Kinetic analysis of antibody binding to integral membrane proteins stabilized in SMALPs.

The fundamental importance of membrane protein (MP) targets in central biological and cellular events has driven a marked increase in the use of membrane mimetics for exploring these proteins as therapeutic targets. The main challenge associated with biophysical analysis of membrane protein is the need for detergent extraction from the bilayer environment, which in many cases causes the proteins to become insoluble, unstable or display altered structure or activity. Recent technological advances have tried to limit the exposure of purified membrane protein to detergents. One such method involves the amphipathic co-polymer of styrene and maleic acid (SMA), which can release lipids and integral membrane proteins into water soluble native particles (or vesicles) termed SMALPs (Styrene Maleic Acid Lipid Particles). In this study, assay conditions that leverage SMA for membrane protein stabilization were developed to perform kinetic analysis of antibody binding to integral membrane protein and complexes in SMALPs in both purified and complex mixture settings using multiple biosensor platforms. To develop a robust and flexible platform using SMALPs technology, we optimized various SPR assay formats to analyze SMALPs produced with cell membrane pellets as well as whole cell lysates from the cell lines overexpressing membrane protein of interest. Here we emphasize the extraction of model membrane proteins of diverse architecture and function from native environments to encapsulate with SMALPs. Given the importance of selected membrane targets in central biological events and therapeutic relevance, MP-specific or tag-specific antibodies were used as a proof-of-principal to validate the SMALPs platform for ligand binding studies to support drug discovery or tool generation processes. MP-SMALPs that retain specific binding capability in multiple assay formats and biosensors, such as waveguide interferometry and surface plasmon resonance, would be a versatile platform for a wide range of downstream applications.

Encapsulated membrane proteins: A simplified system for molecular simulation.

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Current approaches and challenges in targeted absolute quantification of membrane proteins.

Experimental determination of absolute protein amounts is becoming increasingly important for the establishment and validation of biomarkers, and systems biology approaches aimed at a quantitative description of a biological process. Residing at compartmental or cellular barriers, and acting as prominent drug targets, integral membranes proteins, being completely embedded in the lipid bilayer, possess characteristic physicochemical properties and are often in low abundance. These features challenge the quantification with targeted MS and the ability to accurately determine the amount of membrane proteins with high sensitivity. This review summarizes the current status of targeted membrane protein quantification with emphasis on sample preparation beforehand MS. From the beginning to the end of a usual sample preparation workflow, consisting essentially of reference point selection, cell lysis, digestion, and addition of suitable isotope-labeled standards, general and particular challenges for membrane proteins will be discussed step by step. Based on the presentation of current achievements, possible measures to better address these challenges and future avenues of targeted membrane proteomics are presented.