Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy.

Post-translational modifications (PTMs) are essential for the regulation of all cellular processes. The interplay of various PTMs on a single protein or different proteins comprises a complexity that we are far from understanding in its entirety. Reliable strategies for the enrichment and accurate quantification of PTMs are needed to study as many PTMs on proteins as possible.

Quantitative Mass Spectrometry-Based Proteomics: An Overview.

In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry.

Structure and function of SARS-CoV-2 polymerase.

Coronaviruses use an RNA-dependent RNA polymerase (RdRp) to replicate and express their genome. The RdRp associates with additional non-structural proteins (nsps) to form a replication-transcription complex (RTC) that carries out RNA synthesis, capping and proofreading. However, the structure of the RdRp long remained elusive, thus limiting our understanding of coronavirus genome expression and replication.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega-7,9 trans-fat rumenic acid.

Key Points: Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of β-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors.

The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II.

The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae.

Optical coherence transfer mediated by free electrons.

We theoretically investigate the quantum-coherence properties of the cathodoluminescence (CL) emission produced by a temporally modulated electron beam. Specifically, we consider the quantum-optical correlations of CL produced by electrons that are previously shaped by a laser field. Our main prediction is the presence of phase correlations between the emitted CL field and the electron-modulating laser, even though the emission intensity and spectral profile are independent of the electron state.

Liquid-liquid phase separation of tau: From molecular biophysics to physiology and disease.

Biomolecular condensation via liquid-liquid phase separation (LLPS) of intrinsically disordered proteins/regions (IDPs/IDRs), with and without nucleic acids, has drawn widespread interest due to the rapidly unfolding role of phase-separated condensates in a diverse range of cellular functions and human diseases. Biomolecular condensates form via transient and multivalent intermolecular forces that sequester proteins and nucleic acids into liquid-like membrane-less compartments. However, aberrant phase transitions into gel-like or solid-like aggregates might play an important role in neurodegenerative and other diseases.

Rare germline variants in the E-cadherin gene CDH1 are associated with the risk of brain tumors of neuroepithelial and epithelial origin.

The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.

H, C, N and P chemical shift assignment for stem-loop 4 from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required.

Bayesian Markov models improve the prediction of binding motifs beyond first order.

Transcription factors (TFs) regulate gene expression by binding to specific DNA motifs. Accurate models for predicting binding affinities are crucial for quantitatively understanding of transcriptional regulation. Motifs are commonly described by position weight matrices, which assume that each position contributes independently to the binding energy.

A quantitative description of macaque ganglion cell responses to natural scenes: the interplay of time and space.

Key Points: Responses to natural scenes are the business of the retina. We find primate ganglion cell responses to such scenes consistent with those to simpler stimuli. A biophysical model confirmed this and predicted ganglion cell responses with close to retinal reliability.

Challenges Encountered Applying Equilibrium and Nonequilibrium Binding Free Energy Calculations.

Binding free energy calculations have become increasingly valuable to drive decision making in drug discovery projects. However, among other issues, inadequate sampling can reduce accuracy, limiting the value of the technique. In this paper, we apply absolute binding free energy calculations to ligands binding to T4 lysozyme L99A and HSP90 using equilibrium and nonequilibrium approaches.

Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron-Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching.

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron-electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy.

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast and human PIC and a topological model for DNA opening by the general transcription factor TFIIH.

The Persistent Question of Potassium Channel Permeation Mechanisms.

Potassium channels play critical roles in many physiological processes, providing a selective permeation route for K ions in and out of a cell, by employing a carefully designed selectivity filter, evolutionarily conserved from viruses to mammals. The structure of the selectivity filter was determined at atomic resolution by x-ray crystallography, showing a tight coordination of desolvated K ions by the channel. However, the molecular mechanism of K ions permeation through potassium channels remains unclear, with structural, functional and computational studies often providing conflicting data and interpretations.

Analytical Ultracentrifugation for Analysis of Protein-Nucleic Acid Interactions.

Analytical ultracentrifugation is a powerful tool to characterize interactions of macromolecules in solution. In sedimentation velocity experiments, the sedimentation of interaction partners and complexes can be monitored directly and can be used to characterize interactions quantitatively. As an example, we show how the interaction of the clamp loader subcomplex of DNA polymerase III from E.

The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3'-UTRs of yeast mitochondrial mRNAs.

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism.

Origins and mechanisms leading to aneuploidy in human eggs.

The gain or loss of a chromosome-or aneuploidy-acts as one of the major triggers for infertility and pregnancy loss in humans. These chromosomal abnormalities affect more than 40% of eggs in women at both ends of the age spectrum, that is, young girls as well as women of advancing maternal age. Recent studies in human oocytes and embryos using genomics, cytogenetics, and in silico modeling all provide new insight into the rates and potential genetic and cellular factors associated with aneuploidy at varying stages of development.

Spontaneous and stimulated electron-photon interactions in nanoscale plasmonic near fields.

The interplay between free electrons, light, and matter offers unique prospects for space, time, and energy resolved optical material characterization, structured light generation, and quantum information processing. Here, we study the nanoscale features of spontaneous and stimulated electron-photon interactions mediated by localized surface plasmon resonances at the tips of a gold nanostar using electron energy-loss spectroscopy (EELS), cathodoluminescence spectroscopy (CL), and photon-induced near-field electron microscopy (PINEM). Supported by numerical electromagnetic boundary-element method (BEM) calculations, we show that the different coupling mechanisms probed by EELS, CL, and PINEM feature the same spatial dependence on the electric field distribution of the tip modes.

Structural basis of nucleosome transcription mediated by Chd1 and FACT.

Efficient transcription of RNA polymerase II (Pol II) through nucleosomes requires the help of various factors. Here we show biochemically that Pol II transcription through a nucleosome is facilitated by the chromatin remodeler Chd1 and the histone chaperone FACT when the elongation factors Spt4/5 and TFIIS are present. We report cryo-EM structures of transcribing Saccharomyces cerevisiae Pol II-Spt4/5-nucleosome complexes with bound Chd1 or FACT.