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 ß-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.

Exploring Lipid and Membrane Protein Dynamics Using Lipid-Bilayer Nanodiscs and Solution-State NMR Spectroscopy.

The relationship of membrane protein function and the surrounding lipid bilayer goes far beyond simple hydrophobic interactions. At least from the 1980s, it is speculated that a certain fluid lipid state may be important not only for the lateral diffusion of membrane proteins (MPs) but also for modulating the catalytic activity of MPs (Lenaz. Bioscience Rep 7 (11):823-837, 1987). Indeed, acyl chain length, hydrophobic mismatch, and lipid headgroups are determinants for enzymatic and transport activities of MPs (Dumas et al. Biochemistry 39(16):4846-4854, 2000; Johannsson et al. Biochim Biophys Acta 641(2):416-421, 1981; Montecucco et al. FEBS Lett 144(1):145-148, 1982; Martens et al. Nat Struct Mol Biol 23(8):744-751, 2016). Moreover, it is speculated that changes in membrane lipid dynamics are important in the field of thermosensation (Vriens J, Nilius B, Voets T, Nat Rev Neurosci 15:573-589, 2014). Atomic insights into lipid-mediated modulation of membrane protein dynamics would therefore provide new insights with the potential to fundamentally extend our understanding on dynamic lipid-protein interdependencies.This chapter describes the expression and purification of nanodiscs assembled from membrane scaffold protein (MSP) as well as the expression and purification of the outer membrane protein X (OmpX). Subsequently, the incorporation of OmpX into MSP-derived nanodiscs is explained in detail. The chapter concludes with the setup of nuclear magnetic resonance (NMR) relaxation experiments and the extraction of relaxation rates for OmpX and the surrounding lipids.

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR.

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

A guide to quantifying membrane protein dynamics in lipids and other native-like environments by solution-state NMR spectroscopy.

Recent biochemical and technical developments permit residue-specific solution NMR measurements of membrane protein (MP) dynamics in lipidic and chaperone-bound environments. This is possible by combinations of improved sample preparations with suitable NMR relaxation experiments to correlate protein function to backbone dynamics on timescales from picoseconds to seconds, even for large MP-lipid assemblies above 100 kDa in molecular mass. Here, we introduce the basic concepts of different NMR relaxation experiments, individually sensitive to specific timescales. We discuss the general limitations of detergent environments and highlight the importance for native-like environments when studying MPs. We then review three practical studies of fast- and slow-timescale MP dynamics in lipid environments, as well as in a natively unfolded, chaperone-bound state. These examples illustrate the new avenues solution NMR spectroscopy is taking to investigate MP dynamics in native-like environments with atomic resolution.

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.

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

ß-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 ß-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 ß-strand 16. Here, we describe ∼70% complete sequence-specific NMR resonance assignments of the transmembrane region of the BamA ß-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.

Proton-Detected NMR Spectroscopy of Nanodisc-Embedded Membrane Proteins: MAS Solid-State vs Solution-State Methods.

The structural and dynamical characterization of membrane proteins in a lipid bilayer at physiological pH and temperature and free of crystal constraints is crucial for the elucidation of a structure/dynamics-activity relationship. Toward this aim, we explore here the properties of the outer-membrane protein OmpX embedded in lipid bilayer nanodiscs using proton-detected magic angle spinning (MAS) solid-state NMR at 60 and 110 kHz. [1H,15N]-correlation spectra overlay well with the corresponding solution-state NMR spectra. Line widths as well as line intensities in solid and solution both depend critically on the sample temperature and, in particular, on the crossing of the lipid phase transition temperature. MAS (110 kHz) experiments yield well-resolved NMR spectra also for fully protonated OmpX and both below and above the lipid phase transition temperature.

Lipid Internal Dynamics Probed in Nanodiscs.

Nanodiscs offer a very promising tool to incorporate membrane proteins into native-like lipid bilayers and an alternative to liposomes to maintain protein functions and protein-lipid interactions in a soluble nanoscale object. The activity of the incorporated membrane protein appears to be correlated to its dynamics in the lipid bilayer and by protein-lipid interactions. These two parameters depend on the lipid internal dynamics surrounded by the lipid-encircling discoidal scaffold protein that might differ from more unrestricted lipid bilayers observed in vesicles or cellular extracts. A solid-state NMR spectroscopy investigation of lipid internal dynamics and thermotropism in nanodiscs is reported. The gel-to-fluid phase transition is almost abolished for nanodiscs, which maintain lipid fluid properties for a large temperature range. The addition of cholesterol allows fine-tuning of the internal bilayer dynamics by increasing chain ordering. Increased site-specific order parameters along the acyl chain reflect a higher internal ordering in nanodiscs compared with liposomes at room temperature; this is induced by the scaffold protein, which restricts lipid diffusion in the nanodisc area.

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 ß-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 ß-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.

Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I.

High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity.

