Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.

Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases1. However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial cellular functions2. Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood. Here we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that PK forms stress-dependent reversible amyloids through a pH-sensitive amyloid core. Stress- induced cytosolic acidification promotes aggregate formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Our work thus unravels a conserved and potentially widespread mechanism underlying amyloid functionality and reversibility, fine-tuned to the respective physiological cellular pH range.

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

High-density lipoprotein particles (HDLs) are transport containers in the circulatory system that receive cellular cholesterol and lipids destined for the liver and other lipoprotein particles. Because low levels of HDL-cholesterol often indicate an increased risk for cardiovascular diseases, HDL particles are considered as important pharmacological targets for therapeutic strategies. Mature spherical HDLs develop from lipid-free apolipoprotein apoA-I through the formation of intermediate discoidal HDL particles which are the primary acceptors of cellular cholesterol. Although of high biophysical and medical importance heterogeneity in density, size, shape, as well as protein and lipid composition prohibited a detailed molecular and structural description of discoidal HDL particles. Here, we present the three-dimensional solution structure of reconstituted discoidal HDL (rdHDL) particles by combining nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. By using amino acid selective labeling, methyl labeling, Lipid-PREs and long-range EPR data we found that rdHDL particles are composed of two helical apoA-I molecules that dimerise in an antiparallel fashion to form a double belt around a lipid bilayer patch. The integrity of this unique structure is maintained by up to 28 salt bridges and an unusual zipper-like pattern of cation-I€ interactions between helices 4 and 6. In order to accommodate a hydrophobic interior a gross †˜ right to right†™ rotation of the helices upon lipidation is necessary. The structure relevant in our understanding of HDLbiology and metabolism reflects thereby the beauty and complexity of this type of biological shuttling container that is able to hold a fluid lipid/cholesterol interior at a protein lipid ratio of 1:50.

Significance The characteristic feature of Parkinson’s disease is the deposition of α-synuclein into insoluble amyloid fibrils. The so-called secondary nucleation mechanism appears to be key for the aggregation kinetics, because binding of monomers on the fibril surface can autocatalytically induce new amyloid seeds. We show by nuclear magnetic and electron paramagnetic resonance spectroscopy that α-synuclein monomer–fibril binding is primarily mediated by transient electrostatic interactions. These intermolecular contacts result in an unfolding of the loosely packed α-synuclein structures and expose the otherwise protected aggregation-prone non-amyloid-β component of the protein. Our data demonstrate that intramolecular unfolding of α-synuclein is a prerequisite for protein aggregation that leads to rapid multiplication of α-synuclein amyloid fibrils via the secondary nucleation process.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

The Parkinson's disease protein α-synuclein (αSyn) promotes membrane fusion and fission by interacting with various negatively charged phospholipids. Despite postulated roles in endocytosis and exocytosis, plasma membrane (PM) interactions of αSyn are poorly understood. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3), two highly acidic components of inner PM leaflets, mediate PM localization of endogenous pools of αSyn in A2780, HeLa, SK-MEL-2, and differentiated and undifferentiated neuronal SH-SY5Y cells. We demonstrate that αSyn binds to reconstituted PIP2 membranes in a helical conformation in vitro and that PIP2 synthesizing kinases and hydrolyzing phosphatases reversibly redistribute αSyn in cells. We further delineate that αSyn-PM targeting follows phosphoinositide-3 kinase (PI3K)-dependent changes of cellular PIP2 and PIP3 levels, which collectively suggests that phosphatidylinositol polyphosphates contribute to αSyn's function(s) at the plasma membrane.

In potassium (K+) channels, permeation, selectivity, and gating at the selectivity filter are all governed by the thermodynamics and kinetics of the ion-protein interactions. Specific contacts between the carbonyl groups from the Thr-Val-Gly-Tyr-Gly signature filter sequence and the permeant ions generate four equidistant K+ binding sites, thereby defining the high ion selectivity and controlling the transport rate of K+ channels. Here, we used 15N-labeled ammonium (15NH4+) as a proxy for K+ to study ion interaction with the selectivity filter of the prototypical full-length K+ channel KcsA by solution state NMR spectroscopy in order to obtain detailed insights into the physicochemical basis of K+ gating. We found that in the closed inactive state of KcsA (at pH 7) four K+ binding sites are occupied over a wide range of 15NH4+ concentrations, while in intermediate closed-open conformations (at pH ∼6) the number and occupancy of K+ binding sites are reduced to two. However, in the presence of the scorpion toxin agitoxin II a total loss of 15NH4+ binding is observed. 15NH4+ titration studies allowed us to determine the dissociation constants of the four binding sites with values around 10 mM in the closed state of KcsA. Moreover, kinetic NMR experiments measured in the steady state equilibrium detected an off- and on-rate for 15NH4+ of ca. 102 s-1 and 103 s-1 between KcsA-bound 15NH4+ and the bulk. These findings reveal both the thermodynamics and kinetics of the ion binding sites and thus contribute to our understanding of the action of K+ channels.

