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A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Biomacromolecules: Proteins".

Deadline for manuscript submissions: closed (31 December 2020) | Viewed by 51137

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Dr. Wylie Benjamin

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Department of Chemistry, Texas Tech University at Lubbock, Lubbock, TX 79409, USA
Interests: solid-state NMR; membrane proteins; protien aggregates; biolophysics; biological spectroscopy
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The structure and activity of membrane proteins within biological membranes govern key behaviors, from regulating transmembrane voltage and the action potential, to signaling and molecular transport. Understanding the structure–activity relationships of these proteins within the milieu of native-like bilayers is required to fully ascertain the how these systems govern human health, act as drug-targetable systems of pathogenic organisms, and lead to the development of novel membrane-targeted therapies. Established and emerging biophysical techniques, including liquid- and solid-state NMR (ssNMR), Electron Spin Resonance (ESR), cryo Electron Microscopy (cryo-EM), X-ray crystallography, Förster Resonance Energy Transfer (FRET), and Fluorescence Spectroscopy have made invaluable contributions to understanding native membrane protein dynamic structures, lipid–protein and lipid–lipid interactions, and long-range membrane organization. In addition, computational approaches and the molecular-dynamics of membranes have provided many novel, key insights into these systems. Despite this, many open questions remain, and new biophysical techniques along with novel applications of existing methods are required to advance these crucial studies.

In this Special Issue of Biomolecules, we invite your contributions, either in the form of original research articles, reviews, or “perspective” articles on all aspects related to the overarching theme of “Advances in biophysical and computational approaches to studying the native behaviors of membrane proteins and biological membrane components.” Articles with novel applications of spectroscopic or microscopic techniques are especially welcome.

Dr. Wylie Benjamin
Guest Editor

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

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Keywords

Membrane Proteins
Membrane Biophysics
Biological Spectroscopy
Biological Microscopy
Computational Approaches to Study Membrane Proteins
Molecular Dynamics

Published Papers (11 papers)

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Research

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22 pages, 2943 KiB

Open AccessArticle

Dynamic Nuclear Polarization of Biomembrane Assemblies

by Nhi T. Tran, Frédéric Mentink-Vigier and Joanna R. Long

Biomolecules 2020, 10(9), 1246; https://doi.org/10.3390/biom10091246 - 27 Aug 2020

Cited by 12 | Viewed by 3433

Abstract

While atomic scale structural and dynamic information are hallmarks of nuclear magnetic resonance (NMR) methodologies, sensitivity is a fundamental limitation in NMR studies. Fully exploiting NMR capabilities to study membrane proteins is further hampered by their dilution within biological membranes. Recent developments in dynamic nuclear polarization (DNP), which can transfer the relatively high polarization of unpaired electrons to nuclear spins, show promise for overcoming the sensitivity bottleneck and enabling NMR characterization of membrane proteins under native-like conditions. Here we discuss fundamental aspects of DNP-enhanced solid-state NMR spectroscopy, experimental details relevant to the study of lipid assemblies and incorporated proteins, and sensitivity gains which can be realized in biomembrane-based samples. We also present unique insights which can be gained from DNP measurements and prospects for further development of the technique for elucidating structures and orientations of membrane proteins in native lipid environments. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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20 pages, 15251 KiB

Open AccessArticle

The Droserasin 1 PSI: A Membrane-Interacting Antimicrobial Peptide from the Carnivorous Plant Drosera capensis

by Marc A. Sprague-Piercy, Jan C. Bierma, Marquise G. Crosby, Brooke P. Carpenter, Gemma R. Takahashi, Joana Paulino, Ivan Hung, Rongfu Zhang, John E. Kelly, Natalia Kozlyuk, Xi Chen, Carter T. Butts and Rachel W. Martin

Biomolecules 2020, 10(7), 1069; https://doi.org/10.3390/biom10071069 - 17 Jul 2020

Cited by 7 | Viewed by 4911

Abstract

The Droserasins, aspartic proteases from the carnivorous plant Drosera capensis, contain a 100-residue plant-specific insert (PSI) that is post-translationally cleaved and independently acts as an antimicrobial peptide. PSIs are of interest not only for their inhibition of microbial growth, but also because they modify the size of lipid vesicles and strongly interact with biological membranes. PSIs may therefore be useful for modulating lipid systems in NMR studies of membrane proteins. Here we present the expression and biophysical characterization of the Droserasin 1 PSI (D1 PSI.) This peptide is monomeric in solution and maintains its primarily

