Pulse EPR measurements of intramolecular distances in a TOPP-labeled transmembrane peptide in lipids.

We present the performance of nanometer-range pulse electron paramagnetic resonance distance measurements (pulsed electron-electron double resonance/double electron-electron resonance, PELDOR/DEER) on a transmembrane WALP24 peptide labeled with the semirigid unnatural amino acid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP). Distances reported by the TOPP label are compared to the ones reported by the more standard MTSSL spin label, commonly employed in protein studies. Using high-power pulse electron paramagnetic resonance spectroscopy at Q-band frequencies (34 GHz), we show that in contrast to MTSSL, our label reports one-peak, sharp (Δr ≤ 0.4 nm) intramolecular distances. Orientational selectivity is not observed. When spin-labeled WALP24 was inserted in two representative lipid bilayers with different bilayer thickness, i.e., DMPC and POPC, the intramolecular distance reported by TOPP did not change with the bilayer environment. In contrast, the distance measured with MTSSL was strongly affected by the hydrophobic thickness of the lipid. The results demonstrate that the TOPP label is well suited to study the intrinsic structure of peptides immersed in lipids

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy

Chemical synthesis of site-selective advanced glycation end products in α-synuclein and its fragments

\ua9 2024 The Royal Society of Chemistry.Advanced glycation end products (AGEs) arise from the Maillard reaction between dicarbonyls and proteins, nucleic acids, or specific lipids. Notably, AGEs are linked to aging and implicated in various disorders, spanning from cancer to neurodegenerative diseases. While dicarbonyls like methylglyoxal preferentially target arginine residues, lysine-derived AGEs, such as N(6)-(1-carboxymethyl)lysine (CML) and N(6)-(1-carboxyethyl)lysine (CEL), are also abundant. Predicting protein glycation in vivo proves challenging due to the intricate nature of glycation reactions. In vitro, glycation is difficult to control, especially in proteins that harbor multiple glycation-prone amino acids. α-Synuclein (aSyn), pivotal in Parkinson\u27s disease and synucleinopathies, has 15 lysine residues and is known to become glycated at multiple lysine sites. To understand the influence of glycation in specific regions of aSyn on its behavior, a strategy for site-specific glycated protein production is imperative. To fulfill this demand, we devised a synthetic route integrating solid-phase peptide synthesis, orthogonal protection of amino acid side-chain functionalities, and reductive amination strategies. This methodology yielded two disease-related N-terminal peptide fragments, each featuring five and six CML and CEL modifications, alongside a full-length aSyn protein containing a site-selective E46CEL modification. Our synthetic approach facilitates the broad introduction of glycation motifs at specific sites, providing a foundation for generating glycated forms of synucleinopathy-related and other disease-relevant proteins

Spontaneous aggregation of the insulin-derived steric zipper peptide VEALYL results in different aggregation forms with common features.

Recently, several short peptides have been shown to self-assemble into amyloid fibrils with generic cross-beta spines, so-called steric zippers, suggesting common underlying structural features and aggregation mechanisms. Understanding these mechanisms is a prerequisite,for designing fibril-binding compounds and inhibitors of fibril formation. The hexapeptide VEALYL, corresponding to the residues B12-17 of full-length insulin, has been identified as one of these short segments. Here, we analyzed the structures of multiple, morphologically different (fibrillar, microcrystal-like, oligomeric) [C-13,N-15]VEALYL samples by solid-state nuclear magnetic resonance complemented with results from molecular dynamics simulations. By performing NHHC/CHHC experiments, we could determine that the beta-strands within a given sheet of the amyloid-like fibrils formed by the insulin hexapeptide VEALYL are stacked in an antiparallel manner, whereas the sheet-to-sheet packing arrangement was found to be parallel. Experimentally observed secondary chemical shifts for all aggregate forms, as well as empty set and Psi backbone torsion angles calculated with TALOS, are indicative of beta-strand conformation, consistent with the published crystal structure (PDB ID: 2OMQ). Thus, we could demonstrate that the structural features of all the observed VEALYL aggregates are in agreement with the previously observed homosteric zipper spine packing in the crystalline state, suggesting that several distinct aggregate morphologies share the same molecular architecture. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved

Membrane protein sequestering by ionic protein–lipid interactions.

Neuronal exocytosis is catalyzed by the SNARE protein syntaxin-1A(1). Syntaxin-1A is clustered in the plasma membrane at sites where synaptic vesicles undergo exocytosis(2,3). However, how syntaxin-1A is sequestered is unknown. Here, we show that syntaxin clustering is mediated by electrostatic interactions with the strongly anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2). We found with super-resolution STED microscopy on the plasma membrane of PC12 cells that PIP2 is the dominant inner-leaflet lipid in ~73 nm-sized microdomains. This high accumulation of PIP2 was required for syntaxin-1A sequestering, as destruction of PIP2 by the phosphatase synaptojanin-1 reduced syntaxin-1A clustering. Furthermore, co-reconstitution of PIP2 and the C-terminal part of syntaxin-1A in artificial giant unilamellar vesicles resulted in segregation of PIP2 and syntaxin-1A into distinct domains even when cholesterol was absent. Our results demonstrate that electrostatic protein-lipid interactions can result in the formation of microdomains independent of cholesterol or lipid phases

