EPR Methods for Biological Cu(II): L-Band CW and NARS

Abstract: Copper has many roles in biology that involve the change of coordination sphere and/or oxidation state of the copper ion. Consequently, the study of copper in heterogeneous environments is an important area in biophysics. EPR is a primary technique for the investigation of paramagnetic copper, which is usually the isolated Cu(II) ion, but sometimes as Cu(II) in different oxidation states of multitransition ion clusters. The gross geometry of the coordination environment of Cu(II) can often be determined from a simple inspection of the EPR spectrum, recorded in the traditional X-band frequency range (9–10 GHz). Identification and quantitation of the coordinating ligand atoms, however, is not so straightforward. In particular, analysis of the superhyperfine structure on the EPR spectrum, to determine the number of coordinated nitrogen atoms, is fraught with difficulty at X-band, despite the observation that the overwhelming number of EPR studies of Cu(II) in the literature have been carried out at X-band. Greater reliability has been demonstrated at S-band (3–4 GHz), using the low-field parallel (gz) features. However, analysis relies on clear identification of the outermost superhyperfine line, which has the lowest intensity of all the spectral features. Computer simulations have subsequently indicated that the much more intense perpendicular region of the spectrum can be reliably interpreted at L-band (2 GHz). The present work describes the development of L-band EPR of Cu(II) into a routine method that is applicable to biological samples

Characterising Inter-helical Interactions of G Protein-Coupled Receptors with the Fragment Molecular Orbital Method

G-protein coupled receptors (GPCRs) are the largest superfamily of membrane proteins, regulating almost every aspect of cellular activity and serving as key targets for drug discovery. We have identified an accurate and reliable computational method to characterise the strength and chemical nature of the inter-helical interactions between the residues of transmembrane (TM) domains during different receptor activation states, something that cannot be characterised solely by visual inspection of structural information. Using the fragment molecular orbital (FMO) quantum mechanics method to analyse 35 crystal structures representing different branches of the class A GPCR family, we have identified 69 topologically-equivalent TM residues that form a consensus network of 51 inter-TM interactions, providing novel results that are consistent with and help to rationalise experimental data. This discovery establishes a comprehensive picture of how defined molecular forces govern specific inter-helical interactions which, in turn, support the structural stability, ligand binding and activation of GPCRs

The Involvement of amino acid side chains in shielding the nickel coordination site: an NMR study

Coordination of proteins and peptides to metal ions is known to affect their properties, often by a change in their structural organization. Side chains of the residues directly involved in metal binding or very close to the coordination centre may arrange themselves around it, in such a way that they can, for instance, disrupt the protein functions or stabilize a metal complex by shielding it from the attack of water or other small molecules. The conformation of these side chains may be crucial to different biological or toxic processes. In our research we have encountered such behaviour in several cases, leading to interesting results for our purposes. Here we give an overview on the structural changes involving peptide side chains induced by Ni(II) coordination. In this paper we deal with a number of peptides, deriving from proteins containing one or more metal coordinating sites, which have been studied through a series of NMR experiments in their structural changes caused by Ni(II) complexation. Several peptides have been included in the study: short sequences from serum albumin (HSA), Des-Angiotensinogen, the 30-amino acid tail of histone H4, some fragments from histone H2A and H2B, the initial fragment of human protamine HP2 and selected fragments from prion and Cap43 proteins. NMR was the election technique for gathering structural information. Experiments performed for this purpose included 1D ¹H and ¹³C, and 2D HSQC, COSY, TOCSY, NOESY and ROESY acquisitions, which allowed the calculation of the Ni(II) complexes structural models

