Structural Polymorphism of 441-Residue Tau at Single Residue Resolution

Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered β-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins

Die Enträtselung der Struktur von monomeren und fibrillisiertem 441-Reste tau mithilfe der NMR Spektroskopie

Das Mikrotubuli-stabilisierende, intrinisch ungeordnete Protein tau ist für den axonalen Transport in Neuronen verantwortlich. Das binden an und das lösen von den Mikrotubuli wird durch ein komplexes Gleichgewicht von Phosphorylierung und Dephosphorylierung reguliert. Zusätzlich konnte ein klarer Zusammenhang zwischen der Alzheimerschen Krankkheit (AK) und tau etabliert werden. Tau-vermittelte neurodegenerative Erkrankungen sind durch unlösliche tau-Aggregate charakterisiert, sogenannten „paired helical filaments“, welche sich zu „neurofibrillären Geflechten“ zusammenlagern. Es ist jedoch unklar, welche extra- oder intrazellulären Faktoren die Umwandlung von einem physiologisch relevanten Protein zu einem Pathogen induzieren. In der vorliegenden Arbeit werden verschiedene Aspekte des Tau Proteins mittels NMR Spektroskopie untersucht. Im ersten Teil dieser Arbeit wurde die tertiäre Struktur des Tau Proteins mithilfe von paramagnetischen Relaxationsexperimenten untersucht. Die dabei erhaltenen 2288 interatomaren Distanzen wurden für Ensemble-Strukturberechnungen benutzt, welche in ein Reste-spezifisches Modell resultierten. Der zweite Teil der Arbeit untersucht die derzeitige Leistungsfähigkeit von „disorder predictors“, Programme zur Vorhersage von Eigenschaften von ungeordneten Proteinen. Eine gute Vorhersage von intramolekularen Kontakten konnte durch IUPred erreicht werden, welche gut mit der experimentell ermittelten intramolekularen Kontaktkarte korrelierte. Protein-Protein-Bindungssstellen wurden mit ANCHOR vorhergesagt, welche sehr gut mit den Tau-Mikrotubuli Bindungsstellen übereinstimmten. Für die Sekundärstrukturvorhersage wurde ein Kombination von Programmen benutzt, welche letztendlich die korrekten Sekundärstrukturen für die höchsten experimentell ermittelten Sekundärstrukturwahrscheinlichkeiten prognostizierten. Das Auftreten von paired helical filaments ist immer mit deren Hyperphosphorylierung verbunden. Um unspezifische und partielle Phosphorylierung zu vermeiden wurde der hyperphosphorylierte Zustand simuliert, indem Serine und Threonine durch Glutaminsäure ersetzt wurden. Wir pseudophosphorylierten Serine und Threonine in AK-spezifischen Epitopen (AT8, AT100 und PHF1). Wir untersuchten dann, inwiefern sich die globale und lokale Struktur dieses pseudophosphorylierten Tau-Proteins veränderten. Wir konnten zeigen, dass Pseudophosphorylierung zu einem Öffnen der „paper-clip“ Struktur führte. Die Wichtigkeit von transienten langreichweitigen Wechselwirkungen wird dadurch unterstrichen, da genau diese Mutante eine höhere Aggregationsrate besitzt Die Abschwächung der transienten langreichweitigen intramolekularen Wechselwirkungen geschieht vor lokalen Strukturänderungen. Im vierten Teil dieser Arbeit untersuchten wir die fuzzy coat der paired helical filaments mit NMR. Aufgrund der Größe der Aggregate nutzten wir „high-resolution magic angle spinning“. Wir sahen eine hoch dynamische N-terminale (Reste 1M-T212) und C-terminale fuzzy coat (Reste 399V-L441), welche transient mit dem Fibrillenkern wechselwirkt. Der N-terminus zeigte eine verstärkte elektrostatische Wechselwirkung mit dem Fibrillenkern. Das erklärt die Bindung von konformations-spezifischen Antikörpern an das Tau-Protein mit zwei diskontinuierlichen Epitopen am N-terminus und am Fibrillenkern

α-Synuclein lipoprotein nanoparticles

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 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

α-Synuclein Lipoprotein Nanoparticles

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 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.ISSN:2191-9089ISSN:2191-909

Lipid- and Cholesterol-Mediated Time-Scale-Specific Modulation of the Outer Membrane Protein X Dynamics in Lipid Bilayers

Membrane protein function fundamentally depends on lipid-bilayer fluidity and the composition of the biological membrane. Although dynamic interdependencies between membrane proteins and the surrounding lipids are suspected, a detailed description is still missing. To uncover lipid-modulated membrane protein backbone dynamics, time-scale-specific NMR relaxation experiments with residue-resolution were recorded. The data revealed that lipid order, modified either biochemically or biophysically, changes the dynamics of the immersed membrane protein in a specific and time-scale-dependent manner. A temperature-dependent dynamics analysis furthermore suggests a direct coupling between lipid and protein dynamics in the picosecond-nanosecond, microsecond, and millisecond time scales, caused by the lipid's trans-gauche isomerization, the segmental and rotational motion of lipids, and the fluidity of the lipid phase, respectively. These observations provide evidence of a direct modulatory capability of the membrane to regulate protein function through lipid dynamics ranging from picoseconds to milliseconds

Enzyme Selectivity Fine-Tuned through Dynamic Control of a Loop

Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand-selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations

Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded β-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer

Micelles, Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per-residue resolution that while OmpX forms a stable β-barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico- to nanosecond and micro- to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β-strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane-mimetic-dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility

Sequence-Specific Solution NMR Assignments of the beta-Barrel Insertase BamA to Monitor Its Conformational Ensemble at the Atomic Level

β-barrel outer membrane proteins (Omps) are key functional components of the outer membranes of Gram-negative bacteria, mitochondria, and plastids. In bacteria, their biogenesis requires the β-barrel-assembly machinery (Bam) with the central insertase BamA, but the exact translocation and insertion mechanism remains elusive. The BamA insertase features a loosely closed gating region between the first and last β-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA β-barrel in detergent micelles. On the basis of the assignments, NMR spectra show that the BamA barrel populates a conformational ensemble in slow exchange equilibrium, both in detergent micelles and lipid bilayer nanodiscs. Individual conformers can be selected from the ensemble by the introduction of a C-terminal strand extension, single-point mutations, or specific disulfide cross-linkings, and these modifications at the barrel seam are found to be allosterically coupled to sites at the entire barrel circumference. The resonance assignment provides a platform for mechanistic studies of BamA at atomic resolution, as well as for investigating interactions with potential antibiotic drugs and partner proteins

Measuring membrane protein bond orientations in nanodiscs via residual dipolar couplings

Membrane proteins are involved in numerous vital biological processes. To understand membrane protein functionality, accurate structural information is required. Usually, structure determination and dynamics of membrane proteins are studied in micelles using either solution state NMR or X-ray crystallography. Even though invaluable information has been obtained by this approach, micelles are known to be far from ideal mimics of biological membranes often causing the loss or decrease of membrane protein activity. Recently, nanodiscs, which are composed of a lipid bilayer surrounded by apolipoproteins, have been introduced as a more physiological alternative than micelles for NMR investigations on membrane proteins. Here, we show that membrane protein bond orientations in nanodiscs can be obtained by measuring residual dipolar couplings (RDCs) with the outer membrane protein OmpX embedded in nanodiscs using Pf1 phage as an alignment medium. The presented collection of membrane protein RDCs in nanodiscs represents an important step toward more comprehensive structural and dynamical NMR-based investigations of membrane proteins in a natural bilayer environment