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Probing biophysical sequence constraints within the transmembrane domains of rhodopsin by deep mutational scanning.
Penn WD, McKee AG, Kuntz CP, Woods H, Nash V, Gruenhagen TC, Roushar FJ, Chandak M, Hemmerich C, Rusch DB, Meiler J, Schlebach JP
(2020) Sci Adv 6: eaay7505
MeSH Terms: Cell Membrane, Gene Expression, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Lipid Bilayers, Models, Molecular, Mutation, Protein Domains, Protein Folding, Protein Structure, Secondary, Recombinant Proteins, Rhodopsin, Solubility, Thermodynamics
Show Abstract · Added March 21, 2020
Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin's expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin's plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).
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15 MeSH Terms
Delineating the Molecular Basis of the Calmodulin‒bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry-Evidence for a Novel CaM Binding Motif in bMunc13-2.
Piotrowski C, Moretti R, Ihling CH, Haedicke A, Liepold T, Lipstein N, Meiler J, Jahn O, Sinz A
(2020) Cells 9:
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Calmodulin, Cattle, Cross-Linking Reagents, Hydrophobic and Hydrophilic Interactions, Mass Spectrometry, Models, Molecular, Nerve Tissue Proteins, Protein Binding, Protein Domains, Rats, Swine
Show Abstract · Added March 21, 2020
Exploring the interactions between the Ca binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an -terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.
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15 MeSH Terms
Dual carrier-cargo hydrophobization and charge ratio optimization improve the systemic circulation and safety of zwitterionic nano-polyplexes.
Jackson MA, Bedingfield SK, Yu F, Stokan ME, Miles RE, Curvino EJ, Hoogenboezem EN, Bonami RH, Patel SS, Kendall PL, Giorgio TD, Duvall CL
(2019) Biomaterials 192: 245-259
MeSH Terms: Animals, Cations, Cell Line, Tumor, Female, Humans, Hydrophobic and Hydrophilic Interactions, Mice, Inbred BALB C, Mice, Nude, Nanostructures, Neoplasms, Polymers, RNA, Small Interfering, RNAi Therapeutics, Tissue Distribution
Show Abstract · Added April 10, 2019
While polymeric nano-formulations for RNAi therapeutics hold great promise for molecularly-targeted, personalized medicine, they possess significant systemic delivery challenges including rapid clearance from circulation and the potential for carrier-associated toxicity due to cationic polymer or lipid components. Herein, we evaluated the in vivo pharmacokinetic and safety impact of often-overlooked formulation parameters, including the ratio of carrier polymer to cargo siRNA and hydrophobic siRNA modification in combination with hydrophobic polymer components (dual hydrophobization). For these studies, we used nano-polyplexes (NPs) with well-shielded, zwitterionic coronas, resulting in various NP formulations of equivalent hydrodynamic size and neutral surface charge regardless of charge ratio. Doubling nano-polyplex charge ratio from 10 to 20 increased circulation half-life five-fold and pharmacokinetic area under the curve four-fold, but was also associated with increased liver enzymes, a marker of hepatic damage. Dual hydrophobization achieved by formulating NPs with palmitic acid-modified siRNA (siPA-NPs) both reduced the amount of carrier polymer required to achieve optimal pharmacokinetic profiles and abrogated liver toxicities. We also show that optimized zwitterionic siPA-NPs are well-tolerated upon long-term, repeated administration in mice and exhibit greater than two-fold increased uptake in orthotopic MDA-MB-231 xenografts compared to commercial transfection reagent, in vivo-jetPEI. These data suggest that charge ratio optimization has important in vivo implications and that dual hydrophobization strategies can be used to maximize both NP circulation time and safety.
Copyright © 2018 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
Mechanistic insight into the interaction of gastrointestinal mucus with oral diblock copolymers synthesized via ATRP method.
Liu J, Cao J, Cao J, Han S, Liang Y, Bai M, Sun Y
(2018) Int J Nanomedicine 13: 2839-2856
MeSH Terms: Administration, Oral, Animals, Caco-2 Cells, Drug Carriers, Humans, Hydrophobic and Hydrophilic Interactions, Indoles, Intestinal Absorption, Intestinal Mucosa, Male, Methacrylates, Methylmethacrylates, Mice, Nanoparticles, Nylons, Particle Size, Polymers, Propionates, Tissue Distribution
Show Abstract · Added April 2, 2019
Introduction - Nanoparticles are increasingly used as drug carriers for oral administration. The delivery of drug molecules is largely dependent on the interaction of nanocarriers and gastrointestinal (GI) mucus, a critical barrier that regulates drug absorption. It is therefore important to understand the effects of physical and chemical properties of nanocarriers on the interaction with GI mucus. Unfortunately, most of the nanoparticles are unable to be prepared with satisfactory structural monodispersity to comprehensively investigate the interaction. With controlled size, shape, and surface chemistry, copolymers are ideal candidates for such purpose.