pH MODULATION OF FIBRIL DISSOCIATION AND COPPER BINDING PROPERTIES OF THE PRION PROTEIN

The cellular form of prion protein (PrPC) is a cell-surface glycoprotein attached to lipid rafts via its glycosylphosphatidylinositol anchor. Conversion of PrPC to its scrapie conformer (PrPSc, the fibrillar form) constitutes the key event of the etiology of prion diseases. Fibril dissociation is necessary for efficient conversion and continued propagation of the disease state. Recent studies have revealed that conversion occurs along the endocytic pathway. To better understand the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils. We show that under conditions that mimic the endocytic environment, amyloid fibrils made from full-length recombinant prion protein dissociate both laterally and axially to form protofilaments. About 5% of the protofilaments are short enough to be considered soluble and contain ~100-300 monomers per structure; these also retain the biophysical characteristics of the filaments. We propose that protonation of His residues and charge repulsion in the N-terminal domain trigger fibril dissociation. Our data suggest that lysosomes and late endosomes are competent milieus for propagating the misfolded state not only by destabilizing the normal prion protein, but by accelerating fibril dissociation into smaller structures that may act as seeds for further fibril formation. PrPC binds four Cu(II) in its octarepeat region and another at the fifth binding site. Previous work has demonstrated detailed structural information on copper binding to these sites at neutral pH. Both types of binding sites contain ionizable groups, thus the effect of pH on copper binding needs to be clarified. Moreover, much less attention has been devoted to understanding copper binding in PrPSc, which is more pathologically relevant. These two aspects are investigated here using isothermal titration calorimetry and X-band electron paramagnetic spectroscopy. Our results confirm that copper binding to both the octarepeats and the fifth binding site is pH-dependent. We show that both sites bind copper in the fibrillar form with coordination modes similar to their monomeric counterparts. However, the ratios of the different coordination modes have changed in the fibril, which might suggest changes in their affinities after conversion and have potential effects on the redox properties of fibrils

Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection

Atomistic Simulations of Model Amyloid Beta Aggregates, Water Networks and their Optical Properties

Based on the amyloid hypothesis, amyloid oligomers and the fibrils that they aggregate into, have been implicated in neurodegenerative diseases. Most of these amyloid proteins live in a solvent environment. The role of solvent in modulating the structural and dynamical properties of amyloid proteins remains poorly understood. In this thesis, computer simulations are used to reveal the structural properties of the amyloid protein and the coupling between protein and water using model systems. After assessing the validity of the force fields by comparison with high-level quantum chemistry calculations, we examine further the conformational free energy landscape of an amyloid protein. Different conformations characterized in the free energy surface are driven by internal protein interactions as well as interactions between protein and water, resulting in the collective reorganization of protein and water hydrogen bond networks. We show that these proteins are surrounded by water wires that add a roughness to the free energy surface. To better understand the water hydrogen bond network and particularly the water wires around protein, we used data-science algorithms allowing for the dimensionality and free energy landscape of different water coordinates to be determined. These results confirm that using water wire coordinates encodes more information on the underlying secondary structure of the protein. Finally, ab initio calculations are used to investigate the optical properties of amyloid proteins to help rationalize recent experiments suggesting the intrinsic fluorescence in fibrils that can occur without aromatic residues

Innovative Non-PrP-Targeted Drug Strategy Designed to Enhance Prion Clearance

Prion diseases are a group of neurodegenerative disorders characterized by the accumulation of misfolded prion protein (called PrPSc). Although conversion of the cellular prion protein (PrPC) to PrPSc is still not completely understood, most of the therapies developed until now are based on blocking this process. Here, we propose a new drug strategy aimed at clearing prions without any direct interaction with neither PrPC nor PrPSc. Starting from the recent discovery of SERPINA3/SerpinA3n upregulation during prion diseases, we have identified a small molecule, named compound 5 (ARN1468), inhibiting the function of these serpins and effectively reducing prion load in chronically infected cells. Although the low bioavailability of this compound does not allow in vivo studies in prion-infected mice, our strategy emerges as a novel and effective approach to the treatment of prion disease

Development and application of quantitative proteomics strategies to analyze molecular mechanisms of neurodegeneration

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration

Energy decomposition analysis approaches and their evaluation on prototypical protein–drug interaction patterns

The partitioning of the energy in ab initio quantum mechanical calculations into its chemical origins (e.g., electrostatics, exchange-repulsion, polarization, and charge transfer) is a relatively recent development; such concepts of isolating chemically meaningful energy components from the interaction energy have been demonstrated by variational and perturbation based energy decomposition analysis approaches. The variational methods are typically derived from the early energy decomposition analysis of Morokuma [Morokuma, J. Chem. Phys., 1971, 55, 1236], and the perturbation approaches from the popular symmetry-adapted perturbation theory scheme [Jeziorski et al., Methods and Techniques in Computational Chemistry: METECC-94, 1993, ch. 13, p. 79]. Since these early works, many developments have taken place aiming to overcome limitations of the original schemes and provide more chemical significance to the energy components, which are not uniquely defined. In this review, after a brief overview of the origins of these methods we examine the theory behind the currently popular variational and perturbation based methods from the point of view of biochemical applications. We also compare and discuss the chemical relevance of energy components produced by these methods on six test sets that comprise model systems that display interactions typical of biomolecules (such as hydrogen bonding and pi-pi stacking interactions) including various treatments of the dispersion energy