Session PB. There are 27 abstracts in this session.

Session: Biomolecules in the Solid-State, poster number: 040
A New Aβ(1-42) Fibril Type Studied by High Resolution Solid-State MAS NMR
Daniel Schölzel1, 2; Lothar Gremer1, 2; Carla Schenk1; Elke Reinartz2; Jörg Labahn1, 3; Raimond B. G. Ravelli4; Markus Tusche1; Carmen Lopez-Iglesias4; Wolfgang Hoyer1, 2; Dieter Willbold1, 2; Gunnar F. Schröder1, 5; Henrike Heise1, 2
1ICS6 im Forschungszentrum Jülich, Jülich, Germany; 2Institut für Physikalische Biologie, Düsseldorf, Germany; 3Centre for Structural Systems Biology, Hamburg, Germany; 4Maastricht Multimodal Molecular Imaging Institute, Maastricht, The Netherlands; 5Physics Department of Heinrich-Heine-University, Düsseldorf, Germany
We present MAS NMR results together with a cryo-EM 3D structure at 4 Å resolution obtained for one hitherto unobserved type of Aβ(1-42) fibrils(1). NMR spectra are indicative of the high degree of homogeneity, demonstrating that the structure is representative of the majority of fibrils. Site-specific resonance assignments were obtained for all 42 amino acid residues, indicating that, in contrast to previously described fibrillar Aβ(1-42) structures, the full peptide is part of the β-sheet rich fibril core, including the N-terminus. Local dynamic variations in the fibrillar structure were further probed with T1ρ and DNP measurements. (1) Gremer, L. et al. Science 2017, 358, 116-119.

Session: Biomolecules in the Solid-State, poster number: 041
The Influence of Pulse Transients on Pulse Sequences in Solid-State NMR 
Johannes Hellwagner; Johannes J. Wittmann; Nino Wili; Luis Fábregas Ibáñez; Beat Meier; Matthias Ernst
ETH Zurich, Zurich, Switzerland
Pulse transients and rf-field maladjustments are the main source of performance degradation and limited reproducibility in solid-state NMR experiments. We focus on commonly used pulse sequences under MAS including homo- and heteronuclear recoupling sequences as well as homo- and heteronuclear decoupling.
We show experimentally that the main source of loss of recoupling efficiency in symmetry-based recoupling sequences (C- and R-sequences) and dipolar recoupling sequences using isolated π-pulse trains is the detuning of the resonance condition. This detuning can be negated by pulse-transient compensation which results in the removal of effective fields caused by non-unity rf interaction-frame propagators.
We also show that non-resonant pulse sequences under MAS like homo- and heteronuclear decoupling sequences are also be affected by pulse transients.

Session: Biomolecules in the Solid-State, poster number: 042
Dimerisation of Glutathione - Solution and Solid State Structures Studied by NMR
Dillip Kumar Senapati1; Y.Jayasubba Reddy1, 2; S. Raghothama1; K.V. Ramanathan1
1Indian Institute Of Science, Bangalore, India; 2EPFL, Switzerland., Lausanne, Switzerland
Glutathione (GSH) helps in removing reactive oxygen in cells by undergoing REDOX reaction when two GSH molecules form a dimer (GSSG) linked by a disulfide bridge. The dimer can have either parallel or antiparallel orientation. Earlier solution NMR and crystal structure studies indicate contrary relative orientations of the two molecules of GSSG. To address this apparent ambiguity, we have investigated the molecules in both solution and the solid state using NMR. In solution important NOE connectivities indicate an antiparallel dimer. In the solid state, proton double quantum – carbon single quantum correlation experiment clearly indicated a parallel dimer confirming that the crystal packing interactions in the solid play a role in influencing molecular arrangements.

