scholarly journals Membrane protein engineering to the rescue

2018 ◽  
Vol 46 (6) ◽  
pp. 1541-1549
Author(s):  
Andrea E. Rawlings
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Engineering ◽  
Mutation Screening ◽  
Water Soluble ◽  
Scaffold Proteins ◽  
Major Barrier ◽  
Expression Levels ◽  
Aqueous Environments ◽  
Protein Research

The inherent hydrophobicity of membrane proteins is a major barrier to membrane protein research and understanding. Their low stability and solubility in aqueous environments coupled with poor expression levels make them a challenging area of research. For many years, the only way of working with membrane proteins was to optimise the environment to suit the protein, through the use of different detergents, solubilising additives, and other adaptations. However, with innovative protein engineering methodologies, the membrane proteins themselves are now being adapted to suit the environment. This mini-review looks at the types of adaptations which are applied to membrane proteins from a variety of different fields, including water solubilising fusion tags, thermostabilising mutation screening, scaffold proteins, stabilising protein chimeras, and isolating water-soluble domains.

scholarly journals Membrane proteins: always an insoluble problem?

2016 ◽  
Vol 44 (3) ◽  
pp. 790-795 ◽  
Author(s):  
Andrea E. Rawlings
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Drug Targets ◽  
Functional Characterization ◽  
Water Soluble ◽  
High Yield ◽  
Cellular Processes ◽  
Native Sequence ◽  
Protein Research ◽  
New Methodologies

Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence.

Mutations in transmembrane proteins: diseases, evolutionary insights, prediction and comparison with globular proteins

2020 ◽  
Author(s):  
Jan Zaucha ◽  
Michael Heinzinger ◽  
A Kulandaisamy ◽  
Evans Kataka ◽  
Óscar Llorian Salvádor ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Protein Structures ◽  
Experimental Studies ◽  
Single Amino Acid ◽  
Evolutionary Information ◽  
Missense Variants ◽  
Current State ◽  
Aqueous Environments

Abstract Membrane proteins are unique in that they interact with lipid bilayers, making them indispensable for transporting molecules and relaying signals between and across cells. Due to the significance of the protein’s functions, mutations often have profound effects on the fitness of the host. This is apparent both from experimental studies, which implicated numerous missense variants in diseases, as well as from evolutionary signals that allow elucidating the physicochemical constraints that intermembrane and aqueous environments bring. In this review, we report on the current state of knowledge acquired on missense variants (referred to as to single amino acid variants) affecting membrane proteins as well as the insights that can be extrapolated from data already available. This includes an overview of the annotations for membrane protein variants that have been collated within databases dedicated to the topic, bioinformatics approaches that leverage evolutionary information in order to shed light on previously uncharacterized membrane protein structures or interaction interfaces, tools for predicting the effects of mutations tailored specifically towards the characteristics of membrane proteins as well as two clinically relevant case studies explaining the implications of mutated membrane proteins in cancer and cardiomyopathy.

scholarly journals The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Michael Luke Carlson ◽  
John William Young ◽  
Zhiyu Zhao ◽  
Lucien Fabre ◽  
Daniel Jun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Cost Effective ◽  
Water Soluble ◽  
Protein Assemblies ◽  
Simple Method ◽  
Helical Peptide ◽  
Biological Studies ◽  
Multiple Copies ◽  
Exposed Part

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

scholarly journals Methods for the solubilisation of membrane proteins: the micelle-aneous world of membrane protein solubilisation

2021 ◽  
Author(s):  
Giedre Ratkeviciute ◽  
Benjamin F. Cooper ◽  
Timothy J. Knowles
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Stability ◽  
Lipid Membranes ◽  
Lipid Membrane ◽  
Modus Operandi ◽  
Membrane Mimetic ◽  
Aqueous Environments ◽  
Recent Developments

The solubilisation of membrane proteins (MPs) necessitates the overlap of two contradictory events; the extraction of MPs from their native lipid membranes and their subsequent stabilisation in aqueous environments. Whilst the current myriad of membrane mimetic systems provide a range of modus operandi, there are no golden rules for selecting the optimal pipeline for solubilisation of a specific MP hence a miscellaneous approach must be employed balancing both solubilisation efficiency and protein stability. In recent years, numerous diverse lipid membrane mimetic systems have been developed, expanding the pool of available solubilisation strategies. This review provides an overview of recent developments in the membrane mimetic field, with particular emphasis placed upon detergents, polymer-based nanodiscs and amphipols, highlighting the latest reagents to enter the toolbox of MP research.

scholarly journals Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

2021 ◽  
Author(s):  
Kristen Gaffney ◽  
Ruiqiong Guo ◽  
Michael D Bridges ◽  
Daoyang Chen ◽  
Shaima Muhammednazaar ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Water Soluble ◽  
Disordered Proteins ◽  
Resonance Spectroscopy ◽  
Denatured State ◽  
Helical Bundle ◽  
Intrinsically Disordered ◽  
State Ensemble

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured its conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55-90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