Functional recovery from central neurotrauma, such as spinal cord injury, is limited by myelin-associated inhibitory proteins. The most prominent example, Nogo-A, imposes an inhibitory cue for nerve fibre growth via two independent domains: Nogo-A-Δ20 (residues 544-725 of the rat Nogo-A sequence) and Nogo-66 (residues 1026-1091). Inhibitory signalling from these domains causes a collapse of the neuronal growth cone via individual receptor complexes, centred around sphingosine 1-phosphate receptor 2 (S1PR2) for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) for Nogo-66. Whereas the helical conformation of Nogo-66 has been studied extensively, only little structural information is available for the Nogo-A-Δ20 region. We used nuclear magnetic resonance (NMR) spectroscopy to assess potential residual structural propensities of the intrinsically disordered Nogo-A-Δ20. Using triple resonance experiments, we were able to assign 94% of the non-proline backbone residues. While secondary structure analysis and relaxation measurements highlighted the intrinsically disordered character of Nogo-A-Δ20, three stretches comprising residues 561EAIQESL567, 639EAMNVALKALGT650, and 693SNYSEIAK700 form transient α-helical structures. Interestingly, 561EAIQESL567 is situated directly adjacent to one of the most conserved regions of Nogo-A-Δ20 that contains a binding motif for ß1-integrin. Likewise, 639EAMNVALKALGT650 partially overlaps with the epitope recognized by 11C7, a Nogo-A-neutralizing antibody that promotes functional recovery from spinal cord injury. Diffusion measurements by pulse-field gradient NMR spectroscopy suggest concentration- and oxidation state-dependent dimerisation of Nogo-A-Δ20. Surprisingly, NMR and isothermal titration calorimetry (ITC) data could not validate previously shown binding of extracellular loops of S1PR2 to Nogo-A-Δ20.

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.

A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution.

For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60-70 % of the side-chain conformations of cyclophilin appear to be correlated.

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.

Predictive atomic resolution descriptions of intrinsically disordered hTau40 and α-synuclein in solution from NMR and small angle scattering.

The development of molecular descriptions of intrinsically disordered proteins (IDPs) is essential for elucidating conformational transitions that characterize common neurodegenerative disorders. We use nuclear magnetic resonance, small angle scattering, and molecular ensemble approaches to characterize the IDPs Tau and α-synuclein. Ensemble descriptions of IDPs are highly underdetermined due to the inherently large number of degrees of conformational freedom compared with available experimental measurements. Using extensive cross-validation we show that five different types of independent experimental parameters are predicted more accurately by selected ensembles than by statistical coil descriptions. The improvement increases in regions whose local sampling deviates from statistical coil, validating the derived conformational description. Using these approaches we identify enhanced polyproline II sampling in aggregation-nucleation sites, supporting suggestions that this region of conformational space is important for aggregation.

Phosphorylation of human Tau protein by microtubule affinity-regulating kinase 2.

Tau protein plays an important role in neuronal physiology and Alzheimer's neurodegeneration. Its abilities to aggregate abnormally, to bind to microtubules (MTs), and to promote MT assembly are all influenced by phosphorylation. Phosphorylation of serine residues in the KXGS motifs of Tau's repeat domain, crucial for MT interactions and aggregation, is facilitated most efficiently by microtubule-associated protein/microtubule affinity-regulating kinases (MARKs). Here we applied high-resolution nuclear magnetic resonance analysis to study the kinetics of phosphorylation of Tau by MARK2 and its impact on the structure and microtubule binding of Tau. We demonstrate that MARK2 binds to the N-terminal tail of Tau and selectively phosphorylates three major and five minor serine residues in the repeat domain and C-terminal tail. Structural changes induced by phosphorylation of Tau by MARK2 are highly localized in the proximity of the phosphorylation site and do not affect the global conformation, in contrast to phosphorylation in the proline-rich region. Furthermore, single-residue analysis of binding of Tau to MTs provides support for a model in which Tau's hot spots of MT interaction bind independently of each other and are differentially affected by phosphorylation.

Structural impact of proline-directed pseudophosphorylation at AT8, AT100, and PHF1 epitopes on 441-residue tau.

The intrinsically disordered protein tau becomes excessively phosphorylated and aggregates into neurofibrillary tangles in Alzheimer's disease. To obtain insight into the structural consequences of phosphorylation, we characterized a mutant protein of tau in which epitopes recognized by Alzheimer diagnostic antibodies were mimicked by mutation to glutamic acid [AT8 (S199E, S202E, T205E), AT100 (T212E and S214E), and PHF1 (S396E and S404E)]. A large number of distance restraints obtained from NMR paramagnetic relaxation enhancement in combination with ensemble conformer calculations demonstrate that pseudophosphorylation causes an opening of the transient folding of tau. Together with previous studies on the Parkinson-related protein α-synuclein, our data indicate that networks of transient long-range interactions are common properties of intrinsically disordered proteins and that their modulation is important for aggregation.

Automatic assignment of the intrinsically disordered protein Tau with 441-residues.

Intrinsically disordered proteins carry out many important functions in the cell. However, the lack of an ordered structure causes dramatic signal overlap and complicates the NMR-based characterization of their structure and dynamics. Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) and htau23 (352 residues), three prototypes of intrinsically disordered proteins, which bind to microtubules and play a key role in Alzheimer disease, can be obtained within 5 days by a combination of seven-dimensional NMR spectra with optimized methods for automatic assignment. Chemical shift differences between the three isoforms provide evidence for the global folding of Tau in solution.

Conformational changes specific for pseudophosphorylation at serine 262 selectively impair binding of tau to microtubules.

Aggregation of the microtubule-associated protein tau into neurofibrillary tangles is the pathological hallmark of a variety of dementias. For reasons not yet known, tau becomes excessively phosphorylated in Alzheimer's brains and as a result no longer binds properly to microtubules. Here we studied the impact of phosphorylation on the conformational and binding properties of the repeat region of tau (K18) that is necessary for microtubule assembly and forms the core of paired helical filaments. To mimic phosphorylation, we introduced four mutations of serine to glutamate residues at positions 262, 293, 324, and 356. NMR spectroscopy demonstrates that pseudophosphorylation at these sites modifies the structural properties in repeats 1 and 2, in particular for Gln265-Lys267. Gln265-Lys267 are in close proximity to Ser262, the phosphorylation site that most strongly attenuates binding to microtubules. In contrast, the pseudophosphorylation mimic of tau efficiently interacts with the polyanion heparin. Thus, phosphorylation of the repeat region of natively unfolded tau induces specific conformational changes that have a strong impact on its biological function and involvement in disease.