Session: Biomolecules in the Solid-State, poster number: 043
Magic bullets to fight antimicrobial resistance: Understanding how antibiotics break down the bacterial cell envelope
João Medeiros-Silva1; Shehrazade Jekhmane1; Barend Elenbaas1; Kamaleddin Tehrani2; Marc Baldus1; Nathaniel Martin2; Eefjan Breukink3; Markus Weingarth1
1NMR Spectroscopy; Utrecht University, Utrecht, Netherlands; 2Chem. Biology & Drug Discovery; Utrecht University, Utrecht, Netherlands; 3Membrane Biochemistry; Utrecht University, Utrecht, Netherlands
The rise of antimicrobial resistance urgently calls for novel antibiotics. Ideal templates could be peptide-antibiotics that target the membrane-anchored bacterial cell wall precursor lipid II at irreplaceable pyrophosphate groups. Indeed, these drugs kill the most refractory bacteria at nanomolar concentrations without detectable resistance. However, structural information on these antibotics is scare and totally absent in native-like media.<br />Using <b><i>high-field (800-950 MHz) DNP-enhanced and <sup>1</sup>H-detected solid-state NMR</i>,<i> we present extensive high-resolution studies on lipid II binding antibiotics plectasin<sup>1</sup> and nisin<sup>2</sup> in liposomes and directly in cell membranes of pathogenic bacteria</i></b>. Our data provide exciting novel structural insights that upset previous models derived in non-physiological media, and open new avenues for antibiotic design.<br /><br /><i>1&amp;2) Schneider, Science 2010; Breukink, Science 2006</i><br />

Session: Biomolecules in the Solid-State, poster number: 044
Selective 3D pulse sequences with proton detections for side-chain and back-bone assignment experiments in deuterated proteins at fast MAS
Alons Lends1; Francesco Ravotti1; Anja Böckmann2; Matthias Ernst1; Beat Meier1
1ETH, Zurich, Switzerland; 2IBCP, Lyon, France
Proton detection in combination with fast MAS opens up new opportunities for assignment experiments. Typical pulse sequences for sequential assignments using proton-detected spectroscopy under fast MAS use cross polarization (CP) and/or INEPT type of transfers. In 3D experiments the final signal intensity is only between 2.0% and 24% of the theoretical one, for proton-detection experiments. Here, we will present a new optimized selective 3D pulse sequence which consists of 1H-15N CP transfers and a REDOR/TEDOR type polarization transfer to 13C. We show that the optimized selective experiments are up to 60% more efficient for HNCA-type experiment compared to previously described pulse sequences at 55.5 kHz MAS. The selective pulse sequence can also be extended for aliphatic and aromatic side-chain assignments. 

Session: Biomolecules in the Solid-State, poster number: 045
Membrane-bound Structure and Oligomeric Assembly of Viral Fusion Proteins by Solid-State NMR
Myungwoon Lee; Byungsu Kwon; Hongwei Yao; Chandan Singh; Mei Hong
Department of Chemistry, MIT, Cambridge, MA
The conformation and oligomeric structure of the transmembrane domain (TMD) of two fusion proteins, PIV5 F and HIV gp41, are investigated using SSNMR. Chemical shifts and 19F spin diffusion data indicate that the PIV5 fusion protein’s TMD has a central α-helical core for the trimeric structure while the two termini adopt membrane-dependent secondary structures. For HIV gp41, 2D 1H-13C spin diffusion and water-transferred 2D correlation spectra indicate that TMD spans the membrane while the antibody-targeted MPER domain resides on the membrane surface. 19F CODEX and 13C-19F REDOR data revealed the trimeric structure and relative orientation of the MPER and TMD, providing the first SSNMR structural model of this important region of the HIV fusion protein in lipid bilayers.

Session: Biomolecules in the Solid-State, poster number: 046
Mapping the Regulation of Sarcoplasmic Reticulum Ca2+-ATPase by Sarcolipin Using Solid-State NMR
Songlin Wang; Gopinath Tata; Erik Larson; Kaustubh Mote; Gianluigi Veglia
University of Minnesota, Minneapolis, Minnesota
Sarcoplasmic Reticulum Calcium-ATPase (SERCA) plays an important role in muscle relaxation by maintaining Ca2+ homeostatic balance in the sarcoplasmic reticulum lumen. Sarcolipin (SLN), which is expressed in cardiac and skeletal muscles, modulates SERCA’s function by uncoupling ATP-hydrolysis from Ca2+-transport and its phosphorylation results in relieved inhibition, making it an invaluable tool in studying SERCA structure-function relationship. Using Oriented-Sample ssNMR, we captured the residues of SLN interacting with SERCA and mapped the topologic change of SLN upon SERCA binding along the Ca2+-transport cycle. The results suggest that the topology of SLN is very sensitive to the different states of SERCA. These findings will lead to a deeper understanding of the increasingly complex system involved in modulating Ca2+ cycling in the cardiomyocyte.

