G-quadruplexes and their ligands: Biophysical methods to unravel g-quadruplex/ligand interactions

Funding Information: (PD/00065/2013). This work was supported by PESSOA program ref. 5079 and project “Projeto de Investigação Exploratória” ref. IF/00959/2015 entitled “NCL targeting by G-quadruplex aptamers for cervical cancer therapy” financed by Fundo Social Europeu e Programa Operacional Potencial Humano. Thanks are due to FCT/MCT for the financial support of the CICS-UBI UIDB/00709/2020 research unit and to the Portuguese NMR Network (ROTEIRO/0031/2013-PINFRA/22161/2016), through national funds and, where applicable, supported by European Investment Funds FEDER through COMPETE 2020, POCI, PORL and PIDDAC.Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural-and affinity and apparent affinity-based methods are in several cases complex and/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.publishersversionpublishe

Structural transitions in orb2 prion-like domain relevant for functional aggregation in memory consolidation

Grant BBM_TRA_0203 PD/BD/148028/2019 UIDB/04378/2020The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Ca, 13Cb, 1Ha, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated a-helix spanning residues 55–60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca21, but do bind Zn21, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.publishersversionpublishe

Implications for phase separation

Funding Information: The authors would also like to acknowledge Prof. Dr. Jaime Mota, Dra. Irina Franco for the technical assistance with the microscopic experiments, Philip O'Toole for the aid in protein production and Dr. Aldino Viegas and Dr. David Pantoja-Uceda for the support and valuable discussions regarding NMR spectroscopy. This work was supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy – i4HB Project (LA/P/0140/2020). The authors also thank FCT-Portugal for the PhD grant attributed to SF (PD/BD/148028/2019) under the PTNMRPhD Program. JO is a recipient of a Leonardo Grant from the Spanish BBVA Foundation (BBM_TRA_0203) and a Ramón y Cajal Grant (RYC2018-026042-I funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future.”) JO and DVL are supported by the Spanish Grants PID-2019-109276RA-I00 and PID-2019-109306RB-I00, respectively, both funded by MCIN/AEI/10.13039/501100011033. The NMR spectrometers are part of the National NMR Facility supported by FCT-Portugal (ROTEIRO/0031/2013–PINFRA/22161/2016, co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). The 800 MHz spectrometer present in the “Manuel Rico” NMR laboratory (LMR-CSIC) is a node of the Spanish Large-Scale National Facility (ICTS R-LRB-MR). Publisher Copyright: © 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.The mediation of liquid–liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+, and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.publishersversionpublishe

Revisiting ionic liquid structure‐property relationship: A critical analysis

grant number 201863/2014-6 UIDB/04378/2020 UIDB/50025/2020-2023 POCI-01-0145-FEDER-007688 PTDC/QUI-QFI/31508/2017 FCT-(ROTEIRO/0031/2013-PINFRA/22161/2016 UIDB/04378/2020In the last few years, ionic liquids (ILs) have been the focus of extensive studies concerning the relationship between structure and properties and how this impacts their application. Despite a large number of studies, several topics remain controversial or not fully answered, such as: the existence of ion pairs, the concept of free volume and the effect of water and its implications in the modulation of ILs physicochemical properties. In this paper, we present a critical review of state‐of-the‐art literature regarding structure–property relationship of ILs, we re‐examine analytical theories on the structure–property correlations and present new perspectives based on the existing data. The interrelation between transport properties (viscosity, diffusion, conductivity) of IL structure and free volume are analysed and discussed at a molecular level. In addition, we demonstrate how the analysis of microscopic features (particularly using NMR‐derived data) can be used to explain and predict macroscopic properties, reaching new perspectives on the properties and application of ILs.publishersversionpublishe

Epitope Mapping by NMR of a Novel Anti-Aβ Antibody (STAB-MAb)

FP7-SP3-People-606950 POCI-01-0145-FEDER-007728 Project No 022161Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of β-amyloid (Aβ), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-Aβ monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (KD = 80 pM) and fibrils (KD = 130 pM) of Aβ(1–42) and has shown therapeutic efficacy in preclinical mouse models of AD. Our findings reveal a widespread epitope that embraces several key Aβ residues that have been previously described as important in the Aβ fibrillation process. Of note, STAB-MAb exhibits a stronger affinity for the N-terminus of Aβ and stabilizes an α-helix conformation in the central to N-terminal region of the peptide, in addition to disrupting a characteristic salt-bridge of a hairpin structure present in fibrils. The NMR derived epitope supports the observed results from ThT-monitored fluorescence and electron microscopy experiments, in which STAB-MAb was shown to inhibit the formation of aggregates and promote disruption of pre-formed fibrils. In combination with the published in vitro and in vivo assays, our study highlights STAB-MAb as a rare and versatile antibody with analytical, diagnostic and therapeutic efficacy.publishersversionpublishe

