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 Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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 A comparative study of branched and linear mannitol-based amphiphiles on membrane protein stability

The Analyst ◽  
2018 ◽  
Vol 143 (23) ◽  
pp. 5702-5710 ◽  
Author(s):  
Hazrat Hussain ◽  
Tyler Helton ◽  
Yang Du ◽  
Jonas S. Mortensen ◽  
Parameswaran Hariharan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Comparative Study ◽  
Protein Stability ◽  
Alkyl Chain ◽  
Alkyl Chains ◽  
Membrane Protein Stability ◽  
The Comparative Study

The comparative study on linear vs. branched alkyl-chain amphiphiles indicates a favorable role for branched alkyl-chains in stabilizing membrane proteins.

Abstract 142: Biomimetic Recombinant Thrombomodulin Conjugate and Its Anticoagulant Activity

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Lin Wang ◽  
Rui Jiang ◽  
Xue-Long Sun
Keyword(s):  
Membrane Protein ◽  
Lipid Membrane ◽  
Anticoagulant Activity ◽  
E Coli ◽  
C Terminus ◽  
Membrane Mimetic ◽  
Cell Membrane Protein ◽  
Recombinant Thrombomodulin ◽  
Plasma Half Life

Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C (PC) anticoagulant pathway. To closely mimic membrane protein structural feature of TM, we proposed a membrane-mimetic re-expression of recombinant TM onto liposome. The EGF-like domains 4-6 of TM (TM 456 ) are essential for PC activation. A recombinant TM containing TM 456 and an azidohomoalanine at C-terminus was expressed in E. coli . The biomimetic liposomal recombinant TM conjugate was prepared by conjugation of the recombinant TM 456 -azide with liposome via orthogonal chemistry and confirmed with Western blotting and PC activation activity. The liposomal recombinant TM 456 conjugate showed a 2-fold higher PC activation activity than that of the recombinant TM 456 alone , which indicated that the lipid membrane has a beneficiary effect on the recombinant TM 456 ’s activity. Also, the liposomal recombinant TM 456 conjugate showed higher stability and longer plasma half-life than TM 456 . Moreover, the liposomal recombinant TM 456 conjugate showed in vivo anticoagulant activity by decreasing the mortality in a thrombin-induced thromboembolism mouse model. The reported liposomal TM 456 conjugate mimics endothelial TM structure and anticoagulant activity and may serve as an anticoagulant agent candidate for future development.

Crystallization of Membrane Proteins

1999 ◽  
Author(s):  
F. Reiss-Husson ◽  
D. Picot
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein A ◽  
Limited Proteolysis ◽  
Three Dimensional ◽  
Hydrophobic Surface ◽  
Biological Macromolecules ◽  
Recent Developments ◽  
Tight Association ◽  
Membrane Components

Crystallization of membrane proteins is one of the most recent developments in protein crystal growth; in 1980, for the first time, two membrane proteins were successfully crystallized, bacteriorhodopsin (1) and porin (2). Since then, a number of membrane proteins (about 30) yielded three-dimensional crystals. In several cases, the quality of the crystals was sufficient for X-ray diffraction studies. The first atomic structure of a membrane protein, a photosynthetic bacterial reaction centre, was described in 1985 (3), followed by the structure of about ten other membrane protein families. Crystallization of membrane proteins is now an actively growing field, and has been discussed in several recent reviews (4-8). The major difficulty in the study of membrane proteins, which for years hampered their crystallization, comes from their peculiar solubility properties. These originate from their tight association with other membrane components, particularly lipids. Indeed integral membrane proteins contain hydrophobic surface regions buried in the lipid bilayer core, as well as hydrophilic regions with charged or polar residues more or less exposed at the external faces of the membrane. Disruption of the bilayer for isolating a membrane protein can be done in various ways: extraction with organic solvents, use of chaotropic agents, or solubilization by a detergent. The last method is the most frequently used, since it maintains the biological activity of the protein if a suitable detergent is found. This chapter will be restricted to specific aspects of three-dimensional crystallizations done in micellar solutions of detergent. In some cases, it is possible to separate soluble domains from the membrane protein either by limited proteolysis or by genetic engineering. Such protein fragments can then be treated as soluble proteins and so will not be discussed further in this chapter. We refer to Chapter 12 and the review by Kühlbrandt (9) for the methodology of two-dimensional crystallization used for electron diffraction. The general principles discussed in this book for the crystallization of soluble biological macromolecules apply for membrane proteins; the protein solution must be brought to supersaturation by modifying its physical parameters (concentrations of constituents, ionic strength, and so on), so that nucleation may occur.

