scholarly journals Current strategies for protein production and purification enabling membrane protein structural biology

2016 ◽  
Vol 94 (6) ◽  
pp. 507-527 ◽  
Author(s):  
Aditya Pandey ◽  
Kyungsoo Shin ◽  
Robin E. Patterson ◽  
Xiang-Qin Liu ◽  
Jan K. Rainey
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Protein Production ◽  
Protein Structures ◽  
Data Bank ◽  
In Vitro Translation ◽  
Preparation Methods ◽  
Natural Hosts

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles, owing to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and (or) amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through the introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10–15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM).

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 A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

1991 ◽  
Vol 112 (5) ◽  
pp. 809-821 ◽  
Author(s):  
R N Thrift ◽  
D W Andrews ◽  
P Walter ◽  
A E Johnson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Secretory Protein ◽  
In Vitro Translation ◽  
Nascent Chain ◽  
Transmembrane Sequence ◽  
Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

scholarly journals SecY alterations that impair membrane protein folding and generate a membrane stress

2007 ◽  
Vol 176 (3) ◽  
pp. 307-317 ◽  
Author(s):  
Nobuyuki Shimohata ◽  
Shushi Nagamori ◽  
Yoshinori Akiyama ◽  
H. Ronald Kaback ◽  
Koreaki Ito
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
In Vitro Translation ◽  
Membrane Protein Folding ◽  
Protein Biogenesis ◽  
Simple Reduction

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

scholarly journals Structure-Guided Computational Approaches to Unravel Druggable Proteomic Landscape of Mycobacterium leprae

2021 ◽  
Vol 8 ◽  
Author(s):  
Sundeep Chaitanya Vedithi ◽  
Sony Malhotra ◽  
Marta Acebrón-García-de-Eulate ◽  
Modestas Matusevicius ◽  
Pedro Henrique Monteiro Torres ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Schwann Cells ◽  
Protein Structures ◽  
Mycobacterium Leprae ◽  
Data Bank ◽  
Nerve Damage ◽  
Structural Proteomics ◽  
Bacterial Survival ◽  
Functional Sites

Leprosy, caused by Mycobacterium leprae (M. leprae), is treated with a multidrug regimen comprising Dapsone, Rifampicin, and Clofazimine. These drugs exhibit bacteriostatic, bactericidal and anti-inflammatory properties, respectively, and control the dissemination of infection in the host. However, the current treatment is not cost-effective, does not favor patient compliance due to its long duration (12 months) and does not protect against the incumbent nerve damage, which is a severe leprosy complication. The chronic infectious peripheral neuropathy associated with the disease is primarily due to the bacterial components infiltrating the Schwann cells that protect neuronal axons, thereby inducing a demyelinating phenotype. There is a need to discover novel/repurposed drugs that can act as short duration and effective alternatives to the existing treatment regimens, preventing nerve damage and consequent disability associated with the disease. Mycobacterium leprae is an obligate pathogen resulting in experimental intractability to cultivate the bacillus in vitro and limiting drug discovery efforts to repositioning screens in mouse footpad models. The dearth of knowledge related to structural proteomics of M. leprae, coupled with emerging antimicrobial resistance to all the three drugs in the multidrug therapy, poses a need for concerted novel drug discovery efforts. A comprehensive understanding of the proteomic landscape of M. leprae is indispensable to unravel druggable targets that are essential for bacterial survival and predilection of human neuronal Schwann cells. Of the 1,614 protein-coding genes in the genome of M. leprae, only 17 protein structures are available in the Protein Data Bank. In this review, we discussed efforts made to model the proteome of M. leprae using a suite of software for protein modeling that has been developed in the Blundell laboratory. Precise template selection by employing sequence-structure homology recognition software, multi-template modeling of the monomeric models and accurate quality assessment are the hallmarks of the modeling process. Tools that map interfaces and enable building of homo-oligomers are discussed in the context of interface stability. Other software is described to determine the druggable proteome by using information related to the chokepoint analysis of the metabolic pathways, gene essentiality, homology to human proteins, functional sites, druggable pockets and fragment hotspot maps.

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.

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.

