Monitoring motions in relevant biological systems by means of Electron Paramagnetic Resonance

Biochemical patterns in living systems are based in several instances on mechanical events, resulting from proteins undergoing large amplitude motions triggered by chemical signals. Indeed, while performing their function, these proteins undergo some important conformational changes, which modulate the final outcome. A better knowledge of the systems and of the mechanisms regulating these motions is a key point in understanding the function of the proteins under exam. In particular, this thesis work is focused on the study of two relevant biological systems: the investigation on motor system proteins, like class II myosins, involved in the contractile function, and on a maturation protein involved in the correct assembly of the active site of a [FeFe]-hydrogenase. Myosin is one of the three molecular motors that are present in eukaryotic cell genome and, together with actin, it is responsible for muscle contraction. During the completion of its function, myosin undergoes large amplitude motions that result in a change of its biochemical state, causing the powerstroke which is necessary for muscle contraction. Point mutations and post-translational modifications of this protein lead to contractile dysfunctions and the organism can incur in severe diseases and damages. So far, myosin function and dysfunction have mainly been studied by means of parameters related to ATP hydrolysis or to mechanical output, without paying attention to the molecular motion in relation to protein structural alterations. The investigation of the mechanics of myosin at a molecular level, combined with the study of mechanical output such as force and shortening velocity, is required in altered systems for the rational design of drugs. This target, in fact, would be difficult to reach without a deeper understanding of alterations in the structure of the protein involved in the working of the system. [FeFe]-hydrogenases are enzymes that catalyze the reversible production of H2 in many bacteria and unicellular eukaryotes and several efforts are underway to understand how their complex active site (the H-cluster) is assembled. This site is characterized by a [4Fe4S]-2Fe cluster and its biosynthesis requires the cooperation of three conserved maturation proteins: HydE, HydF and HydG. Among them, HydF plays a central role in the whole maturation process, a double function of scaffold for the building of the H-cluster and carrier for the delivery of the mature prosthetic group to the hydrogenase, which is eventually converted in the active holo form. The protein contains a GTP-binding domain, in which the conformational changes related to the protein function still have to be defined at a molecular level, but they are certainly responsible for the interplay between partners in the maturation process. The topic gained a lot of scientific interest in recent years, as the bio-production of hydrogen is considered a clean source of energy, but its use is still limited for the high production costs. For these reasons, a deeper knowledge of the catalytic site assembly and functioning is required to improve the capability of hydrogen production. For the investigation on the conformational changes of both systems, Electron Paramagnetic Resonance (EPR), coupled with Site-Directed Spin Labeling (SDSL), is the election technique, as paramagnetic probes are sensitive to the rotational and internal dynamics in bio-molecules. In particular, Continuous Wave EPR (CW-EPR) enables to obtain information on the mobility and the dynamics of a nitroxide at a desired site, together with solvent accessibility, while Double Electron Electron Resonance (DEER) can be used to measure distances between couples of spins. In this way, possible conformational changes induced by stimuli in the proteins under investigation can be detected and models on the protein functionality may be proposed. In this thesis, EPR techniques have been applied in order to shed light on the molecular changes occurring in the systems upon different stimuli or mutations. To have a more complete overview on the events occurring in the mentioned proteins, complementary biochemical essays have also been carried out. Regarding the investigation on myosin, as a main result we were able to optimize a set-up to perform EPR experiments on muscle fibers, which allowed us to get insight into the structural modifications occurring in diseased systems; in particular, hypertrophy, atrophy and oxidation in mice muscles were examined. In the case of HydF, a detailed investigation on the structural changes promoted by the GTP-binding domain has been carried out. The results clearly show that the GTPase domain of HydF behaves as a small GTP switch, like a family of proteins that are involved in different biochemical pathways and that undergo conformational changes upon the binding of the nucleotide. The work presented in this thesis has been divided into three parts: in the first part, a general introduction on the importance of molecular motions in biological systems and an overview of the EPR techniques employed for the investigations will be given, while in the other two parts, the results obtained on the two systems that have been object of the present study will be discussed

Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase. The X-ray structure of HydF indicates that the protein is a homodimer with both monomers carrying two functional domains: a C-terminal FeS cluster-binding domain, where the precursor is assembled, and a N-terminal GTPase domain, whose exact contribution to cluster biogenesis and hydrogenase activation is still elusive. We previously obtained several hints suggesting that the binding of GTP to HydF could be involved in the interactions of this scaffold protein with the other maturases and with the hydrogenase itself. In this work, by means of site directed spin labeling coupled to EPR/PELDOR spectroscopy, we explored the conformational changes induced in a recombinant HydF protein by GTP binding, and provide the first clue that the HydF GTPase domain could be involved in the H-cluster assembly working as a molecular switch similarly to other known small GTPases

