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.

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.

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.

scholarly journals Why do proteins aggregate? “Intrinsically insoluble proteins” and “dark mediators” revealed by studies on “insoluble proteins” solubilized in pure water

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 94 ◽  
Author(s):  
Jianxing Song
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
High Selectivity ◽  
Pure Water ◽  
Integral Membrane Protein ◽  
Integral Membrane Proteins ◽  
Common Mechanism ◽  
Interacting Proteins ◽  
Primitive Membranes

In 2008, I reviewed and proposed a model for our discovery in 2005 that unrefoldable and insoluble proteins could in fact be solubilized in unsalted water. Since then, this discovery has offered us and other groups a powerful tool to characterize insoluble proteins, and we have further addressed several fundamental and disease-relevant issues associated with this discovery. Here I review these results, which are conceptualized into several novel scenarios. 1) Unlike 'misfolded proteins', which still retain the capacity to fold into well-defined structures but are misled to 'off-pathway' aggregation, unrefoldable and insoluble proteins completely lack this ability and will unavoidably aggregate in vivo with ~150 mM ions, thus designated as 'intrinsically insoluble proteins (IIPs)' here. IIPs may largely account for the 'wastefully synthesized' DRiPs identified in human cells. 2) The fact that IIPs including membrane proteins are all soluble in unsalted water, but get aggregated upon being exposed to ions, logically suggests that ions existing in the background play a central role in mediating protein aggregation, thus acting as 'dark mediators'. Our study with 14 salts confirms that IIPs lack the capacity to fold into any well-defined structures. We uncover that salts modulate protein dynamics and anions bind proteins with high selectivity and affinity, which is surprisingly masked by pre-existing ions. Accordingly, I modified my previous model. 3) Insoluble proteins interact with lipids to different degrees. Remarkably, an ALS-causing P56S mutation transforms the β-sandwich MSP domain into a helical integral membrane protein. Consequently, the number of membrane-interacting proteins might be much larger than currently recognized. To attack biological membranes may represent a common mechanism by which aggregated proteins initiate human diseases. 4) Our discovery also implies a solution to the 'chicken-and-egg paradox' for the origin of primitive membranes embedded with integral membrane proteins, if proteins originally emerged in unsalted prebiotic media.

Glucose transport in erythrocytes of diabetic and healthy children as related to hemoglobin A1c.

1985 ◽  
Vol 31 (8) ◽  
pp. 1387-1389 ◽  
Author(s):  
H B Mortensen ◽  
J Brahm
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Glucose Transport ◽  
Significant Variation ◽  
Hemoglobin A1c ◽  
Integral Membrane Protein ◽  
Healthy Children ◽  
Transport Function ◽  
Physiological Conditions ◽  
Diabetic Children

Abstract We studied glucose transport under physiological conditions (38 degrees C, pH 7.2, 5 mmol of glucose per liter) in erythrocytes of nine diabetic children with hemoglobin A1c values ranging from 6.6 to 13.8%, and in erythrocytes from six healthy children. Glucose transport was determined to be 2.38 (SD 0.16) X 10(-10) mol/cm2 X s (n = 18), and 2.47 (SD 0.18) X 10(-10) mol/cm2 X s (n = 12) in erythrocytes from diabetics and controls, respectively. The corresponding values for hemoglobin A1c were 11.0% (SD 2.3%) for the diabetics and 5.6% (SD 0.3%) for the controls. Thus the concentration of hemoglobin A1c, which reflects the degree of glycation of membrane proteins, differs significantly (p less than 0.001) between the two groups, whereas there was no significant variation (p greater than 0.1) in D-glucose transport. We conclude that glycation of the integral membrane protein that mediates glucose transport has no effect on transport function under physiological conditions.

scholarly journals Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

2020 ◽  
Vol 48 (2) ◽  
pp. 547-558 ◽  
Author(s):  
Cagla Sahin ◽  
Deseree J. Reid ◽  
Michael T. Marty ◽  
Michael Landreh
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Vesicles ◽  
Native Mass Spectrometry ◽  
Integral Membrane Proteins ◽  
Structural Basis ◽  
Lipid Interactions ◽  
Native Ms ◽  
Peripheral Membrane Protein

A growing number of integral membrane proteins have been shown to tune their activity by selectively interacting with specific lipids. The ability to regulate biological functions via lipid interactions extends to the diverse group of proteins that associate only peripherally with the lipid bilayer. However, the structural basis of these interactions remains challenging to study due to their transient and promiscuous nature. Recently, native mass spectrometry has come into focus as a new tool to investigate lipid interactions in membrane proteins. Here, we outline how the native MS strategies developed for integral membrane proteins can be applied to generate insights into the structure and function of peripheral membrane proteins. Specifically, native MS studies of proteins in complex with detergent-solubilized lipids, bound to lipid nanodiscs, and released from native-like lipid vesicles all shed new light on the role of lipid interactions. The unique ability of native MS to capture and interrogate protein–protein, protein–ligand, and protein–lipid interactions opens exciting new avenues for the study of peripheral membrane protein biology.

scholarly journals Nanodiscs: A Controlled Bilayer Surface for the Study of Membrane Proteins

2018 ◽  
Vol 47 (1) ◽  
pp. 107-124 ◽  
Author(s):  
Mark A. McLean ◽  
Michael C. Gregory ◽  
Stephen G. Sligar
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Information Transfer ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Cellular Communication ◽  
Phospholipid Bilayer ◽  
Signaling Cascade ◽  
Biophysical Techniques ◽  
Lipid Specificity

The study of membrane proteins and receptors presents many challenges to researchers wishing to perform biophysical measurements to determine the structure, function, and mechanism of action of such components. In most cases, to be fully functional, proteins and receptors require the presence of a native phospholipid bilayer. In addition, many complex multiprotein assemblies involved in cellular communication require an integral membrane protein as well as a membrane surface for assembly and information transfer to soluble partners in a signaling cascade. Incorporation of membrane proteins into Nanodiscs renders the target soluble and provides a native bilayer environment with precisely controlled composition of lipids, cholesterol, and other components. Likewise, Nanodiscs provide a surface of defined area useful in revealing lipid specificity and affinities for the assembly of signaling complexes. In this review, we highlight several biophysical techniques made possible through the use of Nanodiscs.

scholarly journals An automated platform for structural analysis of membrane proteins through serial crystallography

2021 ◽  
Author(s):  
Robert D Healey ◽  
Shibom Basu ◽  
Anne-Sophie Humm ◽  
Cedric Leyrat ◽  
Xiaojing Cong ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
Drug Targets ◽  
Protein Crystallization ◽  
Integral Membrane Protein ◽  
Structural Level ◽  
Ligand Discovery ◽  
Serial Crystallography ◽  
Automated Platform

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

scholarly journals Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins.

1985 ◽  
Vol 33 (8) ◽  
pp. 813-820 ◽  
Author(s):  
K L Goldenthal ◽  
K Hedman ◽  
J W Chen ◽  
J T August ◽  
M C Willingham
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Membrane Glycoproteins ◽  
Animal Cells ◽  
Cytoplasmic Proteins ◽  
Membrane Antigens ◽  
Endocytic Vesicles ◽  
Triton X ◽  
Nonionic Detergents

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.

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