The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins

Backbone conformational flexibility of the lipid modified membrane anchor of the human N-Ras protein investigated by solid-state NMR and molecular dynamics simulation

AbstractThe lipid modified human N-Ras protein, implicated in human cancer development, is of particular interest due to its membrane anchor that determines the activity and subcellular location of the protein. Previous solid-state NMR investigations indicated that this membrane anchor is highly dynamic, which may be indicative of backbone conformational flexibility. This article aims to address if a dynamic exchange between three structural models exist that had been determined previously. We applied a combination of solid-state nuclear magnetic resonance (NMR) methods and replica exchange molecular dynamics (MD) simulations using a Ras peptide that represents the terminal seven amino acids of the human N-Ras protein. Analysis of correlations between the conformations of individual amino acids revealed that Cys 181 and Met 182 undergo collective conformational exchange. Two major structures constituting about 60% of all conformations could be identified. The two conformations found in the simulation are in rapid exchange, which gives rise to low backbone order parameters and nuclear spin relaxation as measured by experimental NMR methods. These parameters were also determined from two 300 ns conventional MD simulations, providing very good agreement with the experimental data

The Influence of Chemical Modification on Linker Rotational Dynamics in Metal–Organic Frameworks

The robust synthetic flexibility of metal–organic frameworks (MOFs) offers a promising class of tailorable materials, for which the ability to tune specific physicochemical properties is highly desired. This is achievable only through a thorough description of the consequences for chemical manipulations both in structure and dynamics. Magic angle spinning solid‐state NMR spectroscopy offers many modalities in this pursuit, particularly for dynamic studies. Herein, we employ a separated‐local‐field NMR approach to show how specific intraframework chemical modifications to MOF UiO‐66 heavily modulate the dynamic evolution of the organic ring moiety over several orders of magnitude.Intraframework ring rotations in metal–organic frameworks have been sensitively detected by dipolar dephasing over the rotor period in magic angle spinning solid‐state NMR experiments. Information on the dynamics within MOFs is important, because the rate of rotational motions of linkers affects sorption and separation properties of MOFs.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/1/anie201805004.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/2/anie201805004-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144616/3/anie201805004_am.pd

Selective observation of semi-rigid non-core residues in dynamically complex mutant huntingtin protein fibrils

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein's expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils' segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of 'intermediate motion selection' (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter's detection with specific dyes and positron emission tomography tracers

Insights into Crystalline and Material Solids from Ultrafast Magic Angle Spinning NMR Spectroscopy

While tremendous progress has been made in the development of tools used to probe the molecular world, a number of shortcoming persist in many of the commonly used techniques highlighting the importance for the development of new approaches for molecular inquiry. For example, X-ray diffraction, which is considered to be the gold standard for probing molecular structure, is limited in its ability to achieve atomic resolution for non-crystalline or heterogeneous systems including intrinsically disordered proteins, amorphous polymers or other multiphase systems. In addition, only limited dynamic information can be obtained by X-ray diffraction. Solution-state Nuclear Magnetic Resonance (NMR) is a complementary technique which can address a number of these issues including the ability to probe dynamics and detect disordered systems with high resolution. However, this requires good solubility of the sample and is limited by molecular size before spectral line-widths become too broad to be detected for slow-tumbling macromolecules. Magic Angle Spinning (MAS) NMR is not bound by the limitation of molecular size, opening up many new avenues for applications including the ability to detect signals from solid or semi-solid systems. In contrast to solution NMR, orientation dependent contributions to the spin state are present in solids which reflect structural characteristics at the atomic and molecular levels. While the increased complexity of solid-state systems provides an opportunity for rich insight, the spectral resolution is limited by the MAS frequency. Until recently, solid-state NMR spectra have rarely achieved the resolution obtained in solution. Simultaneously addressing sensitivity and resolution is a major goal in modern solid-state NMR spectroscopy. Recent innovations in MAS technology combined with refined approaches for pulse sequence design have made a substantial impact to this end. The topic of this dissertation focuses on the application of these approaches to crystalline and material systems. The main thrust of the work describes 1H-based techniques under fast MAS frequencies. Under these spinning speeds, 1H/1H dipolar couplings are suppressed thereby reducing spectral broadening and achieving chemical shift resolution to render “solution-like” proton NMR spectra. It is advantageous to detect protons as the large gyromagnetic ratio and near 100% natural-abundance dramatically reduce the experimental time or required sample quantity. New approaches utilizing 1H-detected fast MAS are presented for the interrogation of structural differences in crystalline polymorphs and hydrates with a focus on 1H chemical shift anisotropy tensors. Valuable insights are gleaned from experimentally measured NMR parameters reflective of the distinct structural features complementing X-ray data. In the second part, novel dynamic insights were found using 13C-detected slow MAS approaches in Metal-Organic Frameworks (MOFs). The quantification of 13C-1H heteronuclear dipolar couplings is used to probe dynamics in the microporous structure. This was completed for a series of Zr, terephthalate based MOFs (UiO66) with different chemical functionalization on the terephthalate ring. The dynamic evolution of the rings is shown to span several orders of magnitude depending on the nature of the functional group. The dramatic reduction in the required sample quantity and considerable enhancements in spectral resolution and sensitivity by ultrafast-MAS are bound to enable a plethora of investigations on numerous exciting classes of chemical and biological materials without any constraints on the molecular size and nature of the sample. Therefore, we believe that the methods and results reported in this thesis will be useful for a variety of other systems as well.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144002/1/jtda_1.pd