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (alpha Syn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific alpha Syn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered alpha Syn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of alpha Syn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

AbstractApolipoprotein nanodiscs are a versatile tool in nanotechnology as membrane mimetics allowing, for example, the study of membrane proteins. It has recently been discovered that the Parkinson’s disease associated protein α-synuclein (α-Syn) can also form discoid-like lipoprotein nanoparticles. The present review highlights the observation that α-Syn has the properties to define stable and homogeneous populations of nanoparticles with diameters of 7–10 nm and 19–28 nm by modifying lipid vesicles or encapsulating lipid bilayers in a nanodisc-type fashion, respectively. In contrast to apolipoprotein nanodiscs, α-Syn nanoparticles can incorporate entirely negatively charged lipids emphasizing their potential use in nanotechnology as a negatively charged membrane mimetic.

The cellular processes underpinning life are orchestrated by proteins and their interactions. Structural and dynamic heterogeneity, despite being key to protein and drug function, continues to pose a fundamental challenge to existing analytical and structural methodologies used to study these associations. Here, we use interferometric scattering microscopy to mass-image single biomolecules in solution with <2% mass error, up to 19-kDa resolution and 1-kDa precision. Thereby, we resolve oligomeric distributions at high dynamic range, detect small-molecule binding, and mass-image biomolecules composed not only of amino acids, but also heterogeneous species, such as lipo- and glycoproteins. These capabilities enable us to characterize the molecular mechanisms of processes as diverse as oligomeric selfassembly, glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry (iSCAMS) provides spatially resolved access to the dynamics of biomolecular interactions ranging from those involving small molecules to mesoscopic assemblies, one molecule at a time.

alpha-Synuclein (alpha-Syn) is an intrinsically disordered protein in solution whose fibrillar aggregates are the hallmark of Parkinson's disease (PD). Although the specific function of alpha-Syn is still unclear, its high structural plasticity is key for the interactions of alpha-Syn with biological membranes. Recently, it has been observed that alpha-Syn is able to form high-density lipoprotein-like (HDL-like) particles that are reminiscent of self-assembling phospholipid bilayer nanodiscs. Here, we extended our preparation method for the production of alpha-Syn lipoprotein particles to the beta- and theta-Syn variants, and the PD-related familial alpha-Syn mutants. We show that all human Syns can form stable and homogeneous populations of HDL-like particles with distinct morphologies. Our results characterize the impact of the individual Syns on the formation capacity of these particles and indicate that Syn HDL-like particles are neither causing toxicity nor a toxicity-related loss of alpha-Syn in PD.

Apolipoprotein nanodiscs are a versatile tool in nanotechnology as membrane mimetics allowing, for example, the study of membrane proteins. It has recently been discovered that the Parkinson's disease associated protein alpha-synuclein (alpha-Syn) can also form discoid-like lipoprotein nanoparticles. The present review highlights the observation that a-Syn has the properties to define stable and homogeneous populations of nanoparticles with diameters of 7-10 nm and 19-28 nm by modifying lipid vesicles or encapsulating lipid bilayers in a nanodisctype fashion, respectively. In contrast to apolipoprotein nanodiscs, alpha-Syn nanoparticles can incorporate entirely negatively charged lipids emphasizing their potential use in nanotechnology as a negatively charged membrane mimetic.

S-Nitrosylation is well established as an important post-translational regulator in protein function and signaling. However, relatively little is known about its structural and dynamical consequences. We have investigated the effects of S-nitrosylation on the rhodanese domain of the Escherichia coli integral membrane protein YgaP by NMR, X-ray crystallography, and mass spectrometry. The results show that the active cysteine in the rhodanese domain of YgaP is subjected to two competing modifications: S-nitrosylation and S-sulfhydration, which are naturally occurring in vivo. It has been observed that in addition to inhibition of the sulfur transfer activity, S-nitrosylation of the active site residue Cys63 causes an increase in slow motion and a displacement of helix 5 due to a weakening of the interaction between the active site and the helix dipole. These findings provide an example of how nitrosative stress can exert action at the atomic level.

