Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Structural Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Membrane Dynamics ◽  
Signaling Proteins ◽  
Structural Constraints ◽  
Juxtamembrane Domain ◽  
Hydrophobic Membrane ◽  
Anionic Lipids ◽  
Lipid Diffusion

<br>Effective transmembrane signaling is vital to the life of the cell. However, plasma membrane dynamics pose numerous challenges for signaling proteins and attempts to study them. The receptor tyrosine kinase family transmits signals via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Detailed<br>studies of JMD-membrane interactions are challenging due to naturally slow lipid diffusion. We employ a combination of all-atom molecular dynamics of highly mobile membrane mimetics with in vitro and in cell experiments to capture critical roles of electrostatic and hydrophobic membrane-JMD interactions of<br>tropomyosin receptor kinase A (TrkA). We show that a conserved three-residue hydrophobic patch enables JMD anchoring in the membrane, a single residue deletion can dramatically reverse it, and a single charged to hydrophobic residue mutation can enhance binding affinity. The highly charged anionic lipids are effective<br>at promoting binding and restraining JMD flexibility. Our work highlights versatility of molecular controls of these interactions.<br>

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

scholarly journals Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

2020 ◽  
Author(s):  
Sean A. Newmister ◽  
Kinshuk Raj Srivastava ◽  
Rosa V. Espinoza ◽  
Kersti Caddell Haatveit ◽  
Yogan Khatri ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Molecular Basis ◽  
Hydrophobic Interactions ◽  
Cytochromes P450 ◽  
Targeted Mutagenesis ◽  
P450 Monooxygenase ◽  
Bond Functionalization ◽  
Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

scholarly journals In Vitro Cellular Uptake Studies of Self-Assembled Fluorinated Nanoparticles Labelled with Antibodies

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1906
Author(s):  
Mona Atabakhshi-Kashi ◽  
Mónica Carril ◽  
Hossein Mahdavi ◽  
Wolfgang J. Parak ◽  
Carolina Carrillo-Carrion ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cellular Uptake ◽  
Hydrophobic Interactions ◽  
Intrinsic Properties ◽  
Wide Range ◽  
Self Assembled ◽  
Uptake Studies ◽  
Targeting Ability ◽  
Fluorinated Nanoparticles

Nanoparticles (NPs) functionalized with antibodies (Abs) on their surface are used in a wide range of bioapplications. Whereas the attachment of antibodies to single NPs to trigger the internalization in cells via receptor-mediated endocytosis has been widely studied, the conjugation of antibodies to larger NP assemblies has been much less explored. Taking into account that NP assemblies may be advantageous for some specific applications, the possibility of incorporating targeting ligands is quite important. Herein, we performed the effective conjugation of antibodies onto a fluorescent NP assembly, which consisted of fluorinated Quantum Dots (QD) self-assembled through fluorine–fluorine hydrophobic interactions. Cellular uptake studies by confocal microscopy and flow cytometry revealed that the NP assembly underwent the same uptake procedure as individual NPs; that is, the antibodies retained their targeting ability once attached to the nanoassembly, and the NP assembly preserved its intrinsic properties (i.e., fluorescence in the case of QD nanoassembly).

scholarly journals Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

1987 ◽  
Vol 105 (3) ◽  
pp. 1463-1469 ◽  
Author(s):  
C Fornieri ◽  
M Baccarani-Contri ◽  
D Quaglino ◽  
I Pasquali-Ronchetti
Keyword(s):  
Hydrophobic Interactions ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Elastic Fibers ◽  
Amino Groups ◽  
Lysine Residues ◽  
Oxidase Inhibition

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

scholarly journals Screening marine algae metabolites as high-affinity inhibitors of SARS-CoV-2 main protease (3CLpro): an in silico analysis to identify novel drug candidates to combat COVID-19 pandemic

2020 ◽  
Vol 63 (1) ◽  
Author(s):  
Ghazala Muteeb ◽  
Adil Alshoaibi ◽  
Mohammad Aatif ◽  
Md. Tabish Rehman ◽  
M. Zuhaib Qayyum
Keyword(s):  
Hydrophobic Interactions ◽  
In Silico Analysis ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Binding Energies ◽  
Dynamics Simulation ◽  
In Vivo Studies ◽  
Energy Calculation