α

-helical secondary structure over a wide range of temperatures and pH values, even under conditions where its three disulfide bonds are reduced. Vesicle fusion assays indicate that the D1 PSI strongly interacts with bacterial and fungal lipids at pH 5 and lower, consistent with the physiological pH of D. capensis mucilage. It binds lipids with a variety of head groups, highlighting its versatility as a potential stabilizer for lipid nanodiscs. Solid-state NMR spectra collected at a field strength of 36 T, using a unique series-connected hybrid magnet, indicate that the peptide is folded and strongly bound to the membrane. Molecular dynamics simulations indicate that the peptide is stable as either a monomer or a dimer in a lipid bilayer. Both the monomer and the dimer allow the passage of water through the membrane, albeit at different rates. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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20 pages, 8243 KiB

Open AccessArticle

Fibrillization of 40-residue β-Amyloid Peptides in Membrane-Like Environments Leads to Different Fibril Structures and Reduced Molecular Polymorphisms

by Qinghui Cheng, Zhi-Wen Hu, Yuto Tobin-Miyaji, Amy E. Perkins, Terrence Deak and Wei Qiang

Biomolecules 2020, 10(6), 881; https://doi.org/10.3390/biom10060881 - 8 Jun 2020

Cited by 11 | Viewed by 2751

Abstract

The molecular-level polymorphism in β-Amyloid (Aβ) fibrils have recently been considered as a pathologically relevant factor in Alzheimer’s disease (AD). Studies showed that the structural deviations in human-brain-seeded Aβ fibrils potentially correlated with the clinical histories of AD patients. For the 40-residue Aβ (Aβ₄₀) fibrils derived from human brain tissues, a predominant molecular structure was proposed based on solid-state nuclear magnetic resonance (ssNMR) spectroscopy. However, previous studies have shown that the molecular structures of Aβ₄₀ fibrils were sensitive to their growth conditions in aqueous environments. We show in this work that biological membranes and their phospholipid bilayer mimics serve as environmental factors to reduce the structural heterogeneity in Aβ₄₀ fibrils. Fibrillization in the presence of membranes leads to fibril structures that are significantly different to the Aβ₄₀ fibrils grown in aqueous solutions. Fibrils grown from multiple types of membranes, including the biological membranes extracted from the rats’ synaptosomes, shared similar ssNMR spectral features. Our studies emphasize the biological relevance of membranes in Aβ₄₀ fibril structures and fibrillization processes. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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17 pages, 3298 KiB

Open AccessArticle

PIRT the TRP Channel Regulating Protein Binds Calmodulin and Cholesterol-Like Ligands

by Nicholas J. Sisco, Dustin D. Luu, Minjoo Kim and Wade D. Van Horn

Biomolecules 2020, 10(3), 478; https://doi.org/10.3390/biom10030478 - 21 Mar 2020

Cited by 5 | Viewed by 3764

Abstract

Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP₂) lipid. As many PIP₂ modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP₂. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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27 pages, 4583 KiB

Open AccessFeature PaperArticle

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in Pichia pastoris for Solid-State NMR

by Rachel Munro, Jeffrey de Vlugt, Vladimir Ladizhansky and Leonid S. Brown

Biomolecules 2020, 10(3), 434; https://doi.org/10.3390/biom10030434 - 11 Mar 2020

Cited by 8 | Viewed by 4215

Abstract

Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein. Some membrane proteins, such as human aquaporin 2 (hAQP2), exhibit poor expression, which can make producing a sample for SSNMR in an economic fashion extremely difficult, as growth in minimal media adds additional strain on expression hosts. We have developed an optimized growth protocol for eukaryotic membrane proteins in the methylotrophic yeast Pichia pastoris. Our new growth protocol uses the combination of sorbitol supplementation, higher cell density, and low temperature induction (LT-SEVIN), which increases the yield of full-length, isotopically labeled hAQP2 ten-fold. Combining mass spectrometry and SSNMR, we were able to determine the nature and the extent of post-translational modifications of the protein. The resultant protein can be functionally reconstituted into lipids and yields excellent resolution and spectral coverage when analyzed by two-dimensional SSNMR spectroscopy. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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20 pages, 6103 KiB

Open AccessArticle

Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

by Evan Van Aalst, Maryam Yekefallah, Anil K. Mehta, Isaac Eason and Benjamin Wylie

Biomolecules 2020, 10(3), 430; https://doi.org/10.3390/biom10030430 - 10 Mar 2020

Cited by 11 | Viewed by 4453

Abstract

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K⁺ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K⁺ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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17 pages, 2946 KiB