The nanodisc: a novel tool for membrane protein studies

2009 ◽  
Vol 390 (8) ◽  
Author(s):  
Jonas Borch ◽  
Thomas Hamann
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Biological Membrane ◽  
Integral Membrane Protein ◽  
Water Soluble ◽  
High Density Lipoproteins ◽  
And Control ◽  
Membrane Scaffold ◽  
Integral Proteins

Abstract A major challenge in the research on membrane-anchored and integral membrane protein complexes is to obtain these in a functionally active, water-soluble, and monodisperse form. This requires the incorporation of the membrane proteins into a native-like membrane or detergent micelle that mimics the properties of the original biological membrane. However, solubilization in detergents or reconstitution in liposomes or supported monolayers sometimes suffers from loss of activity and problematic analyses due to heterogeneity and aggregation. A developing technology termed nanodiscs exploits discoidal phospholipid bilayers encircled by a stabilizing amphipatic helical membrane scaffold protein to reconstitute membranes with integral proteins. After reconstitution, the membrane nanodisc is soluble, stable, and monodisperse. In the present review, we outline the biological inspiration for nanodiscs as discoidal high-density lipoproteins, the assembly and handling of nanodiscs, and finally their diverse biochemical applications. In our view, major advantages of nanodisc technology for integral membrane proteins is homogeneity, control of oligomerization state, access to both sides of the membrane, and control of lipids in the local membrane environment of the integral protein.

scholarly journals Selection of Biophysical Methods for Characterisation of Membrane Proteins

2019 ◽  
Vol 20 (10) ◽  
pp. 2605 ◽  
Author(s):  
Tristan O. C. Kwan ◽  
Rosana Reis ◽  
Giuliano Siligardi ◽  
Rohanah Hussain ◽  
Harish Cheruvara ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Early Stage ◽  
Biological Membrane ◽  
Basic Research ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Functional Protein ◽  
Protein Research ◽  
Selection Of

Over the years, there have been many developments and advances in the field of integral membrane protein research. As important pharmaceutical targets, it is paramount to understand the mechanisms of action that govern their structure–function relationships. However, the study of integral membrane proteins is still incredibly challenging, mostly due to their low expression and instability once extracted from the native biological membrane. Nevertheless, milligrams of pure, stable, and functional protein are always required for biochemical and structural studies. Many modern biophysical tools are available today that provide critical information regarding to the characterisation and behaviour of integral membrane proteins in solution. These biophysical approaches play an important role in both basic research and in early-stage drug discovery processes. In this review, it is not our objective to present a comprehensive list of all existing biophysical methods, but a selection of the most useful and easily applied to basic integral membrane protein research.

scholarly journals Artificial membranes for membrane protein purification, functionality and structure studies

2016 ◽  
Vol 44 (3) ◽  
pp. 877-882 ◽  
Author(s):  
Mayuriben J. Parmar ◽  
Carine De Marcos Lousa ◽  
Stephen P. Muench ◽  
Adrian Goldman ◽  
Vincent L.G. Postis
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Purification ◽  
New Technologies ◽  
Pharmaceutical Companies ◽  
Artificial Membranes ◽  
Significant Step ◽  
Protein Research

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method.

scholarly journals A new azobenzene-based design strategy for detergents in membrane protein research

2020 ◽  
Vol 11 (13) ◽  
pp. 3538-3546 ◽  
Author(s):  
Leonhard H. Urner ◽  
Maiko Schulze ◽  
Yasmine B. Maier ◽  
Waldemar Hoffmann ◽  
Stephan Warnke ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Gas Phase ◽  
Design Strategy ◽  
Protein Research ◽  
Phase Properties

Here, L. H. Urner and co-workers identify a new detergent design strategy for the non-denaturing structural analysis of membrane proteins by studying the gas-phase properties of azobenzene-based oligoglycerol detergents.

Comparison of lipidic carrier systems for integral membrane proteins – MsbA as case study

2019 ◽  
Vol 400 (11) ◽  
pp. 1509-1518 ◽  
Author(s):  
Dominique-Maurice Kehlenbeck ◽  
Inokentijs Josts ◽  
Julius Nitsche ◽  
Sebastian Busch ◽  
V. Trevor Forsyth ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Proteins ◽  
Structural Studies ◽  
Long Term Stability ◽  
Activity Measurements ◽  
Protein Research ◽  
Protein Instability ◽  
Lipid Nanodiscs ◽  
Carrier Systems

Abstract Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead to protein instability and reduced activity. Recently, lipid nanodiscs (NDs) and saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems that keep membrane proteins in a native-like lipidic solution environment and are suitable for biophysical and structural studies. Here, we systematically compare nanodiscs and Salipros with respect to long-term stability as well as activity and stability of the incorporated membrane protein using the ABC transporter MsbA as model system. Our results show that both systems are suitable for activity measurements as well as structural studies in solution. Based on our results we suggest screening of different lipids with respect to activity and stability of the incorporated IMP before performing structural studies.

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