Specific protein-RNA interactions are mostly preserved in biomolecular condensates.

Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.

Protein-RNA interactions: from mass spectrometry to drug discovery.

Proteins and RNAs are fundamental parts of biological systems, and their interactions affect many essential cellular processes. Therefore, it is crucial to understand at a molecular and at a systems level how proteins and RNAs form complexes and mutually affect their functions. In the present mini-review, we will first provide an overview of different mass spectrometry (MS)-based methods to study the RNA-binding proteome (RBPome), most of which are based on photochemical cross-linking. As we will show, some of these methods are also able to provide higher-resolution information about binding sites, which are important for the structural characterisation of protein-RNA interactions. In addition, classical structural biology techniques such as nuclear magnetic resonance (NMR) spectroscopy and biophysical methods such as electron paramagnetic resonance (EPR) spectroscopy and fluorescence-based methods contribute to a detailed understanding of the interactions between these two classes of biomolecules. We will discuss the relevance of such interactions in the context of the formation of membrane-less organelles (MLOs) by liquid-liquid phase separation (LLPS) processes and their emerging importance as targets for drug discovery.

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

Cross-linking and mass spectrometry as a tool for studying the structural biology of ribonucleoproteins.

Cross-linking and mass spectrometry (XL-MS) workflows represent an increasingly popular technique for low-resolution structural studies of macromolecular complexes. Cross-linking reactions take place in the solution state, capturing contact sites between components of a complex that represent the native, functionally relevant structure. Protein-protein XL-MS protocols are widely adopted, providing precise localization of cross-linking sites to single amino acid positions within a pair of cross-linked peptides. In contrast, protein-RNA XL-MS workflows are evolving rapidly and differ in their ability to localize interaction regions within the RNA sequence. Here, we review protein-protein and protein-RNA XL-MS workflows, and discuss their applications in studies of protein-RNA complexes. The examples highlight the complementary value of XL-MS in structural studies of protein-RNA complexes, where more established high-resolution techniques might be unable to produce conclusive data.

"Thermal Peroxidation" of Dietary Pentapeptides Yields N-Terminal 1,2-Dicarbonyls.

In this contribution we investigate the thermal degradation of dietary-relevant pentapeptides. Most unsaturated lipids degrade by the well-known peroxidation mechanism. Here we show a degradation mechanism of peptides analogous to lipid peroxidation, forming a series of novel degradation products with possible toxicological relevance. At elevated temperatures above 180°C, pentapeptides with an N-terminal phenylalanine moiety react via a debenzylation to form 1,2-dicabonyl compounds, replacing the N-terminal primary amine. We propose a radical-based reaction mechanism that leads via a common peroxoaminal intermediate to two distinct types of reaction products with a terminal α-1,2 diamide or an α-amide-aldehyde functionality.

Heat induced hydrolytic cleavage of the peptide bond in dietary peptides and proteins in food processing.

We investigate the hypothesis that proteins and peptides are thermally degraded by hydrolytic bond cleavage of amide bonds, hence yielding shorter peptides as main degradation products. A series of fifteen pentapeptides with varying sequences was subjected to heating. Products were investigated by targeted UHPLC-ESI-tandem mass spectrometry and targeted analysis revealed formation of 2,5-diketopiperazines, di- and tri-peptides. Relative quantities of the thermal degradation were determined to show that hydrolytic cleavage is an important, however not dominant degradation pathway. A series of dietary intact proteins were subjected to heating and products formed analyzed by MALDI-TOF mass spectrometry. For the majority of proteins larger degradation products with m/z values between 900 and 2500 could be observed, which we tentatively assign as hydrolytic cleavage products. For coffee globulin a series of eleven short peptides formed through thermal hydrolytic cleavage could be unambiguously identified formed through thermal proteolysis. The identical products could as well be identified in samples of roasted coffee clearly illustrating the occurrence and relevance of thermally induced proteolysis of proteins.

A proteolytic nanobiocatalyst with built-in disulphide reducing properties.

We report a method to equip proteolytic nanobiocatalysts with intrinsic disulphide bond reducing properties. After immobilisation onto silica particles, selected protease enzymes are partially shielded in a nanometre-thick mercaptosilica layer acting not only as a protective system but also as a substrate reducing agent. The biocatalysts produced efficiently perform simultaneous disulphide bond reduction and protein digestion. Besides a significant simplification of the proteolysis process, this strategy allows for a drastic increase of the enzyme stability.

Identification of Products from Thermal Degradation of Tryptophan Containing Pentapeptides: Oxidation and Decarboxylation.

In this contribution, we investigate the thermal decomposition of four pentapeptides containing a tryptophan moiety. Pentapeptides were heated at 220 °C, and the resulting reaction mixtures were investigated by HPLC coupled to high-resolution mass spectrometry and tandem mass spectrometry. A total of 95 thermal decomposition products could be observed and resolved by chromatography. In detail, we report on the structure assignment of two types of reaction products common to investigated peptides and introduce two decomposition mechanisms. Pentapeptides react with oxygen to produce hydroxyl-tryptophan derivatives. In addition, we observe the C-terminal decarboxylation of two peptides to form N-acyl tryptamine derivatives.