Folding Of Xylan Onto Cellulose Fibrils In Plant Cell Walls Revealed By Solid-state Nmr

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. C-13 solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two-and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.7BBSRC Grant via BBSRC Sustainable Bioenergy Cell Wall Sugars Programme [BB/G016240/1]CNPq [159341/2011-6, 206278/2014-4]Royal SocietyLeverhulme Trust grant for the Centre for Natural Material InnovationEPSRCBBSRCUniversity of WarwickBirmingham Science City Advanced Materials ProjectsAdvantage West Midlands (AWM)European Regional Development Fund (ERDF)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Xylan Structure and Dynamics in Native <i>Brachypodium</i> Grass Cell Walls Investigated by Solid-State NMR Spectroscopy.

The polysaccharide composition and dynamics of the intact stem and leaf cell walls of the model grass Brachypodium distachyon are investigated to understand how developmental stage affects the polysaccharide structure of grass cell walls. 13C enrichment of the entire plant allowed detailed analysis of the xylan structure, side-chain functionalization, dynamics, and interaction with cellulose using magic-angle-spinning solid-state NMR spectroscopy. Quantitative one-dimensional 13C NMR spectra and two-dimensional 13C-13C correlation spectra indicate that stem and leaf cell walls contain less pectic polysaccharides compared to previously studied seedling primary cell walls. Between the stem and the leaf, the secondary cell wall-rich stem contains more xylan and more cellulose compared to the leaf. Moreover, the xylan chains are about twofold more acetylated and about 60% more ferulated in the stem. These highly acetylated and ferulated xylan chains adopt a twofold conformation more prevalently and interact more extensively with cellulose. These results support the notion that acetylated xylan is found more in the twofold screw conformation, which preferentially binds cellulose. This in turn promotes cellulose-lignin interactions that are essential for the formation of the secondary cell wall

UV Pretreatment Impairs the Enzymatic Degradation of Polyethylene Terephthalate

The biocatalytic degradation of polyethylene terephthalate (PET) emerged recently as a promising alternative plastic recycling method. However, limited activity of previously known enzymes against post-consumer PET materials still prevents the application on an industrial scale. In this study, the influence of ultraviolet (UV) irradiation as a potential pretreatment method for the enzymatic degradation of PET was investigated. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and 1H solution nuclear magnetic resonance (NMR) analysis indicated a shortening of the polymer chains of UV-treated PET due to intra-chain scissions. The degradation of UV-treated PET films by a polyester hydrolase resulted in significantly lower weight losses compared to the untreated sample. We also examined site-specific and segmental chain dynamics over a time scale of sub-microseconds to seconds using centerband-only detection of exchange, rotating-frame spin-lattice relaxation (T1), and dipolar chemical shift correlation experiments which revealed an overall increase in the chain rigidity of the UV-treated sample. The observed dynamic changes are most likely associated with the increased crystallinity of the surface, where a decreased accessibility for the enzymecatalyzed hydrolysis was found. Moreover, our NMR study provided further knowledge on how polymer chain conformation and dynamics of PET can mechanistically influence the enzymatic degradation

The Influence of Chemical Modification on Linker Rotational Dynamics in Metal–Organic Frameworks

The robust synthetic flexibility of metal–organic frameworks (MOFs) offers a promising class of tailorable materials, for which the ability to tune specific physicochemical properties is highly desired. This is achievable only through a thorough description of the consequences for chemical manipulations both in structure and dynamics. Magic angle spinning solid‐state NMR spectroscopy offers many modalities in this pursuit, particularly for dynamic studies. Herein, we employ a separated‐local‐field NMR approach to show how specific intraframework chemical modifications to MOF UiO‐66 heavily modulate the dynamic evolution of the organic ring moiety over several orders of magnitude.Ringrotationen in MOFs wurden in Festkörper‐NMR‐Experimenten unter Probenrotation um den magischen Winkel durch dipolare Dephasierung über die Rotorperiode detektiert. Informationen zur Dynamik in Metall‐organischen Gerüsten sind wichtig, weil die Geschwindigkeit der Rotationsbewegung des Linkers die Sorptions‐ und Trenneigenschaften von MOFs beeinflusst.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144665/1/ange201805004_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144665/2/ange201805004-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144665/3/ange201805004.pd

Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR

Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. $^{13}$C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.This work was part supported by BBSRC Grant BB/G016240/1 via The BBSRC Sustainable Bioenergy Cell Wall Sugars Programme. ODB and ERdA are grateful to CNPq for financial support for this work via Grants # 159341/2011-6 and 206278/2014-4. ACP is grateful to the Royal Society for a Newton International Fellowship. PD is supported by the Leverhulme Trust grant for the Centre for Natural Material Innovation. The UK 850 MHz solid-state NMR Facility used in this research was funded by EPSRC and BBSRC, as well as the University of Warwick including via part funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF). (Contract reference PR140003 for work after 5 January 2015). DFT calculations of NMR parameters were performed at the Centre for Scientific Computing at the University of Warwick