Diagnosis of obstructive coronary artery disease using computed tomography angiography in patients with stable chest pain depending on clinical probability and in clinically important subgroups: meta-analysis of individual patient data

OBJECTIVE: To determine whether coronary computed tomography angiography (CTA) should be performed in patients with any clinical probability of coronary artery disease (CAD), and whether the diagnostic performance differs between subgroups of patients. DESIGN: Prospectively designed meta-analysis of individual patient data from prospective diagnostic accuracy studies. DATA SOURCES: Medline, Embase, and Web of Science for published studies. Unpublished studies were identified via direct contact with participating investigators. ELIGIBILITY CRITERIA FOR SELECTING STUDIES: Prospective diagnostic accuracy studies that compared coronary CTA with coronary angiography as the reference standard, using at least a 50% diameter reduction as a cutoff value for obstructive CAD. All patients needed to have a clinical indication for coronary angiography due to suspected CAD, and both tests had to be performed in all patients. Results had to be provided using 2×2 or 3×2 cross tabulations for the comparison of CTA with coronary angiography. Primary outcomes were the positive and negative predictive values of CTA as a function of clinical pretest probability of obstructive CAD, analysed by a generalised linear mixed model; calculations were performed including and excluding non-diagnostic CTA results. The no-treat/treat threshold model was used to determine the range of appropriate pretest probabilities for CTA. The threshold model was based on obtained post-test probabilities of less than 15% in case of negative CTA and above 50% in case of positive CTA. Sex, angina pectoris type, age, and number of computed tomography detector rows were used as clinical variables to analyse the diagnostic performance in relevant subgroups. RESULTS: Individual patient data from 5332 patients from 65 prospective diagnostic accuracy studies were retrieved. For a pretest probability range of 7-67%, the treat threshold of more than 50% and the no-treat threshold of less than 15% post-test probability were obtained using CTA. At a pretest probability of 7%, the positive predictive value of CTA was 50.9% (95% confidence interval 43.3% to 57.7%) and the negative predictive value of CTA was 97.8% (96.4% to 98.7%); corresponding values at a pretest probability of 67% were 82.7% (78.3% to 86.2%) and 85.0% (80.2% to 88.9%), respectively. The overall sensitivity of CTA was 95.2% (92.6% to 96.9%) and the specificity was 79.2% (74.9% to 82.9%). CTA using more than 64 detector rows was associated with a higher empirical sensitivity than CTA using up to 64 rows (93.4% v 86.5%, P=0.002) and specificity (84.4% v 72.6%, P<0.001). The area under the receiver-operating-characteristic curve for CTA was 0.897 (0.889 to 0.906), and the diagnostic performance of CTA was slightly lower in women than in with men (area under the curve 0.874 (0.858 to 0.890) v 0.907 (0.897 to 0.916), P<0.001). The diagnostic performance of CTA was slightly lower in patients older than 75 (0.864 (0.834 to 0.894), P=0.018 v all other age groups) and was not significantly influenced by angina pectoris type (typical angina 0.895 (0.873 to 0.917), atypical angina 0.898 (0.884 to 0.913), non-anginal chest pain 0.884 (0.870 to 0.899), other chest discomfort 0.915 (0.897 to 0.934)). CONCLUSIONS: In a no-treat/treat threshold model, the diagnosis of obstructive CAD using coronary CTA in patients with stable chest pain was most accurate when the clinical pretest probability was between 7% and 67%. Performance of CTA was not influenced by the angina pectoris type and was slightly higher in men and lower in older patients. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42012002780

Longer and better lives for patients with atrial fibrillation: the 9th AFNET/EHRA consensus conference.

AIMS: Recent trial data demonstrate beneficial effects of active rhythm management in patients with atrial fibrillation (AF) and support the concept that a low arrhythmia burden is associated with a low risk of AF-related complications. The aim of this document is to summarize the key outcomes of the 9th AFNET/EHRA Consensus Conference of the Atrial Fibrillation NETwork (AFNET) and the European Heart Rhythm Association (EHRA). METHODS AND RESULTS: Eighty-three international experts met in Münster for 2 days in September 2023. Key findings are as follows: (i) Active rhythm management should be part of the default initial treatment for all suitable patients with AF. (ii) Patients with device-detected AF have a low burden of AF and a low risk of stroke. Anticoagulation prevents some strokes and also increases major but non-lethal bleeding. (iii) More research is needed to improve stroke risk prediction in patients with AF, especially in those with a low AF burden. Biomolecules, genetics, and imaging can support this. (iv) The presence of AF should trigger systematic workup and comprehensive treatment of concomitant cardiovascular conditions. (v) Machine learning algorithms have been used to improve detection or likely development of AF. Cooperation between clinicians and data scientists is needed to leverage the potential of data science applications for patients with AF. CONCLUSIONS: Patients with AF and a low arrhythmia burden have a lower risk of stroke and other cardiovascular events than those with a high arrhythmia burden. Combining active rhythm control, anticoagulation, rate control, and therapy of concomitant cardiovascular conditions can improve the lives of patients with AF