Session: Biomolecules in the Solid-State, poster number: 047
A novel labelling method to introduce alpha-protons in deuterated proteins 
Kumar Tekwani Movellan
Max Planck Institute - BPC, Goettingen, Germany
Even with fast spinning to 60kHz, high levels of deuteration are required for proton detected NMR, which is often implemented by producing perdeuterated protein, and then reintroducing amide protons by exchange with H2O. Here we introduce a new strategy for 1H labelling at the alpha position, which we expect will be useful for the study of structure and dynamics in both the solid-state and in solutions. The approach results in near complete amide and alpha protonation, with a high level of deuteration at other positions, and has the advantage that E. Coli growth occurs in H2O. This circumvents problems with amide proton exchange and adaptation to deuterated media that occurs when using D2O. 

Session: Biomolecules in the Solid-State, poster number: 048
Insight into Small Molecule Binding to the Neonatal Fc Receptor by X-ray Crystallography and 100 kHz Magic-Angle-Spinning NMR
Daniel Stöppler1; Alex Macpherson2; Susanne Smith-Penzel3; Lorna Waters4; David Fox III5; Mark Carr4; Richard Taylor2; Beat Meier3; Hartmut Oschkinat1; Alastair Lawson2
1Leibniz Institute of Molecular Pharmacology (FMP), Berlin, Germany; 2UCB Celltech, Slough, United Kingdom; 3ETH, Zürich, Switzerland; 4University of Leicester, Leicester, United Kingdom; 5Beryllium Discovery, Bedford, MA, USA
The neonatal Fc receptor has been suggested as a drug target for the treatment of autoimmune diseases. We herein present the discovery of a small molecule that binds into a conserved cavity of its extra-cellular domain (FcRnECD). X-ray crystallography and 100 kHz Magic-Angle-Spinning (MAS) NMR was applied to explore possibilities for refining the compound towards allosteric interference. Proton-detected MAS NMR experiments on soluble, fully protonated FcRnECD yielded ligand-induced chemical shift perturbations. Generating a chemical shift-informed overlay of X-ray structures with and without ligand reveals a potential for large movements upon binding of an optimized compound. Our study establishes a method to structurally characterize small molecule binding to non-deuterated, large proteins that may prove highly valuable in structure-based drug discovery campaigns.

Session: Biomolecules in the Solid-State, poster number: 049
Integrated 19F, 2H and 13C NMR approach for determining the cholesterol binding site of a membrane protein  
Matthew Elkins; Jonathan Williams; Martin Gelenter; Byungsu Kwon; Mei Hong
MIT Department of Chemistry, Cambridge, MA
Cholesterol is important for membrane protein function, but cholesterol-bound structures of membrane proteins have been difficult to determine by crystallography and microscopy. We have developed a SSNMR approach to probe cholesterol-binding sites of membrane proteins in bilayers. The approach combines 13C-19F distance experiments, multi-spin analysis of motionally averaged dipolar couplings, and 2H spectra for orientation determination. Applied to the influenza M2 protein, the measured  distances and orientation indicate that cholesterol binds M2 at a sub-stoichiometric ratio, flanking methyl-rich TM residues near an amphipathic helix. These results give insights into how cholesterol clusters M2 to the neck where it mediates curvature formation and membrane scission. This approach is applicable to membrane proteins for understanding roles of cholesterol in structure and function.

Session: Biomolecules in the Solid-State, poster number: 050

MAS NMR Studies of Peripheral Membrane Protein Cytochrome c and Its Cardiolipin Interaction in Triggering Mitochondrial Apoptosis

Mingyue Li1, 2; Abhishek Mandal1; Maria DeLucia1; Vladimir A. Tyurin3; Valerian E. Kagan3; Jinwoo Ahn1; Patrick C.A. Van der Wel1
1University of Pittsburgh School of Medicine, Pittsburgh, PA; 2The Center for Protein Conformational Diseases, Pittsburgh, PA; 3Center for Free Radical and Antioxidant Health, Pittsburgh, PA

The interaction of peripheral membrane protein cytochrome c (cytc) and cardiolipin (CL), a mitochondrial specific lipid, has been identified as critical to initialize mitochondrial apoptosis. Understanding the structural specificity of the cytc/CL complex is crucial to drug development targeting early apoptosis in cancer and neurodegenerative diseases. In our studies, the structure and dynamics of peroxidase-active cytc bound to CL are investigated by both through-space and through-bond correlation MAS NMR experiments. Residues showing structural perturbations upon binding to CL are identified. Elaborate dynamics of cytc in complex with CL are observed which indicates structural plasticity. Remarkably, a specific region of cytc is found to exhibit both significant structural and dynamics perturbations and is thought to be crucial for its proapoptotic activity.

