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.

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 Testing Parameters for Two-Dimensional Crystallization and Electron Crystallography on Eukaryotic Membrane Proteins with Liposomes as Controls

2008 ◽  
Vol 16 (4) ◽  
pp. 30-33
Author(s):  
Gengxiang Zhao ◽  
Vasantha Mutucumarana ◽  
Darrel W. Stafford ◽  
Yoshihide Kanaoka ◽  
K. Frank Austen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Drug Targets ◽  
Structural Information ◽  
Protein Solubility ◽  
Three Dimensional ◽  
Phospholipid Bilayer ◽  
Structural Level ◽  
Electron Crystallography

Membrane proteins comprise the majority of known and potential drug targets, yet have been immensely difficult to analyze at the structural level due to their location in the membrane bilayer. Removal from the membrane necessitates replacement of the phospholipid bilayer by detergents in order to maintain protein solubility. However, the absence of lipids and the presence of detergents can render non-physiological conformational changes of the membrane protein (Tate, 2006). Electron crystallography is an important method for studying membrane proteins that usually takes advantage of reconstituting the protein in a phospholipid bilayer and removal of the detergent. Richard Henderson and Nigel Unwin used this technique to elucidate the three-dimensional (3D) arrangement of the transmembrane α-helices of bacteriorhodopsin, which was the first 3D structural information on a membrane protein (Henderson and Unwin, 1975).

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

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 Static light scattering studies of OmpF porin: Implications for integral membrane protein crystallization

2000 ◽  
Vol 9 (8) ◽  
pp. 1559-1566 ◽  
Author(s):  
Carl Hitscherich ◽  
Margaret Allaman ◽  
John Wiencek ◽  
Jeffrey Kaplan ◽  
Patrick J. Loll
Keyword(s):  
Light Scattering ◽  
Membrane Protein ◽  
Protein Crystallization ◽  
Integral Membrane Protein ◽  
Static Light Scattering ◽  
Ompf Porin ◽  
Membrane Protein Crystallization

scholarly journals Development of an Automated High Throughput LCP-FRAP Assay to Guide Membrane Protein Crystallization in Lipid Mesophases

2011 ◽  
Vol 11 (4) ◽  
pp. 1193-1201 ◽  
Author(s):  
Fei Xu ◽  
Wei Liu ◽  
Michael A. Hanson ◽  
Raymond C. Stevens ◽  
Vadim Cherezov
Keyword(s):  
Membrane Protein ◽  
High Throughput ◽  
Protein Crystallization ◽  
Frap Assay ◽  
Membrane Protein Crystallization

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

scholarly journals A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase

2016 ◽  
Vol 72 (4) ◽  
pp. 307-312 ◽  
Author(s):  
Stefan Andrew Kolek ◽  
Bastian Bräuning ◽  
Patrick Douglas Shaw Stewart
Keyword(s):  
Membrane Proteins ◽  
Soluble Protein ◽  
Protein Crystallization ◽  
Protein Structures ◽  
Integral Membrane Protein ◽  
Seed Stock ◽  
New Method ◽  
Cubic Phase ◽  
Seed Crystals ◽  
Order Of Magnitude

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

scholarly journals Characterization of lipid matrices for membrane protein crystallization by high-throughput small angle X-ray scattering

Methods ◽  
2011 ◽  
Vol 55 (4) ◽  
pp. 342-349 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Wei Liu ◽  
Joshua Kunken ◽  
Thomas M. Weiss ◽  
Hiro Tsuruta ◽  
...  
Keyword(s):  
Membrane Protein ◽  
High Throughput ◽  
Small Angle ◽  
Protein Crystallization ◽  
X Ray ◽  
X Ray Scattering ◽  
Membrane Protein Crystallization ◽  
Ray Scattering

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.

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