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells

Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.This work was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2033—Projektnummer 390677874, the DFG Priority Program SPP1601 “New Frontiers in Sensitivity in EPR Spectroscopy” (to E.B.), DFG BO 3000/5-1 (to E.B.), SFB958 – Z04 (to E.B.), DFG grant INST 130/972-1 FUGG (to E.B.). P.E.C. is supported by an Australian NHMRC fellowship (1079700

Neural networks in pulsed dipolar spectroscopy : a practical guide

This work was funded by a grant from Leverhulme Trust (RPG-2019-048). Studentship funding and technical support from MathWorks are gratefully acknowledged. This research was supported by grants from NVIDIA and utilised NVIDIA Tesla A100 GPUs through the Academic Grants Programme. We also acknowledge funding from the Royal Society (University Research Fellowship for JEL) and EPSRC (EP/R513337/1 studentship for HR and EP/L015110/1 studentship for MJT).This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their “robust black box” reputation hides the complexity of their design and training – particularly when the training dataset is effectively infinite. The objective of this paper is to give insight into training against simulated databases, to discuss network architecture choices, to describe options for handling DEER (double electron-electron resonance) and RIDME (relaxation-induced dipolar modulation enhancement) experiments, and to provide a practical data processing flowchart.Publisher PDFPeer reviewe

Monitoring motions in relevant biological systems by means of Electron Paramagnetic Resonance

