scholarly journals The high-throughput production of membrane proteins

2021 ◽  
Author(s):  
James Birch ◽  
Andrew Quigley
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
Protein Function ◽  
Electron Tomography ◽  
Time Resolved ◽  
Functional Characterisation ◽  
Host Membrane ◽  
Expression Host ◽  
Structural Applications

Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

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.

Mechanisms underlying drug-mediated regulation of membrane protein function

2021 ◽  
Vol 118 (46) ◽  
pp. e2113229118
Author(s):  
Radda Rusinova ◽  
Changhao He ◽  
Olaf S. Andersen
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Conformational Changes ◽  
Protein Function ◽  
Nonspecific Binding ◽  
Drug Induced ◽  
Protein Conformational Changes ◽  
Potassium Channel Kcsa ◽  
Membrane Protein Function

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

scholarly journals Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A2A Receptor as a Case Study

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 400 ◽  
Author(s):  
Rosana I. Reis ◽  
Isabel Moraes
Keyword(s):  
Light Scattering ◽  
Membrane Proteins ◽  
Dynamic Light Scattering ◽  
Membrane Protein ◽  
Cell Communication ◽  
Cell Physiology ◽  
Functional Characterisation

Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins

2003 ◽  
Vol 36 (6) ◽  
pp. 1372-1377 ◽  
Author(s):  
Vadim Cherezov ◽  
Martin Caffrey
Keyword(s):  
Optical Properties ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Low Cost ◽  
Protein Crystallization ◽  
Batch Crystallization ◽  
Membrane Protein Crystallization ◽  
Crystallization Trial

A simple convenient and low-cost glass-based plate for high-throughput screening of membrane protein crystallization is described. The plates are robust and reduce dramatically the amount of protein and precipitant solution used per crystallization trial, while offering excellent optical properties for the detection of micro-crystals and crystals of colorless proteins. The plates were developed primarily for crystallization of membrane proteins in lipidic mesophases. They can also be used in batch crystallization of soluble and membrane proteins.

scholarly journals Tracking Membrane Protein Dynamics in Real Time

2021 ◽  
Author(s):  
Fredrik Orädd ◽  
Magnus Andersson
Keyword(s):  
Membrane Protein ◽  
Protein Dynamics ◽  
Structural Dynamics ◽  
Protein Function ◽  
Md Simulation ◽  
Membrane Lipids ◽  
Time Resolved ◽  
Biological Time ◽  
Membrane Protein Dynamics ◽  
Membrane Protein Function

Abstract Membrane proteins govern critical cellular processes and are central to human health and associated disease. Understanding of membrane protein function is obscured by the vast ranges of structural dynamics—both in the spatial and time regime—displayed in the protein and surrounding membrane. The membrane lipids have emerged as allosteric modulators of membrane protein function, which further adds to the complexity. In this review, we discuss several examples of membrane dependency. A particular focus is on how molecular dynamics (MD) simulation have aided to map membrane protein dynamics and how enhanced sampling methods can enable observing the otherwise inaccessible biological time scale. Also, time-resolved X-ray scattering in solution is highlighted as a powerful tool to track membrane protein dynamics, in particular when combined with MD simulation to identify transient intermediate states. Finally, we discuss future directions of how to further develop this promising approach to determine structural dynamics of both the protein and the surrounding lipids. Graphic Abstract

From atoms to cells: bridging the gap between potency, efficacy, and safety of small molecules directed at a membrane protein

2021 ◽  
Author(s):  
Rodrigo Aguayo-Ortiz ◽  
Jeffery Creech ◽  
Eric N. Jimenez-Vazquez ◽  
Guadalupe Guerrero-Serna ◽  
Nulang Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Drug Targets ◽  
Transmembrane Protein ◽  
Protein Domains ◽  
Cardiac Cells ◽  
Therapeutic Drug ◽  
Efficacy And Safety

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

MPI tray: a versatile crystallization plate for membrane proteins

2017 ◽  
Vol 50 (1) ◽  
pp. 327-330 ◽  
Author(s):  
Barbara Rathmann ◽  
David Quirnheim Pais ◽  
Yvonne Thielmann
Keyword(s):  
Surface Tension ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Surface Properties ◽  
High Throughput ◽  
Biological Macromolecules ◽  
Electrostatic Surface Potential ◽  
Extensive Analysis ◽  
Visible Imaging ◽  
The Common

High-throughput crystallization of biological macromolecules is usually performed on multi-well plates, the design of which needs to address different and sometimes conflicting requirements. In this regard, handling of membrane proteins presents a particular challenge owing to the common use of detergents with associated effects on surface tension. Reported here is the design of a new crystallization plate, termed the MPI tray, which is optimized for UV and visible imaging with membrane protein samples. Following basic considerations regarding geometry and material, the surface properties of the plate were subjected to extensive analysis and modification in order to improve the performance in a robotic environment. An electrostatic surface potential was identified as the major problem affecting the automated setup of experiments, and it was found that treatment of the crystallization plate with ethanol is effective in removing this potential.

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.

scholarly journals The high-resolution structure of cell membranes revealed by in situ cryo-electron tomography

2021 ◽  
Author(s):  
Guanfang Zhao ◽  
Sihang Cheng ◽  
Yang Yu ◽  
Tianyi Zou ◽  
Huili Wang ◽  
...  
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Interactions ◽  
Cell Membranes ◽  
Electron Tomography ◽  
3D Structure ◽  
Structure Characterization ◽  
Cryo Electron Tomography

As the structural unit of life, cell is defined by the membrane system. The cell membrane separates the internal and external environment of the cell, and the endomembrane system defines the organelles to perform different functions1-3. However, lack of tools to in situ observe membrane proteins at a molecular resolution has limited our understanding of membrane organization and membrane protein interactions. Here we characterize the high-resolution 3D structure of human red blood cell (hRBC) membranes and the membrane proteins for the first time in situ by cryo-electron tomography (CryoET)4-7. By analyzing tomograms, we have obtained the first fine three-dimensional (3D) structure of hRBC membranes and found the asymmetrical distribution of membrane proteins on both sides of the membranes. We found that the membrane proteins are mainly located on the cytoplasmic side of hRBC membranes, with protein sizes ranging from 6nm to 8nm, in contrast to the ectoplasmic side with basically no proteins. Quantitative analysis of the density of hRBC membrane proteins shows that the membranes with higher protein occupancy have less phospholipid, making the membranes more rigid. Meanwhile, we obtained the channel protein-like structures by preliminary analysis of the membrane protein. Our results represent the first in situ structure characterization of the cell membranes and membrane proteins through cryoET and opens the door for understanding the biological functions of cell membranes in their physiological environments.

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