AbstractThe recent dissemination of SARS-CoV-2 from Wuhan city to all over the world has created a pandemic. COVID-19 has cost many human lives and created an enormous economic burden. Although many drugs/vaccines are in different stages of clinical trials, still none is clinically available. We have screened a marine seaweed database (1110 compounds) against 3CLpro of SARS-CoV-2 using computational approaches. High throughput virtual screening was performed on compounds, and 86 of them with docking score <  − 5.000 kcal mol−1 were subjected to standard-precision docking. Based on binding energies (< − 6.000 kcal mol−1), 9 compounds were further shortlisted and subjected to extra-precision docking. Free energy calculation by Prime-MM/GBSA suggested RC002, GA004, and GA006 as the most potent inhibitors of 3CLpro. An analysis of ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties of RC002, GA004, and GA006 indicated that only RC002 (callophysin A, from red alga Callophycus oppositifolius) passed Lipinski’s, Veber’s, PAINS and Brenk’s filters and displayed drug-like and lead-like properties. Analysis of 3CLpro-callophysin A complex revealed the involvement of salt bridge, hydrogen bonds, and hydrophobic interactions. callophysin A interacted with the catalytic residues (His41 and Cys145) of 3CLpro; hence it may act as a mechanism-based competitive inhibitor. Docking energy and docking affinity of callophysin A towards 3CLpro was − 8.776 kcal mol−1 and 2.73 × 106 M−1, respectively. Molecular dynamics simulation confirmed the stability of the 3CLpro-callophysin A complex. The findings of this study may serve as the basis for further validation by in vitro and in vivo studies.

scholarly journals The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

1998 ◽  
Vol 9 (6) ◽  
pp. 1235-1252 ◽  
Author(s):  
Owais Saifee ◽  
Liping Wei ◽  
Michael L. Nonet
Keyword(s):  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Acetylcholinesterase Inhibitor ◽  
Coiled Coil ◽  
Vesicle Fusion ◽  
Loss Of Function ◽  
Hydrophobic Membrane ◽  
Coiled Coil Domain ◽  
Synaptic Vesicle Fusion

We describe the molecular cloning and characterization of theunc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those ofDrosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

Dynamic organization of the mitochondrial protein import machinery

2016 ◽  
Vol 397 (11) ◽  
pp. 1097-1114 ◽  
Author(s):  
Sebastian P. Straub ◽  
Sebastian B. Stiller ◽  
Nils Wiedemann ◽  
Nikolaus Pfanner
Keyword(s):  
Mitochondrial Membrane ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Membrane Dynamics ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Precursor Proteins ◽  
Dynamic Cooperation

Abstract Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

scholarly journals Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters.

2004 ◽  
Vol 51 (1) ◽  
pp. 107-113 ◽  
Author(s):  
Irena Berezowska ◽  
Carole Lemieux ◽  
Nga N Chung ◽  
Bogumil Zelent ◽  
Peter W Schiller
Keyword(s):  
Hydrophobic Interactions ◽  
Fluorescence Emission ◽  
Opioid Peptide ◽  
Quantum Yields ◽  
Binding Studies ◽  
Dansyl Group ◽  
Distribution Studies ◽  
Peptide Agonist ◽  
Diaminopropionic Acid

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.

scholarly journals (De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants

2021 ◽  
Vol 22 (22) ◽  
pp. 12509
Author(s):  
Joana Angélica Loureiro ◽  
Stéphanie Andrade ◽  
Lies Goderis ◽  
Ruben Gomez-Gutierrez ◽  
Claudio Soto ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Hydrophobic Interactions ◽  
Neurodegenerative Disorder ◽  
Sodium Dodecyl ◽  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Cetyltrimethylammonium Chloride ◽  
Α Helix ◽  
Β Sheet

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or β-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that β-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl β-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that β-sheet structures comprise the assembly of the fibrils.

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