Biochemical patterns in living systems are based in several instances on mechanical events, resulting from proteins undergoing large amplitude motions triggered by chemical signals. Indeed, while performing their function, these proteins undergo some important conformational changes, which modulate the final outcome. A better knowledge of the systems and of the mechanisms regulating these motions is a key point in understanding the function of the proteins under exam. In particular, this thesis work is focused on the study of two relevant biological systems: the investigation on motor system proteins, like class II myosins, involved in the contractile function, and on a maturation protein involved in the correct assembly of the active site of a [FeFe]-hydrogenase. Myosin is one of the three molecular motors that are present in eukaryotic cell genome and, together with actin, it is responsible for muscle contraction. During the completion of its function, myosin undergoes large amplitude motions that result in a change of its biochemical state, causing the powerstroke which is necessary for muscle contraction. Point mutations and post-translational modifications of this protein lead to contractile dysfunctions and the organism can incur in severe diseases and damages. So far, myosin function and dysfunction have mainly been studied by means of parameters related to ATP hydrolysis or to mechanical output, without paying attention to the molecular motion in relation to protein structural alterations. The investigation of the mechanics of myosin at a molecular level, combined with the study of mechanical output such as force and shortening velocity, is required in altered systems for the rational design of drugs. This target, in fact, would be difficult to reach without a deeper understanding of alterations in the structure of the protein involved in the working of the system. [FeFe]-hydrogenases are enzymes that catalyze the reversible production of H2 in many bacteria and unicellular eukaryotes and several efforts are underway to understand how their complex active site (the H-cluster) is assembled. This site is characterized by a [4Fe4S]-2Fe cluster and its biosynthesis requires the cooperation of three conserved maturation proteins: HydE, HydF and HydG. Among them, HydF plays a central role in the whole maturation process, a double function of scaffold for the building of the H-cluster and carrier for the delivery of the mature prosthetic group to the hydrogenase, which is eventually converted in the active holo form. The protein contains a GTP-binding domain, in which the conformational changes related to the protein function still have to be defined at a molecular level, but they are certainly responsible for the interplay between partners in the maturation process. The topic gained a lot of scientific interest in recent years, as the bio-production of hydrogen is considered a clean source of energy, but its use is still limited for the high production costs. For these reasons, a deeper knowledge of the catalytic site assembly and functioning is required to improve the capability of hydrogen production. For the investigation on the conformational changes of both systems, Electron Paramagnetic Resonance (EPR), coupled with Site-Directed Spin Labeling (SDSL), is the election technique, as paramagnetic probes are sensitive to the rotational and internal dynamics in bio-molecules. In particular, Continuous Wave EPR (CW-EPR) enables to obtain information on the mobility and the dynamics of a nitroxide at a desired site, together with solvent accessibility, while Double Electron Electron Resonance (DEER) can be used to measure distances between couples of spins. In this way, possible conformational changes induced by stimuli in the proteins under investigation can be detected and models on the protein functionality may be proposed. In this thesis, EPR techniques have been applied in order to shed light on the molecular changes occurring in the systems upon different stimuli or mutations. To have a more complete overview on the events occurring in the mentioned proteins, complementary biochemical essays have also been carried out. Regarding the investigation on myosin, as a main result we were able to optimize a set-up to perform EPR experiments on muscle fibers, which allowed us to get insight into the structural modifications occurring in diseased systems; in particular, hypertrophy, atrophy and oxidation in mice muscles were examined. In the case of HydF, a detailed investigation on the structural changes promoted by the GTP-binding domain has been carried out. The results clearly show that the GTPase domain of HydF behaves as a small GTP switch, like a family of proteins that are involved in different biochemical pathways and that undergo conformational changes upon the binding of the nucleotide. The work presented in this thesis has been divided into three parts: in the first part, a general introduction on the importance of molecular motions in biological systems and an overview of the EPR techniques employed for the investigations will be given, while in the other two parts, the results obtained on the two systems that have been object of the present study will be discussed.Molto spesso modelli biochimici in sistemi viventi sono basati su eventi meccanici, i quali sono il risultato di moti di grande ampiezza a cui sono soggette diverse proteine in seguito a stimoli chimici. Nel corso dell'esplicazione delle loro funzioni, infatti, queste proteine vanno incontro a variazioni conformazionali anche importanti, che ne regolano il comportamento finale. Un punto chiave per la comprensione della funzione di tali sistemi, pertanto, risulta essere una conoscenza più profonda degli stessi e dei meccanismi che regolano i loro moti. Il particolare, il lavoro contenuto all'interno di questa tesi è focalizzato allo studio di due sistemi biologici rilevanti: l'indagine su sistemi motore, come la classe della miosina di tipo II, coinvolta nella contrazione muscolare, e la ricerca riguardante una proteina di maturazione coinvolta nel corretto assemblaggio del sito attivo di una [FeFe]-idrogenasi. La miosina è uno dei tre motori molecolari che risultano essere presenti nel genoma delle cellule eucariotiche e, insieme all'actina, è responsabile della contrazione muscolare. Nel corso dell'esplicamento della sua funzione, la miosina va incontro a variazioni conformazionali che si traducono in una variazione del suo stato biochimico, provocando il motore biologico necessario alla contrazione. Mutazioni puntuali e modifiche post-traduzionali della miosina portano come conseguenza a disfunzioni contrattili e l'organismo può andare incontro a serie patologie e gravi danni. Finora, il corretto e l'alterato funzionamento della miosina sono stati principalmente studiati dal punto di vista di variazioni legate all'attività di idrolisi dell'ATP o al risultato meccanico, senza prestare particolare attenzione al dettaglio molecolare in relazione ad alterazioni strutturali della proteina. Lo studio della meccanica della miosina ad un livello molecolare, combinata all'indagine di parametri meccanici come produzione di forza e velocità di accorciamento delle fibre, risulta pertanto necessario in sistemi alterati per una progettazione oculata di farmaci per la cura di tali disfunzioni. Tale obiettivo, infatti, risulterebbe pressochè impossibile da raggiungere in assenza di una più profonda conoscenza delle alterazioni a livello strutturale della proteina coinvolta nel globale funzionamento del sistema. Le [FeFe]-idrogenasi sono enzimi che catalizzano la produzione reversibile di idrogeno in molti batteri ed eucarioti unicellulari, e numerosi sforzi vengono attualmente spesi per comprendere come il complesso sito attivo (denominato cluster H) sia assemblato. Il sito è caratterizzato dalla presenza di un cluster [4Fe4S]-2Fe la cui biosintesi richiede la cooperazione di tre proteine di maturazione conservate: HydFE, HydF e HydG. Tra queste, HydF ha un ruolo centrale nell'intero processo di maturazione, possiede infatti un doppio ruolo di scaffold per l'assemblaggio del sito attivo e di trasportatore del gruppo prostetico maturo all'idrogenasi vera e propria, che viene così convertita nella forma attiva. La proteina contiene un dominio per il legame del nucleotide GTP; le variazioni conformazionali di questo dominio, in relazione alla funzione espletata dalla proteina, devono ancora essere chiarite a livello molecolare, ma sono certamente responsabili dell'interazione tra le diverse proteine coinvolte nel processo di maturazione. Tale studio ha guadagnato un notevole interesse scientifico nel corso degli ultimi anni, poichè la bioproduzione di idrogeno è considerata una fonte pulita di energia, limitata tuttavia dagli alti costi di produzione. Per tali ragioni, una conoscenza più approfondita riguardo l'assemblaggio ed il funzionamento del sito catalitico è richiesta al fine di migliorare la capacità di produzione di idrogeno. La tecnica di risonanza paramagnetica elettronica (EPR), accoppiata a marcatura sito-specifica con sonde paramagnetiche, risulta essere la tecnica principe per quanto riguarda l'indagine delle variazioni conformazionali in entrambi i sistemi, dal momento che queste sonde sono sensibili alla dinamica rotazionale ed interna in molecole di interesse biologico. In particolare, EPR in onda continua permette di ottenere informazioni circa la dinamica di un nitrossido in un sito di interesse, oltre che informazioni sull'accessibilità dello stesso al solvente, mentre la tecnica DEER può essere utilizzata per misurare distanze tra coppie di spin. In questo modo, eventuali variazioni conformazionali indotte da diversi stimoli nei sistemi studiati possono essere studiate e si possono avanzare proposte riguardo un modello di funzionalità di queste proteine. In questa tesi, le diverse tecniche EPR sono state applicate in modo da gettare luce sulle variazioni a livello molecolare che avvengono in questi sistemi in seguito all'applicazione di diversi stimoli o mutazioni. Per avere una visione più generale sugli eventi che avvengono in queste proteine, le indagini sono state affiancate da test di funzionalità biochimica. Per quanto concerne lo studio sulla miosina, come risultato principale abbiamo ottimizzato un set-up per l'indagine spettroscopica di fibre muscolari, che ci ha permesso di approfondire lo studio di sistemi alterati; in particolare, lo studio si è concentrato su ipertrofia, atrofia ed ossidazione nei muscoli di topo. Nel caso di HydF, invece, si è svolta un'indagine dettagliata delle variazioni strutturali causate dal legame del GTP alla proteina. I risultati mostrano in modo inequivocabile che la maturasi in questione si comporta come altre proteine denominate GTP switches, le quali subiscono importanti variazioni conformazionali, fondamentali per la loro funzionalità, in seguito al legame del nucleotide. Il lavoro presentato in questa tesi è stato diviso in tre parti: nella prima parte, verranno date un'introduzione generale sull'importanza di moti molecolari che avvengono in sistemi biologici ed una panoramica sulle tecniche EPR utilizzate nello studio; nelle altre due parti, invece, verranno discussi i risultati ottenuti per i due sistemi che sono stati oggetto del presente lavoro