Materials and methods - We synthesized a series of diblock copolymers via the atom transfer radical polymerization method and investigated the GI mucus permeability in vitro and in vivo.
Results - Our results indicated that uncharged and hydrophobic copolymers exhibited enhanced GI absorption.
Conclusion - These results provide insights into developing optimal nanocarriers for oral administration.
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Structure and Function of the Transmembrane Domain of NsaS, an Antibiotic Sensing Histidine Kinase in Staphylococcus aureus.
Bhate MP, Lemmin T, Kuenze G, Mensa B, Ganguly S, Peters JM, Schmidt N, Pelton JG, Gross CA, Meiler J, DeGrado WF
(2018) J Am Chem Soc 140: 7471-7485
MeSH Terms: Anti-Bacterial Agents, Bacitracin, Bacterial Proteins, Gene Knockout Techniques, Histidine Kinase, Hydrophobic and Hydrophilic Interactions, Magnetic Resonance Spectroscopy, Membrane Proteins, Microbial Sensitivity Tests, Molecular Dynamics Simulation, Nisin, Protein Conformation, alpha-Helical, Protein Domains, Staphylococcus aureus
Show Abstract · Added March 21, 2020
NsaS is one of four intramembrane histidine kinases (HKs) in Staphylococcus aureus that mediate the pathogen's response to membrane active antimicrobials and human innate immunity. We describe the first integrative structural study of NsaS using a combination of solution state NMR spectroscopy, chemical-cross-linking, molecular modeling and dynamics. Three key structural features emerge: First, NsaS has a short N-terminal amphiphilic helix that anchors its transmembrane (TM) bundle into the inner leaflet of the membrane such that it might sense neighboring proteins or membrane deformations. Second, the transmembrane domain of NsaS is a 4-helix bundle with significant dynamics and structural deformations at the membrane interface. Third, the intracellular linker connecting the TM domain to the cytoplasmic catalytic domains of NsaS is a marginally stable helical dimer, with one state likely to be a coiled-coil. Data from chemical shifts, heteronuclear NOE, H/D exchange measurements and molecular modeling suggest that this linker might adopt different conformations during antibiotic induced signaling.
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Computational design of membrane proteins using RosettaMembrane.
Duran AM, Meiler J
(2018) Protein Sci 27: 341-355
MeSH Terms: Computer Simulation, Hydrophobic and Hydrophilic Interactions, Membrane Proteins, Protein Multimerization, Software
Show Abstract · Added March 17, 2018
Computational membrane protein design is challenging due to the small number of high-resolution structures available to elucidate the physical basis of membrane protein structure, multiple functionally important conformational states, and a limited number of high-throughput biophysical assays to monitor function. However, structural determination of membrane proteins has made tremendous progress in the past years. Concurrently the field of soluble computational design has made impressive inroads. These developments allow us to tackle the formidable challenge of designing functional membrane proteins. Herein, Rosetta is benchmarked for membrane protein design. We evaluate strategies to cope with the often reduced quality of experimental membrane protein structures. Further, we test the usage of symmetry in design protocols, which is particularly important as many membrane proteins exist as homo-oligomers. We compare a soluble scoring function with a scoring function optimized for membrane proteins, RosettaMembrane. Both scoring functions recovered around half of the native sequence when completely redesigning membrane proteins. However, RosettaMembrane recovered the most native-like amino acid property composition. While leucine was overrepresented in the inner and outer-hydrophobic regions of RosettaMembrane designs, it resulted in a native-like surface hydrophobicity indicating that it is currently the best option for designing membrane proteins with Rosetta.
© 2017 The Protein Society.
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5 MeSH Terms
Prp40 Homolog A Is a Novel Centrin Target.
Díaz Casas A, Chazin WJ, Pastrana-Ríos B
(2017) Biophys J 112: 2529-2539
MeSH Terms: Binding Sites, Calorimetry, Carrier Proteins, Chlamydomonas reinhardtii, Circular Dichroism, Humans, Hydrophobic and Hydrophilic Interactions, Protein Unfolding, Recombinant Proteins, Sequence Homology, Amino Acid, Spectroscopy, Fourier Transform Infrared, Thermodynamics, Trimethoprim, Sulfamethoxazole Drug Combination, Two-Hybrid System Techniques
Show Abstract · Added March 24, 2018
Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad WLL that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.
Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.