Structural biology and structure–function relationships of membrane proteins

2018 ◽  
Vol 47 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Rosana Reis ◽  
Isabel Moraes
Keyword(s):  
Membrane Proteins ◽  
Structure Function ◽  
Structural Biology ◽  
Drug Targets ◽  
Three Dimensional ◽  
Data Bank ◽  
Technical Advances ◽  
Medical Importance ◽  
G Protein Coupled ◽  
Important Drug

Abstract The study of structure–function relationships of membrane proteins (MPs) has been one of the major goals in the field of structural biology. Many Noble Prizes regarding remarkable accomplishments in MP structure determination and biochemistry have been awarded over the last few decades. Mutations or improper folding of these proteins are associated with numerous serious illnesses. Therefore, as important drug targets, the study of their primary sequence and three-dimensional fold, combined with cell-based assays, provides vital information about their structure–function relationships. Today, this information is vital to drug discovery and medicine. In the last two decades, many have been the technical advances and breakthroughs in the field of MP structural biology that have contributed to an exponential growth in the number of unique MP structures in the Protein Data Bank. Nevertheless, given the medical importance and many unanswered questions, it will never be an excess of MP structures, regardless of the method used. Owing to the extension of the field, in this brief review, we will only focus on structure–function relationships of the three most significant pharmaceutical classes: G protein-coupled receptors, ion channels and transporters.

scholarly journals Changes in Membrane Protein Structural Biology

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 401
Author(s):  
James Birch ◽  
Harish Cheruvara ◽  
Nadisha Gamage ◽  
Peter J. Harrison ◽  
Ryan Lithgo ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Protein Targets ◽  
Diverse Range ◽  
Biochemical Processes ◽  
Low Protein ◽  
Essential Components ◽  
Future Challenges ◽  
Difficult Area

Membrane proteins are essential components of many biochemical processes and are important pharmaceutical targets. Membrane protein structural biology provides the molecular rationale for these biochemical process as well as being a highly useful tool for drug discovery. Unfortunately, membrane protein structural biology is a difficult area of study due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Despite this instability, membrane protein structural biology has made great leaps over the last fifteen years. Today, the landscape is almost unrecognisable. The numbers of available atomic resolution structures have increased 10-fold though advances in crystallography and more recently by cryo-electron microscopy. These advances in structural biology were achieved through the efforts of many researchers around the world as well as initiatives such as the Membrane Protein Laboratory (MPL) at Diamond Light Source. The MPL has helped, provided access to and contributed to advances in protein production, sample preparation and data collection. Together, these advances have enabled higher resolution structures, from less material, at a greater rate, from a more diverse range of membrane protein targets. Despite this success, significant challenges remain. Here, we review the progress made and highlight current and future challenges that will be overcome.

scholarly journals 2P103 Direct monitoring of membrane protein folding process during in-vitro expression by Surface Enhanced IR spectroscopy(03. Membrane proteins,Poster)

2013 ◽  
Vol 53 (supplement1-2) ◽  
pp. S176
Author(s):  
Kenichi Ataka ◽  
Joachim Heberle ◽  
Axel Baumann ◽  
Silke Kerruth ◽  
Ramona Schlesinger ◽  
...  
Keyword(s):  
Protein Folding ◽  
Membrane Proteins ◽  
Ir Spectroscopy ◽  
Membrane Protein ◽  
In Vitro Expression ◽  
Folding Process ◽  
Membrane Protein Folding ◽  
Surface Enhanced

scholarly journals A growth arrest-specific (gas) gene codes for a membrane protein.

1990 ◽  
Vol 10 (6) ◽  
pp. 2924-2930 ◽  
Author(s):  
G Manfioletti ◽  
M E Ruaro ◽  
G Del Sal ◽  
L Philipson ◽  
C Schneider
Keyword(s):  
Nucleotide Sequence ◽  
Membrane Protein ◽  
Detailed Analysis ◽  
Growth Arrest ◽  
Protein Product ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Transmembrane Glycoprotein ◽  
Kinetics Of

A set of growth arrest-specific (gas) genes whose expression is negatively regulated by serum has recently been identified. We report on the detailed analysis of one of these genes (gas3). The kinetics of regulation by the presence and absence of serum were investigated, and it was found that this gene is regulated at the post-transcriptional level. The encoded protein deduced from the nucleotide sequence showed some similarity to a mitochondrial oxyreductase, and in vitro translation established that the protein product is a transmembrane glycoprotein.

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