A comparison between pure active pharmaceutical ingredients and therapeutic deep eutectic solvents: solubility and permeability studies

THEDES, so called therapeutic deep eutectic solvents are here defined as a mixture of two components, which at a particular molar composition become liquid at room temperature and in which one of them is an active pharmaceutical ingredient (API). In this work, THEDES based on menthol complexed with three different APIs, ibuprofen (ibu), BA (BA) and phenylacetic acid (PA), were prepared. The interactions between the components that constitute the THEDES were studied by NMR, confirming that the eutectic system is formed by H-bonds between menthol and the API. The mobility of the THEDES components was studied by PFGSE NMR spectroscopy. It was determined that the self-diffusion of the species followed the same behavior as observed previously for ionic liquids, in which the components migrate via jumping between voids in the suprastructure created by punctual thermal fluctuations. The solubility and permeability of the systems in an isotonic solution was evaluated and a comparison with the pure APIs was established through diffusion and permeability studies carried out in a Franz cell. The solubility of the APIs when in the THEDES system can be improved up to 12 fold, namely for the system containing ibu. Furthermore, for this system the permeability was calculated to be 14 105 cm/s representing a 3 fold increase in comparison with the pure API. With the exception of the systems containing PA an increase in the solubility, coupled with an increase in permeability was observed. In this work, we hence demonstrate the efficiency of THEDES as a new formulation for the enhancement of the bioavailability of APIs by changing the physical state of the molecules from a solid dosage to a liquid system.he European Union’s Seventh Framework Programme (FP7/2007- 2013) under grant agreement nREGPOT-CT2012-316331- POLARIS and from Project ‘‘Novel smart and biomimetic materials for innovative regenerative medicine approaches (Ref.: RL1 - ABMR - NORTE-01-0124-FEDER-000016)” co-financed by North Portugal Regional Operational Programme (ON.2 – O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Funding was also provided by Fundação para a Ciência e a Tecnologia, through contracts LAQV-REQUIMTE: UID/QUI/50006/2013 and UCIBIOREQUIMTE: UID/Multi/04378/2013. This work was co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01- 0145-FEDER-007728).info:eu-repo/semantics/publishedVersio

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

UID/Multi/04378/2019 Grant no. BB/P010660/1 grant number BB/M011216/1Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.publishersversionpublishe

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin- dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dualbinding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.publishersversionpublishe

Pre-miRNA-149 G-quadruplex as a molecular agent to capture nucleolin

PD/BD/142851/2018 PD/00065/2013 MIT-EXPL/BIO/0008/2017 IF/00959/2015One of the most significant challenges in capturing and detecting biomarkers is the choice of an appropriate biomolecular receptor. Recently, RNA G-quadruplexes emerged as plausible receptors due to their ability to recognize with high-affinity proteins. Herein, we have unveiled and characterized the capability of the precursor microRNA 149 to form a G-quadruplex structure and determined the role that some ligands may have in its folding and binding capacity to nucleolin. The G-quadruplex formation was induced by K+ ions and stabilized by ligands, as demonstrated by nuclear magnetic resonance and circular dichroism experiments. Surface plasmon resonance measurements showed a binding affinity of precursor microRNA 149 towards ligands in the micromolar range (10−5–10−6 M) and a strong binding affinity to nucleolin RNA-binding domains 1 and 2 (8.38 × 10−10 M). Even in the presence of the ligand PhenDC3, the binding remains almost identical and in the same order of magnitude (4.46 × 10−10 M). The molecular interactions of the RNA G-quadruplex motif found in precursor miRNA 149 (5′-GGGAGGGAGGGACGGG- 3′) and nucleolin RNA-binding domains 1 and 2 were explored by means of molecular docking and molecular dynamics studies. The results showed that RNA G-quadruplex binds to a cavity between domains 1 and 2 of the protein. Then, complex formation was also evaluated through polyacrylamide gel electrophoresis. The results suggest that precursor microRNA 149/ligands and precursor microRNA 149/nucleolin RNA-binding domains 1 and 2 form stable molecular complexes. The in vitro co-localization of precursor microRNA 149 and nucleolin in PC3 cells was demonstrated using confocal microscopy. Finally, a rapid and straightforward microfluidic strategy was employed to check the ability of precursor microRNA 149 to capture nucleolin RNA-binding domains 1 and 2. The results revealed that precursor microRNA 149 can capture nucleolin RNA-binding domains 1 and 2 labeled with Fluorescein 5-isothiocyanate in a concentration-dependent manner, but PhenDC3 complexation seems to decrease the ability of precursor microRNA 149 to capture the protein. Overall, our results proved the formation of the G-quadruplex structure in the precursor microRNA 149 and the ability to recognize and detect nucleolin. This proof-of-concept study could open up a new framework for developing new strategies to design improved molecular receptors for capture and detection of nucleolin in complex biological samples.publishersversionpublishe