Session: Biomolecules in the Solid-State, poster number: 051
Structural characterization  of a 29 kDa enzyme-inhibitor complex at 111 kHz
Suresh Kumar Vasa; Himanshu Singh; Rasmus Linser
Ludwig-Maximilians-Universität München, München, Germany
Proton-detection under Magic-Angle Spinning (MAS) has been revolutionizing solid-state NMR studies of micro-crystalline samples, amyloids and membrane proteins by efficient detection with improved resolution using minimal amounts of samples and ultra fast spinning (> 100 kHz). However, due to limitations in sensitivity and resolution, the size of the individual monomeric units has rarely exceeded 20 kDa. Carbonic anhydrases (CA) are fastest metalloenzymes that catalyzes the inter-conversion between dissolved CO2 and bicarbonate ion. Here, we report atomic-resolution structure of human carbonic anhydrase isozyme (29 kDa) complex with acetazolamide at ultra-fast MAS using proton detection. Our results demonstrate that structural studies of proteins in the 30 kDa range, in particular enzyme-ligand complexes, are well doable with less than a mg of material.

Session: Biomolecules in the Solid-State, poster number: 052

MAS NMR Study of Kinesin-1 Motor Domain in Complex with Polymeric Microtubules

Chunting Zhang1; Changmiao Guo1; Mingyue Li2; John Williams3; Tatyana Polenova1
1University of Delaware, Newark, DE; 2University of Delaware, Pittsburgh, PA; 3Beckman Research Institute of City of Hope, Duarte, CA

In this work, we present an MAS NMR study of KIF5B motor domain in complex with polymeric microtubules. We applied multidimensional homo- and heteronuclear experiments in U-13C,15N-kinesin/MT complex using non-uniform sampling4 (NUS) for time-saving and enhanced sensitivity. Chemical shift predictions5 were performed by ShiftX2 using the X-ray structure of KIF5B bound to α/β tubulin dimer. Based on the 2D/3D spectra, together with SHIFTX2 prediction, characteristic spin systems have been identified and segments of residue specific assignments have been completed. In addition, we applied fast MAS along with 1H detection in U-2H,13C,15N-kinesin/MT complex to reduce spectral complexity and attain resolution enhancement.

Session: Biomolecules in the Solid-State, poster number: 053

Dynamic Nuclear Polarization NMR Below 6 Kelvin within Human Cells Using Fluorescent Trimodal Polarizing Agents

Chukun Gao
Washington University in St. Louis, St. Louis, Missouri

Dynamic nuclear polarization (DNP) transfers electronic spin polarization to nuclei and boosts NMR sensitivity by orders of magnitude. Our preliminary results characterize sensitivity enhancement of 13C enriched HEK293F cells which overexpress PKC using newly designed and synthesized fluorescent trimodal polarizing agents, TotaFAM. It’s composed of three moieties: fluorescein—FAM, DNP polarizing agent—TOTAPol, and cell penetrating peptide—TAT (47-57). We proved that TotaFAM was uptaken by HEK293F cells and achieved sensitivity enhancement over 60. This, to our best knowledge, is the best enhancement ever reported on human cells. As DNP technology continues to improve, it will give us a better understanding of PKC activation in intact human cells for drug development.

Session: Biomolecules in the Solid-State, poster number: 054
The functional interaction between the KirBac1.1 K+ channel and the lipid bilayer as seen by SSNMR 
Collin Borcik1; Marella Canny1; Derek Versteeg1; Hoa Quyhn Do1; Luis Cuello2; Benjamin Wylie1
1Texas Tech University, Lubbock, Texas; 2Texas Tech University Health Sciences Center, Lubbock, TX
The mutual interaction between membrane proteins and lipids in biological membranes govern essential biological functions. Inward-rectifier K+ (Kir) channels are ion channels that regulate the resting membrane potential. Kir channels are activated by anionic lipids and their activity is attenuated by cholesterol and other polycyclic lipids. However, structural details are limited. Solid-state NMR (SSNMR) spectroscopy, partnered with functional assays in identical lipid environments, reveals how bilayer composition modulates the activity and conformation of the prokaryotic Kir channel KirBac1.1. Using 13C-labeled biological lipids, it is further discovered this channel shifts the gel-liquid crystalline phase transition of the bilayer and alters the rate of lateral diffusion and internal dynamics of the surrounding lipids.