HDL particles transport cholesterol and contain apolipoprotein A-I as their major protein. The solution structure of discoidal HDL particles reconstituted with a shortened apoA-I is now solved via a combination of NMR and EPR analyses.

The S-sulfhydration of cysteine residues in proteins has emerged as a common modification that can modulate the activity of a protein. The ubiquity of the rhodanese domain and its occurrence in a wide variety of protein families indicates that it has diverse roles in physiology. Contrary to common expectations, previous structural studies of several rhodanese domains concluded that S-sulfhydration does not induce a structural change in the protein. The presented x-ray structure of a thiosulfatetreated crystal of the rhodanese domain of the E. coli integral membrane protein YgaP reveals two important findings: (1) The S-sulfhydrated catalytic cysteine C63 adopts an atypical conformation. (2) S-sulfhydration leads to a destabilization of the N-terminal part of the helix adjacent to the catalytic loop. These findings assert that S-sulfhydration is accompanied by a specific and complex dynamic process. Introduction Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), is an important gasotransmitter [1] [2] and plays an essential role in cell physiology by signaling through sulfhydration of cysteine residues in proteins [3]. Rhodaneses/sulfurtransferases form a group of enzymes widely distributed in prokaryotic and eukaryotic cells that via sulfhydration are able to catalyze the transfer of sulfur from thiosulfate (S2O3) to cyanide (CN-) that is important for detoxification of cells [4]. The catalysis is a two-step reaction in which the thiol group of the cysteine first reacts with the thiosulfate anion to form an enzyme-persulfide intermediate (CYS-SH), which then reacts with the cyanide ion to produce the much less toxic thiocyanate (SCN-) [4]. The most well-studied rhodanese domain is that of bovine liver rhodanese [5] [6] [7] [8]. The active cysteine is located in the cradle-shaped catalytic loop formed by the backbone atoms of the loop residues in such a way that the reactive sulfur, most likely negatively charged, is pointing to the center of the cradle. It has been stipulated that the S-sulfhydrated enzyme undergoes a significant conformational change. However, the crystal structures of the sulfur-free as well as S-sulfhydrated enzyme are very similar [9]. Even more puzzling results came from the study that investigated in detail the impact of S-sulfhydration on the structure and dynamics of the rhodanese domain of the E. coli integral membrane protein YgaP using solution NMR as the main technique [10]. The study revealed that the S-sulfhydration of YgaP is a transient process: A titration with 1–4 mM sodium thiosulfate to the solution containing 13C, 15N–labeled YgaP rhodanese domain revealed that rather than two distinct sets of cross-peaks corresponding to the S-bound and -unbound states, a single set of cross-peaks that shift upon titration is observed in two-dimensional [15N, 1H]-TROSY experiments indicating a fast exchange (i.e., in the micro– to millisecond range) between the S-bound and -unbound states [10]. These results have been independently confirmed in another study of YgaP [11]. Despite this peculiar finding, the crystal structure of the rhodanease domain of YgaP revealed that the active loop superimposes very well with the analogous bovine rhodanese loop. Furthermore, the structure also revealed that the protein overexpressed in E. coli is partially both S-sulfhydrated and S-nitrosylated and that the S-sulfhydration is likely present in two conformations [12]. It has been known that S-nitrosylation and S-sulfhydration are mutually inhibitory processes, which may play important roles in H2S/NO signaling [13]. It has also been suspected that the ubiquity and frequent presence of the rhodanese domains in multidomain proteins implies physiological functions other than cell detoxification [14]. Therefore, it is important to understand the effects of these posttranslational modifications at atomic resolution. In this paper, we further inS-sulfhydration of the catalytic cysteine in the rhodanese domain of YgaP is complex dynamic process DOI: 10.19185/matters.201602000004 Matters (ISSN: 2297-8240) | 2 vestigated the impact of S-sulfhydration on the rhodanese domain of the E. coli integral membrane protein YgaP and found that S-sulfhydration triggers a dynamical process by destabilizing the α4 helix in the domain. Objective Themain objective of this study is to characterize the structural changes, if any, that are induced in the YgaP rhodanese domain upon S-sulfhydration.