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 Endosomal trafficking of yeast membrane proteins

2018 ◽  
Vol 46 (6) ◽  
pp. 1551-1558 ◽  
Author(s):  
Kamilla M. E. Laidlaw ◽  
Chris MacDonald
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Membrane Trafficking ◽  
Surface Membrane ◽  
Multivesicular Body ◽  
Endosomal Trafficking ◽  
Yeast Saccharomyces Cerevisiae ◽  
Endosomal Membrane ◽  
Recent Developments ◽  
Intracellular Compartments

Various membrane trafficking pathways transport molecules through the endosomal system of eukaryotic cells, where trafficking decisions control the localisation and activity of a diverse repertoire of membrane protein cargoes. The budding yeast Saccharomyces cerevisiae has been used to discover and define many mechanisms that regulate conserved features of endosomal trafficking. Internalised surface membrane proteins first localise to endosomes before sorting to other compartments. Ubiquitination of endosomal membrane proteins is a signal for their degradation. Ubiquitinated cargoes are recognised by the endosomal sorting complex required for transport (ESCRT) apparatus, which mediate sorting through the multivesicular body pathway to the lysosome for degradation. Proteins that are not destined for degradation can be recycled to other intracellular compartments, such as the Golgi and the plasma membrane. In this review, we discuss recent developments elucidating the mechanisms that drive membrane protein degradation and recycling pathways in yeast.

scholarly journals Overcoming bottlenecks in the membrane protein structural biology pipeline

2016 ◽  
Vol 44 (3) ◽  
pp. 838-844 ◽  
Author(s):  
David Hardy ◽  
Roslyn M. Bill ◽  
Anass Jawhari ◽  
Alice J. Rothnie
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Stability ◽  
Structural Biology ◽  
Protein Structures ◽  
Data Bank ◽  
Neopentyl Glycol ◽  
X Ray Crystallography ◽  
Technological Advances ◽  
Near Future

Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

scholarly journals Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics

2020 ◽  
Vol 22 (1) ◽  
pp. 50
Author(s):  
Johannes Thoma ◽  
Björn M. Burmann
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Cubic Phase ◽  
Advantages And Disadvantages ◽  
Membrane Mimetic ◽  
Lipid Environment ◽  
Protein Biophysics ◽  
And Function ◽  
Biological Methods

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics.

scholarly journals Synthesis of Extended, Self-Assembled Biological Membranes containing Membrane Proteins from Gas Phase

2019 ◽  
Author(s):  
Matthias Wilm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Self Assembly ◽  
Protein Function ◽  
Lipid Membranes ◽  
Protein Complexes ◽  
Biological Membranes ◽  
The Self ◽  
In Vitro Systems

1.AbstractMembrane proteins carry out a wide variety of biological functions. The reproduction of specific properties that have evolved over millions of years of biological membranes in a technically controlled environment is of significant interest. Here a method is presented that allows the self-assembly of a macroscopically large, freely transportable membrane with Outer membrane porin G from Escherichia Coli. The technique does not use protein specific characteristics and therefore, could represent a method for the generation of extended layers of membranes with arbitrary membrane protein content. Such in-vitro systems are relevant in the study of membrane-protein function and structure and the self-assembly of membrane-based protein complexes. They might become important for the incorporation of the lipid-membranes in technological devices.

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