on the thermal stability of membrane proteins in ven to be of particular value in producing samples for high resolution these environments. It provides a system that enables us to monitor the specific unfolding/refolding of helical secondary structural elements
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