Session: Biomolecules in the Solid-State, poster number: 055
Polarization optimized experiments (POE) in biomolecular solid-state NMR using multiple receivers at ultrafast magic angle spinning
Gopinath Tata1; Songlin Wang1; Sarah Nelson1; Shane Pawsey2; Jochem Struppe2; Gianluigi Veglia1
1University of Minnesota, Minneapolis, MN; 2Bruker BioSpin Corp, Billerica, MA 01821, USA
Solid-state NMR spectroscopy of proteins is a notoriously low-throughput technique. To speed up data acquisition, we developed a family of experiments called Polarization Optimized Experiments (POE), in which we utilized the orphan spin operators that are discarded in classical multidimensional NMR experiments, recovering them to allow simultaneous acquisition of multiple 2D and 3D experiments. Here we investigate the application of POE experiments at ultrafast MAS rates using 1H and 13C detected multi-receiver experiments. Application of these pulse sequences to crystalline and membrane proteins at various experimental conditions will be demonstrated.

Session: Biomolecules in the Solid-State, poster number: 056
Structure-Dynamic-Function Relationship Studies of Metallated Host Defense Peptides that Bind Lipid Bilayers and Activate Chemotaxis through Formyl Peptide Receptors
Yawei Xiong1; Qiaoyue Kuang1; Steven Paredes1; Dana Moore1; Romtin Nadjafi1; Ji Ming Wang2; Riqiang Fu3; Alexander Greenwood1; Myriam Cotten1
1College of William and Mary, Williamsburg, Virginia ; 2National Cancer Institute, Frederick, MD; 3National High Magnetic Field Laboratory, Tallahassee, FL
This research features host defense peptides from the piscidin family that have antimicrobial and immunomodulatory properties. We previously demonstrated that the isoforms p1 and p3 bind Cu2+ through a conserved amino-terminal-copper-and-nickel-binding motif. Redox cycling of Cu2+ leads to the formation of radicals that enhance bacterial death by oxidizing lipids and nicking DNA. Here, we used solid-state NMR to characterize the structure and dynamics of several piscidin peptides bound to metal ions and model membranes, some of which contained oxidized lipids. To investigate the immunomodulatory effects of the piscidins, we tested their chemotactic properties. Structural studies of the metallated peptides relied on 15N-1H HETCOR experiments on oriented samples.

Session: Biomolecules in the Solid-State, poster number: 057
Spot the difference - chemotyping of Gram-negative bacteria by HR-MAS NMR 
Sabina Koj; Karolina Ucieklak; Tomasz Niedziela
Polish Academy of Sciences, Wroclaw, Poland
The standard NMR “in solution” approach is not directly applicable for bacterial samples. HR-MAS NMR spectra of intact Gram-negative bacterial cells are complex, but the profiles include signals corresponding to the major membrane biomolecule - lipopolysaccharide (LPS). The HR-MAS NMR screening of Plesiomonas shigelloides and Klebsiella pneumoniae LPSs generated the unique profiles of O-antigens. The analysis revealed also similarities between profiles of two distinct bacterial strains. The in-depth studies demonstrated that P.shigelloides 78/89 shares the O-antigenic structural element with ᴅ-galactan I of K.pneumoniae Kp20 LPS. The spectral features observed in HR-MAS NMR profiles indicated the structure reporter groups of this unknown O-antigen. The HR-MAS technique has a great potential for chemotyping and O-antigen determination among different species of Gram-negative bacteria.

Session: Biomolecules in the Solid-State, poster number: 058
Cross Polarization at 111kHz MAS in a 1 GHz Magnet
Joseph Sachleben
Univ of Chicago, Chicago, IL

            Cross Polarization is critical for acquiring spectra of low γ nuclei in the solid state. Ultra-fast Magic Angle Spinning has been shown not to significantly interfere with CP, and allow the use of Double Quantum Cross Polarization as well as the more typical Zero Quantum CP  available at lower spinning frequencies.In this poster, we examine the efficiency of CP at even greater spinning frequencies and stronger magnetic field strengths. We map the conditions for 1H 13C, 1H 15N, and 13C 15N CP, and examine the effects of rf inhomogeneity and effective fields. We show that CPMAS is still effective at these high field strength and ultrafast MAS frequencies, but the effects of rf-inhomogeneity are significant.

