scholarly journals Evaluating Inositol phospholipid interactions with Inward Rectifier Potassium Channels and characterising their role in Disease

2020 ◽  
Author(s):  
Tanadet Pipatpolkai ◽  
Robin A. Corey ◽  
Peter Proks ◽  
Frances M. Ashcroft ◽  
Phillip J. Stansfeld
Keyword(s):  
Amino Acid ◽  
Neonatal Diabetes ◽  
Specific Protein ◽  
Coarse Grained ◽  
Congenital Hyperinsulinism ◽  
Amino Acid Residues ◽  
Inward Rectifier Potassium Channels ◽  
Lipid Species ◽  
Energy Perturbation ◽  
Good Agreement

AbstractMembrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

scholarly journals Evaluating inositol phospholipid interactions with inward rectifier potassium channels and characterising their role in disease

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Tanadet Pipatpolkai ◽  
Robin A. Corey ◽  
Peter Proks ◽  
Frances M. Ashcroft ◽  
Phillip J. Stansfeld
Keyword(s):  
Amino Acid ◽  
Neonatal Diabetes ◽  
Specific Protein ◽  
Coarse Grained ◽  
Congenital Hyperinsulinism ◽  
Amino Acid Residues ◽  
Inward Rectifier Potassium Channels ◽  
Lipid Species ◽  
Energy Perturbation ◽  
Good Agreement

Abstract Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

scholarly journals Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Susan M. Mitchell ◽  
Morven Graham ◽  
Xinran Liu ◽  
Ralf M. Leonhardt
Keyword(s):  
Amino Acid ◽  
Pigment Cell ◽  
Specific Protein ◽  
Single Amino Acid ◽  
Amyloid Formation ◽  
Amino Acid Residues ◽  
Proteolytic Fragment ◽  
The Core ◽  
N Terminus ◽  
In Cis

AbstractThe pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked in cis to the CAF in order to drive amyloid formation. The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.

scholarly journals The amino acid sequence of plastocyanin from Cucurbita pepo L. (vegetable marrow)

1974 ◽  
Vol 143 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Michael D. Scawen ◽  
Donald Boulter
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Phenyl Isothiocyanate ◽  
Amino Acid Residues ◽  
Higher Plant ◽  
Single Polypeptide Chain ◽  
Vegetable Marrow ◽  
Single Polypeptide ◽  
Phylogenetic Affinities ◽  
Good Agreement

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A ‘tree’ of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.

A coarse-grained model of the effective interaction for charged amino acid residues and its application to formation of GCN4-pLI tetramer

2017 ◽  
Vol 116 (5-6) ◽  
pp. 649-657 ◽  
Author(s):  
Kazutomo Kawaguchi ◽  
Satoshi Nakagawa ◽  
Isman Kurniawan ◽  
Koichi Kodama ◽  
Muhammad Saleh Arwansyah ◽  
...  
Keyword(s):  
Amino Acid ◽  
Effective Interaction ◽  
Coarse Grained ◽  
Amino Acid Residues ◽  
Coarse Grained Model

scholarly journals Cartilage proteoglycans. Structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate

1976 ◽  
Vol 157 (1) ◽  
pp. 127-143 ◽  
Author(s):  
T E Hardingham ◽  
R J F Ewins ◽  
H Muir
Keyword(s):  
Amino Acid ◽  
Chondroitin Sulphate ◽  
Specific Protein ◽  
Fluorescence Measurement ◽  
Variable Degree ◽  
Amino Acid Residues ◽  
Binding Region ◽  
Protein Core ◽  
Average Size ◽  
Cartilage Proteoglycans

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.

scholarly journals Discovery of New Classes of Glycine transporter 2 (GlyT2) Inhibitors and Study of GlyT2 Selectivity by Combination of Novel Structural Based Virtual Screening Approach and Free Energy Perturbation (FEP+) Calculations

2019 ◽  
Author(s):  
Filip Fratev ◽  
Manuel Miranda-Arango ◽  
Elvia Padilla ◽  
Suman Sirimulla
Keyword(s):  
Chronic Pain ◽  
Free Energy ◽  
Amino Acid ◽  
Virtual Screening ◽  
Basic Amino Acid ◽  
Free Energy Perturbation ◽  
Amino Acid Residues ◽  
Strong Inhibitor ◽  
Energy Perturbation

In recent years, the mammalian GlyT2 transporter have emerged as a promising target for the development of anti-chronic pain agents. In our current work, we discovered a new set of promising hits that inhibit the glycine transport at nano and micromolar activity and have excellent selectivity over GlyT1 (as shown by in vitro studies), using a newly designed virtual screening (VS) protocol that combines a structure-based pharmacophore and docking screens. Furthermore, the free energy perturbation (FEP+ protocol) calculations and molecular dynamics (MD) studies revealed the GlyT2 amino acid residues critical for the binding and selectivity of both Glycine and our Lead1 compound. The FEP+ results well-matched available literature mutational data proving the quality of generated GlyT2 structure. Based on these calculations we propose that Lead1 may also be a strong inhibitor of the neutral and basic amino acid transporter B (0+) (SLC6A14). Thus, the subsequent lead optimization and characterization of refined compounds may lead to both chronic pain and pancreatic cancer agents addressing an unmet and challenging clinical needs.

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