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14 MeSH Terms
Combinatorial optimization of PEG architecture and hydrophobic content improves ternary siRNA polyplex stability, pharmacokinetics, and potency in vivo.
Werfel TA, Jackson MA, Kavanaugh TE, Kirkbride KC, Miteva M, Giorgio TD, Duvall C
(2017) J Control Release 255: 12-26
MeSH Terms: Animals, Cell Line, Cell Line, Tumor, Female, Humans, Hydrophobic and Hydrophilic Interactions, Luciferases, Methacrylates, Mice, Mice, Nude, Polyethylene Glycols, RNA, Small Interfering, Tissue Distribution
Show Abstract · Added March 14, 2018
A rationally-designed library of ternary siRNA polyplexes was developed and screened for gene silencing efficacy in vitro and in vivo with the goal of overcoming both cell-level and systemic delivery barriers. [2-(dimethylamino)ethyl methacrylate] (DMAEMA) was homopolymerized or copolymerized (50mol% each) with butyl methacrylate (BMA) from a reversible addition - fragmentation chain transfer (RAFT) chain transfer agent, with and without pre-conjugation to polyethylene glycol (PEG). Both single block polymers were tested as core-forming units, and both PEGylated, diblock polymers were screened as corona-forming units. Ternary siRNA polyplexes were assembled with varied amounts and ratios of core-forming polymers to PEGylated corona-forming polymers. The impact of polymer composition/ratio, hydrophobe (BMA) placement, and surface PEGylation density was correlated to important outcomes such as polyplex size, stability, pH-dependent membrane disruptive activity, biocompatibility, and gene silencing efficiency. The lead formulation, DB4-PDB12, was optimally PEGylated not only to ensure colloidal stability (no change in size by DLS between 0 and 24h) and neutral surface charge (0.139mV) but also to maintain higher cell uptake (>90% positive cells) than the most densely PEGylated particles. The DB4-PDB12 polyplexes also incorporated BMA in both the polyplex core- and corona-forming polymers, resulting in robust endosomolysis and in vitro siRNA silencing (~85% protein level knockdown) of the model gene luciferase across multiple cell types. Further, the DB4-PDB12 polyplexes exhibited greater stability, increased blood circulation time, reduced renal clearance, increased tumor biodistribution, and greater silencing of luciferase compared to our previously-optimized, binary parent formulation following intravenous (i.v.) delivery. This polyplex library approach enabled concomitant optimization of the composition and ratio of core- and corona-forming polymers (indirectly tuning PEGylation density) and identification of a ternary nanomedicine optimized to overcome important siRNA delivery barriers in vitro and in vivo.
Copyright © 2017 Elsevier B.V. All rights reserved.
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13 MeSH Terms
A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y R Antagonists.
Burkert K, Zellmann T, Meier R, Kaiser A, Stichel J, Meiler J, Mittapalli GK, Roberts E, Beck-Sickinger AG
(2017) ChemMedChem 12: 75-85
MeSH Terms: Animals, Arginine, Benzazepines, Binding Sites, COS Cells, Cells, Cultured, Chlorocebus aethiops, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Molecular Docking Simulation, Molecular Structure, Receptors, Neuropeptide Y, Structure-Activity Relationship
Show Abstract · Added April 8, 2017
The neuropeptide Y receptor (Y R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Regulation of KCNQ/Kv7 family voltage-gated K channels by lipids.
Taylor KC, Sanders CR
(2017) Biochim Biophys Acta Biomembr 1859: 586-597
MeSH Terms: Amino Acid Sequence, Binding Sites, Cell Membrane, Epilepsy, Benign Neonatal, Fatty Acids, Unsaturated, Hearing Loss, Bilateral, Humans, Hydrophobic and Hydrophilic Interactions, KCNQ1 Potassium Channel, Long QT Syndrome, Membrane Lipids, Models, Molecular, Phosphatidylinositol 4,5-Diphosphate, Protein Binding, Protein Isoforms, Protein Structure, Secondary
Show Abstract · Added November 21, 2018
Many years of studies have established that lipids can impact membrane protein structure and function through bulk membrane effects, by direct but transient annular interactions with the bilayer-exposed surface of protein transmembrane domains, and by specific binding to protein sites. Here, we focus on how phosphatidylinositol 4,5-bisphosphate (PIP) and polyunsaturated fatty acids (PUFAs) impact ion channel function and how the structural details of the interactions of these lipids with ion channels are beginning to emerge. We focus on the Kv7 (KCNQ) subfamily of voltage-gated K channels, which are regulated by both PIP and PUFAs and play a variety of important roles in human health and disease. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Copyright © 2016 Elsevier B.V. All rights reserved.
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