Session: Biomolecules in the Solid-State, poster number: 059
Conformation of the N-terminus of CrgA with Gd3+ chelated lipids for observing Paramagnetic Relaxation Enhancements by solid state NMR
Yiseul Shin1; Riqiang Fu2; Huajun Qin1; Timothy A. Cross3
1Florida State University/NHMFL, Tallahassee, Florida; 2National High Magnetic Field Laboratory, Tallahassee, FL; 3Natl High Magnetic Field Lab, Tallahassee, FL
Solid state NMR is one of the most powerful techniques to study membrane proteins in lipid bilayers. Two TOBSY pulse sequences are utilized to characterize the dynamic N-terminus in the cytoplasmic domain. To preserve the native interaction between the N-terminus and its lipid environment, full length CrgA in a liquid crystalline preparation of bilayers is prepared. Additionally, the bilayer is doped with lipids chelating Gd3+ to the head group. By comparing two sets of experiments, it is observed that the positively charged residues are more susceptible to the PRE effect due to charge-charge interaction with the lipid head group. It is concluded that 9 residues form a tether to the membrane surface by the electrostatic interactions.

Session: Biomolecules in the Solid-State, poster number: 060
Gramicidin A Ion Binding and Conductance: New Insights from 17O solid state NMR at 35T SCH Magnet
Joana Paulino1; Ivan Hung1; Zhehong Gan1; Eduard Chekmenev2; Timothy A. Cross1
1NHMFL, Tallahassee, FL; 2Vanderbilt University Institute of Imaging Science, Nashville, TN
Gramicidin A (gA) forms a dimeric ion channel with a pore lined by the backbone carbonyl oxygens. We have performed 17O NMR of gA using the SCH Magnet at 35.2T (203 MHz 17O field) and report here dramatic gain in spectral sensitivity from the high field and better resolution. Increase in resolution reveals two peaks of gA 17O_Leu10 that have never been observed before. The 17O spectra of other gA labeled sites, L12 and L14, also showed gain in sensitivity, but the peak doubling is not present. 17O NMR is very sensitive to the environment. The carbonyl oxygens at different positions in the channel pore could be experiencing different hydrogen bonding with the water wire that permeates the channel.

Session: Biomolecules in the Solid-State, poster number: 061
Determining the Structure and Dynamics of Protein Kinase C δ with Time Domain Dynamic Nuclear Polarization and Molecular Dynamics Simulations
Patrick Judge; Erika Sesti; Edward Saliba; Nicholas Alaniva; Ned Fischer; Lauren Price; Faith Scott; Brice Albert; Alexander Barnes
Washington University, St. Louis, MO
Phorbol esters and bryostatin analogs are modulators of protein kinase C δ (PKC-δ). Despite their similar binding affinities to the same location, they exhibit vastly different cellular reactions. Determining the structure and dynamics of these protein-modulator complexes is necessary for understanding how they initiate these cellular reactions. MD simulations of the C1B domain of PKC-δ bound to phorbol-13-acetate in homogeneous and heterogeneous environments show that membrane composition significantly affect the topology of the complex. Using our custom-built, frequency-agile gyrotron, we can use solid-state NMR employing dynamic nuclear polarization (DNP) to add constraints to these simulations. Heteronuclear correlation and REDOR experiments coupled with DNP will be used to interrogate the structure of these modulators bound to selectively- and uniformly-labelled C1B domain.

Session: Biomolecules in the Solid-State, poster number: 062
Mechanistic Studies of Histidine-rich Host Defense Peptides

Qiaoyue Kuang; Kia Taylor; Kameron Sullivan; Myriam Cotten
College of William and Mary, Williamsburg, Virginia
Host defense peptides (HDPs) are important immune molecules involved in not only directly bacterial killing but also immunomodulatory effects on host cells. Remarkably, fish HDPs called piscidins act at acidic pH in the phagosome of mast cells and granulocytes, as well as extracellularly at basic pH. Since piscidins are active under changing conditions, we speculated that either individually or collectively, they exhibit pH resiliency. To test our hypothesis, we have focused on four membrane-active piscidins: piscidin 1 (FFHHIFRGIVHVGKTIHRLVTG) and piscidin 3 (FIHHIFRGIVHAGRSIGRFLTG) from hybrid striped seabass, and TP3 (FIHHIIGGLFSVGKHIHSLIHGH) and TP4 (FIHHIIGGLFSAGKAIHRLIRRRRR) from tilapia. We investigated the structures and membrane disruptive effects of these HDPs through dye leakage assays and CD, solution and solid-state NMR experiments under varying pH conditions.

