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8.B.6.  The Reagent-mediated Membrane Protein Extraction/Renaturation (RMPER) Family 

Reproducible techniques for the fractionation of proteins prior to two-dimensional gel electrophoresis are essential for increasing the number of unique proteins that can be identified and assayed following biological experimentation. A simple and robust technique for separating hydrophilic cytoplasmic proteins from hydrophobic membrane-associated proteins uses differential solubility in a progressive series of extraction buffers, each containing more potent solubilizing surfactants, chaotropes and detergents. This 'sequential extraction' procedure is based on protein solubility in Tris buffer for the initial removal of highly soluble proteins, whereas proteins from the insoluble pellet are then extracted in buffers containing urea and CHAPS. The final step of the procedure uses detergents or thiourea and amidosulfobetaine-14 (ASB-14) to solubilize CHAPS-insoluble proteins. This procedure has been optimized for the analysis of outer membrane porins from Gram negative bacteria as well as the separation of plasma membrane proteins from mammalian cells (Cordwell 2008). 

Common denaturing agents such as chaotropes usually do not act on helical membrane proteins, and ionic detergents are often denaturants. Refolding a membrane protein is relatively straightforward for transmembrane β-barrel proteins but more challenging for α-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins. Roman and González Flecha 2014 described efforts to understand the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information is discussed in the context of paradigms in the protein folding field.

β-Barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert (see TC# 1.B.33). Model systems have been established to investigate the mechanisms of insertion and folding of these proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers (Kleinschmidt 2015). Insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies allow the investigation of membrane protein folding and insertion. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with cellular molecular chaperones. Kleinschmidt 2015 provides an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid-protein interactions and the functions of molecular chaperones and folding assistants.

Surfactants are surface-active materials that interact with membrane proteins and lipids (Guagliardo et al. 2018). Castillo-Sánchez et al. 2021 dissected the complexity of the structure-function relationships of surfactants with lipid-protein complexes and the membrane structures that sustain protein synthesis, secretion, interfacial performance and recycling. They reviewed models and the biophysical techniques employed to study the membranous architecture of surfactants. The structural and functional properties of surfactants are often studied in bulk or under static conditions, although surfactant function is strongly connected with highly dynamic behaviour, sustained by polymorphic structures and lipid-lipid, lipid-protein and protein-protein interactions that reorganize in precise spatio-temporal coordinates (Castillo-Sánchez et al. 2021).

References associated with 8.D.6 family:

Castillo-Sánchez, J.C., A. Cruz, and J. Pérez-Gil. (2021). Structural hallmarks of lung surfactant: Lipid-protein interactions, membrane structure and future challenges. Arch Biochem Biophys 703: 108850. 33753033
Cordwell, S.J. (2008). Sequential extraction of proteins by chemical reagents. Methods Mol Biol 424: 139-146. 18369859
Guagliardo, R., J. Pérez-Gil, S. De Smedt, and K. Raemdonck. (2018). Pulmonary surfactant and drug delivery: Focusing on the role of surfactant proteins. J Control Release 291: 116-126. 30321577
Kleinschmidt, J.H. (2015). Folding of β-barrel membrane proteins in lipid bilayers - Unassisted and assisted folding and insertion. Biochim. Biophys. Acta. 1848: 1927-1943. 25983306
Roman, E.A. and F.L. González Flecha. (2014). Kinetics and thermodynamics of membrane protein folding. Biomolecules 4: 354-373. 24970219