Open AccessArticle

Comparing Interfacial Trp, Interfacial His and pH Dependence for the Anchoring of Tilted Transmembrane Helical Peptides

by Fahmida Afrose and Roger E. Koeppe II

Biomolecules 2020, 10(2), 273; https://doi.org/10.3390/biom10020273 - 11 Feb 2020

Cited by 4 | Viewed by 2224

Abstract

Charged and aromatic amino acid residues, being enriched toward the terminals of membrane-spanning helices in membrane proteins, help to stabilize particular transmembrane orientations. Among them, histidine is aromatic and can be positively charge at low pH. To enable investigations of the underlying protein-lipid interactions, we have examined the effects of single or pairs of interfacial histidine residues using the constructive low-dynamic GWALP23 (acetyl-GG²ALW⁵LALALALALALALW¹⁹LAG²²A-amide) peptide framework by incorporating individual or paired histidines at locations 2, 5, 19 or 22. Analysis of helix orientation by means of solid-state ²H NMR spectra of labeled alanine residues reveals marked differences with H^2,22 compared to W^2,22. Nevertheless, the properties of membrane-spanning H^2,22WALP23 helices show little pH dependence and are similar to those having Gly, Arg or Lys at positions 2 and 22. The presence of H5 or H19 influences the helix rotational preference but not the tilt magnitude. H5 affects the helical integrity, as residue 7 unwinds from the core helix; yet once again the helix orientation and dynamic properties show little sensitivity to pH. The overall results reveal that the detailed properties of transmembrane helices depend upon the precise locations of interfacial histidine residues. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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20 pages, 5211 KiB

Open AccessArticle

Structure Modeling of the Norepinephrine Transporter

by Izabella Góral, Kamil Łątka and Marek Bajda

Biomolecules 2020, 10(1), 102; https://doi.org/10.3390/biom10010102 - 7 Jan 2020

Cited by 13 | Viewed by 5590

Abstract

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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13 pages, 1924 KiB

Open AccessArticle

Molecular Dynamics Simulation of Transmembrane Transport of Chloride Ions in Mutants of Channelrhodopsin

by Wenying Zhang, Ting Yang, Shuangyan Zhou, Jie Cheng, Shuai Yuan, Glenn V. Lo and Yusheng Dou

Biomolecules 2019, 9(12), 852; https://doi.org/10.3390/biom9120852 - 10 Dec 2019

Cited by 3 | Viewed by 2975

Abstract

Channelrhodopsins (ChRs) are light-gated transmembrane cation channels which are widely used for optogenetic technology. Replacing glutamate located at the central gate of the ion channel with positively charged amino acid residues will reverse ion selectivity and allow anion conduction. The structures and properties of the ion channel, the transport of chloride, and potential of mean force (PMF) of the chimera protein (C1C2) and its mutants, EK-TC, ER-TC and iChloC, were investigated by molecular dynamics simulation. The results show that the five-fold mutation in E122Q-E129R-E140S-D195N-T198C (iChloC) increases the flexibility of the transmembrane channel protein better than the double mutations in EK-TC and ER-TC, and results in an expanded ion channel pore size and decreased steric resistance. The iChloC mutant was also found to have a higher affinity for chloride ions and, based on surface electrostatic potential analysis, provides a favorable electrostatic environment for anion conduction. The PMF free energy curves revealed that high affinity Cl⁻ binding sites are generated near the central gate of the three mutant proteins. The energy barriers for the EK-TC and ER-TC were found to be much higher than that of iChloC. The results suggest that the transmembrane ion channel of iChloC protein is better at facilitating the capture and transport of chloride ions. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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Review

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26 pages, 4734 KiB

Open AccessReview

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

by Indra D. Sahu and Gary A. Lorigan

Biomolecules 2020, 10(5), 763; https://doi.org/10.3390/biom10050763 - 13 May 2020

Cited by 32 | Viewed by 6906

Abstract

Membrane proteins possess a variety of functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is extremely difficult to probe the structure and dynamic properties of membrane proteins using traditional biophysical techniques, particularly in their native environments. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a very powerful and rapidly growing biophysical technique to study pertinent structural and dynamic properties of membrane proteins with no size restrictions. In this review, we will briefly discuss the most commonly used EPR techniques and their recent applications for answering structure and conformational dynamics related questions of important membrane protein systems. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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20 pages, 6032 KiB

Open AccessReview

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase

by Smita Mohanty, Bharat P Chaudhary and David Zoetewey

Biomolecules 2020, 10(4), 624; https://doi.org/10.3390/biom10040624 - 17 Apr 2020

Cited by 40 | Viewed by 9124

Abstract

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal. Full article

(This article belongs to the Special Issue Advances in Membrane Proteins)

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