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.

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

scholarly journals Structures and Interactions of Transmembrane Targets in Native Nanodiscs

2019 ◽  
Vol 24 (10) ◽  
pp. 943-952 ◽  
Author(s):  
Michael Overduin ◽  
Mansoore Esmaili
Keyword(s):  
Posttranslational Modifications ◽  
Protein Complexes ◽  
Biological Membrane ◽  
Water Soluble ◽  
Ex Situ ◽  
Linear Polymers ◽  
Paramagnetic Resonance ◽  
X Ray ◽  
Biologically Relevant

Transmembrane proteins function within a continuous layer of biologically relevant lipid molecules that stabilizes their structures and modulates their activities. Structures and interactions of biological membrane–protein complexes or “memteins” can now be elucidated using native nanodiscs made by poly(styrene co-maleic anhydride) derivatives. These linear polymers contain a series of hydrophobic and polar subunits that gently fragment membranes into water-soluble discs with diameters of 5–50 nm known as styrene maleic acid lipid particles (SMALPs). High-resolution structures of memteins that include endogenous lipid ligands and posttranslational modifications can be resolved without resorting to synthetic detergents or artificial lipids. The resulting ex situ structures better recapitulate the in vivo situation and can be visualized by methods including cryo-electron microscopy (cryoEM), electron paramagnetic resonance (EPR), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, small angle x-ray scattering (SAXS), and x-ray diffraction (XRD). Recent progress including 3D structures of biological bilayers illustrates how polymers and native nanodiscs expose previously inaccessible membrane assemblies at atomic resolution and suggest ways in which the SMALP system could be exploited for drug discovery.

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 The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

Oligomerization of polytopic α-helical membrane proteins: causes and consequences

2012 ◽  
Vol 393 (11) ◽  
pp. 1215-1230 ◽  
Author(s):  
Florian Cymer ◽  
Dirk Schneider
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Present Article ◽  
Experimental Information ◽  
Protein Oligomerization ◽  
Open Questions ◽  
Specific Activities ◽  
Physiological Impact ◽  
And Control

Abstract Several polytopic α-helical membrane-integrated proteins appear to be organized in higher-ordered oligomeric complexes. While many aspects are still enigmatic, in recent years, the physiological impact of membrane protein oligomerization has been analyzed to some extent. In the present article, oligomerization of structurally well-defined membrane proteins is discussed. The available experimental information indicates the causes and physiological consequences of membrane protein oligomerization, including stabilization, cooperative functions, and control of specific activities. Based on the currently available observations, we aim to derive some general principles and discuss open questions.

scholarly journals YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

2004 ◽  
Vol 166 (6) ◽  
pp. 769-774 ◽  
Author(s):  
Ross E. Dalbey ◽  
Andreas Kuhn
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Family Members ◽  
Membrane Insertion ◽  
Conducting Channel ◽  
Energy Devices ◽  
Domains Of Life ◽  
Membrane Protein Complexes ◽  
Sensor Proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein–conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.

scholarly journals Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

2015 ◽  
Vol 396 (8) ◽  
pp. 903-915 ◽  
Author(s):  
Nazhat Shirzad-Wasei ◽  
Jenny van Oostrum ◽  
Petra H.M. Bovee-Geurts ◽  
Lisanne J.A. Kusters ◽  
Giel J.C.G.M. Bosman ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Partial Purification ◽  
Functional Studies ◽  
Host Membrane ◽  
Lipid Nanodiscs ◽  
Membrane Scaffold ◽  
Rapid Transfer

Abstract Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

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.

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