Session: Biomolecules in the Solid-State, poster number: 063
1H-Detected REDOR with Fast Magic-Angle Spinning of a Deuterated Protein
Manali Ghosh1; Chad Rienstra2
1University of Illinois at Urbana-Champaign, Urbana, IL; 2University of Illinois, Urbana, N/A
In this study, we demonstrate a powerful approach to measure distance restraints in deuterated proteins by combining 1H detection at fast magic-angle spinning (MAS) (>30 kHz) and frequency-selective rotational echo double resonance (REDOR) technique in a single pulse sequence. Involving 1H as one of the spins for measuring REDOR effects increases the distance measurement range and sensitivity of the NMR spectrum. Deuteration strategies and fast MAS improve the 1H resolution of the spectrum. We performed 1H-detected 13C-dephased frequency selective REDOR at various spectral regions in uniformly 13C,15N,2H-labeled alanine and uniformly 13C,15N,2H labeled GB1 protein, back-exchanged with 30% H2O, to achieve resonance assignments, restraints for secondary and tertiary structures and preferred orientations of aromatic rings in the protein core.

Session: Biomolecules in the Solid-State, poster number: 064
Solid State NMR Characterization of a Disease Mutation in hnRNPA2 Protein Fibrils

Dylan Murray1; Xiaoming Zhou2; Masato Kato2; Steven McKnight2; Robert Tycko1
1Laboratory of Chemical Physics, NIH, Bethesda, MD; 2Biochemistry Department, UTSWMC, Dallas, TX
An aspartic acid to valine mutation in the heterogeneous ribonucleoprotein A2 is responsible for degenerative neuromuscular disease with pathological aggregation like that seen in patients with amyotrophic lateral sclerosis. It has been proposed that the mutation eliminates destabilizing electrostatic repulsion between protein monomers in fibril structures. Solid state NMR measurements characterize the structure of the fibrils and show that the aspartic acid is immobilized in the core of the fibrils, is charged at physiological pH, and that the mutant fibrils are more stable than the wild-type. The results show that replacement of electrostatic repulsion with favorable hydrophobic interactions in parallel, in-register, cross-β protein fibrils is responsible for the increased protein aggregation.

Session: Biomolecules in the Solid-State, poster number: 065
Proton-detected Ultrafast MAS Solid-State NMR Spectroscopy of Aβ42 Amyloid Fibrils
Robert Silvers1; Salima Bahri1; Brian Michael1; Daniela Lalli2; Sara Linse3; Guido Pintacuda2; Robert Griffin1
1MIT, Cambridge, MA; 2CNRS - ENS Lyon, Lyon, N/A; 3Lund University, Lund, Sweden
Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein, which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer’s disease (AD). We recently published an atomic resolution structure of the M01−42 amyloid fibril (PDB: 5KK3) and the availability of ultrafast MAS solid-state NMR spectroscopy (>100kHz) allows for the detection of proton chemical shifts in fully protonated M01−42 amyloid fibril samples. Here, we present the progress on expanding our heteronuclear dataset of the M01−42 amyloid fibril. Assignment strategies include state-of-the-art proton-detected 3D experiments such as the hCONH, hCANH, and hcoCAcoNH, among others. Furthermore, strategies to extract long-range proton-proton distances are explored.

Session: Biomolecules in the Solid-State, poster number: 066
Advances in oriented-sample solid-state NMR result from implementation of perdeuteration and 1H detection in stationary aligned samples of proteins.
Zheng Long1; Chin H. Wu2; Sang Ho Park1; Stanley Opella2
1UC San Diego, La Jolla, California; 2University of California, San Diego, La Jolla, CA
Advances in oriented-sample solid-state NMR result from implementation of perdeuteration and 1H detection in stationary aligned samples of proteins.