From in vitro towards in situ: structure-based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

ATP-binding cassette (ABC) exporters have been studied now for more than four decades, and recent structural investigation has produced a large number of protein database entries. Yet, important questions about how ABC exporters function at the molecular level remain debated, such as which are the molecular recognition hotspots and the allosteric couplings dynamically regulating the communication between the catalytic cycle and the export of substrates. This conundrum mainly arises from technical limitations confining all research to in vitro analysis of ABC transporters in detergent solutions or embedded in membrane-mimicking environments. Therefore, a largely unanswered question is how ABC exporters operate in situ, namely in the native membrane context of a metabolically active cell. This review focuses on novel mechanistic insights into type I ABC exporters gained through a unique combination of structure determination, biochemical characterization, generation of conformation-specific nanobodies/sybodies and double electron-electron resonance

Electron paramagnetic resonance spectroscopy in structural-dynamic studies of large protein complexes

Macromolecular protein assemblies are of fundamental importance for many processes inside the cell, as they perform complex functions and constitute central hubs where reactions occur. Generally, these assemblies undergo large conformational changes and cycle through different states that ultimately are connected to specific functions further regulated by additional small ligands or proteins. Unveiling the 3D structural details of these assemblies at atomic resolution, identifying the flexible parts of the complexes, and monitoring with high temporal resolution the dynamic interplay between different pro- tein regions under physiological conditions is key to fully understanding their properties and to fostering biomedical applications. In the last decade, we have seen remarkable advances in cryo-electron microscopy (EM) techniques, which deeply transformed our vision of structural biology, especially in the field of macromolecular assemblies. With cryo-EM, detailed 3D models of large macromolecular complexes in different conformational states became readily available at atomic resolution. Concomitantly, nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy (EPR) have benefited from methodological innovations which also improved the quality of the information that can be achieved. Such enhanced sensitivity widened their applicability to macromolecular complexes in environments close to physiological conditions and opened a path towards in-cell applications. In this review we will focus on the advantages and challenges of EPR techniques with an integrative approach towards a complete understanding of macromolecular structures and functions.</p

Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies

Different types of spin labels are currently available for structural studies of biomolecules both in vitro and in cells using Electron Paramagnetic Resonance (EPR) and pulse dipolar spectroscopy (PDS). Each type of label has its own advantages and disadvantages, that will be addressed in this chapter. The spectroscopically distinct properties of the labels have fostered new applications of PDS aimed to simultaneously extract multiple inter-label distances on the same sample. In fact, combining different labels and choosing the optimal strategy to address their inter-label distances can increase the information content per sample, and this is pivotal to better characterize complex multi-component biomolecular systems. In this review, we provide a brief background of the spectroscopic properties of the four most common orthogonal spin labels for PDS measurements and focus on the various methods at disposal to extract homo- and hetero-label distances in proteins. We also devote a section to possible artifacts arising from channel crosstalk and provide few examples of applications in structural biology.</p