Session WOD. There are 6 abstracts in this session.

Session: Biosolids, time: 11:00-11:20

Reveal the Functional Structure of Complex Carbohydrates in Plants and Fungi Using DNP Solid-State NMR      

Xue Kang1; Alex Kirui1; Malitha Dickwella Widanage1; Frederic Mentink-Vigier3; Ping Wang2; Daniel Cosgrove4; Tuo Wang1
1Louisiana State University, Baton Rouge, LA; 2Louisiana State University Health Sciences Center, New Orleans, LA; 3NHMFL, CIMAR, Tallahassee, FL; 4Pennsylvania State University, University Park, PA
Two-dimensional 13C-13C/15N correlation SSNMR and DNP-assisted spectral editing methods are used to investigate the functional structure and macromolecular assembly of complex carbohydrates, proteins and lignin in native plant and fungal cell walls. The cell walls of pathogenic fungi contain a hydrophobic scaffold of chitin and α-1,3-glucan, which is surrounded by a hydrated matrix of diversely linked β-glucans and capped by a dynamic, outer layer rich in glycoproteins. The two hydrophobic domains of plant biomass, lignin and cellulose, are found to be bridged by the hemicellulose xylan in a conformation-dependent manner and via electrostatic interactions. These findings will promote the development of novel antifungal medications and better bioenergy crops.

Session: Biosolids, time: 11:20-11:40

Gating of a divalent cation channel by NMR at >100 kHz MAS   

Marta Bonaccorsi; Tobias Schubeis; Tanguy Le Marchand; Andrea Bertarello; Guido Pintacuda
Centre RMN à Très Hauts Champs-CNRS/UCBL/ENS Lyon, Villeurbanne, France
Here we demonstrate that MAS rates of 100 kHz and above, coupled to ultra-high magnetic fields,  permit the site-specific measurement of a variety of observables connected to local and global dynamics in proteins of diverse molecular sizes and aggregation states, from microcrystalline to membrane proteins.
These methods are first benchmarked on the model microcrystalline protein GB1 and then illustrated on the bacterial divalent cation channel CorA reconstituted in lipid bilayers, a pentamer of 5 x 42 kDa, comprised of two transmembrane helices and a large cytoplasmic domain hosting a metal binding side (usually Mg2+ or Co2+). In this system, the residue specific information obtained by MAS-NMR challenges the ion transport mechanism previously formulated on the basis of X-ray and cryo-EM structures.

Session: Biosolids, time: 11:40-12:05

A beta barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL

Tobias Schubeis1; Tanguy Le Marchand1; Wojciech Kopec2; Kumar Movellan3; Jan Stanek1; Tom Schwarzer4; Kathrin Castiglione4; Bert de Groot2; Guido Pintacuda1; Loren Andreas3
1CNRS - ENS Lyon, Lyon, France; 2Biomol. Dyn. Grp., MPI for Biophysical Chemistry, Göttingen, Germany; 3bioNMR dept., MPI for Biophysical Chemistry, Göttingen, Germany; 4Technische Universität München, Garching, Germany
The outer membrane protein AlkL is known to conduct hydrophobic molecules across the outer membrane of bacteria, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through MD simulations and by characteristic NMR relaxation parameters. A dynamic lateral exit site occurs in an unexpected location through restructuring of a barrel extension formed by the extracellular loops.

Session: Biosolids, time: 12:00-12:20

Elucidating membrane protein – cholesterol binding using distance measurements and DNP-enhanced solid-state NMR   

Matthew Elkins1; Ivan Sergeyev2; Mei Hong1
1MIT, Cambridge, MA; 2Bruker BioSpin, Billerica, MA
Cholesterol plays a major role in membrane protein function, but direct determination of cholesterol-bound structures of proteins in lipid bilayers has not been shown. We have developed several solid-state NMR techniques and biosynthetic isotope labeling for determining cholesterol-bound structures of membrane proteins. Our approaches include 13C-19F distance measurements to constrain the cholesterol tail position from the protein, biosynthetic 13C labeling of cholesterol and 2D 13C-13C correlation NMR with DNP sensitivity enhancement to identify intermolecular contacts, and 2H NMR to determine cholesterol orientation. Applied to the influenza M2 protein, which binds cholesterol to mediate virus budding, our data led to a structural model of the cholesterol-M2 complex, which gives unique insights into how cholesterol promotes the membrane scission function of M2.

Session: Biosolids, time: 12:20-12:40

Proton R1ρ relaxation dispersion as a reporter on µs protein motion

Petra Rovó1; Colin Smith2, 4; Diego Gauto3; Bert De Groot4; Paul Schanda3; Rasmus Linser1, 5
1Ludwig-Maximilians-University Munich, Munich, Germany; 2Wesleyan University, Middletown, CT; 3Institut de Biologie Structurale, Grenoble, France; 4Max-Planck Institute for physical chemistry, Göttingen, Germany; 5TU Dortmund, Dortmund, Germany
R1rho relaxation dispersion has been used successfully for characterization of microsecond-to-millisecond-timescale dynamics. 15N R1rho relaxation dispersion is most widely known as Bloch-McConnell-type RD, as used in solution NMR, characterizing isotropic shift changes. In the solid state, near-rotary-resonance RD (NERRD) offers a second regime for assessing amide-bond fluctuations via anisotropic interactions CSA and heteronuclear dipolar couplings.
Proton R1rho assessment has been avoided due to the difficulties to quantify the various interactions present. Nevertheless, we show that on a qualitative level, proton NERRD is indeed a sensitive reporter on microsecond-timescale motion. While it correlates reasonably well with 15N BMRD data, proton NERRD at the HORROR condition, which recouples proton-proton homonuclear interactions, is shown to be useful as a complement to 15N NERRD.

Session: Biosolids, time: 12:40-1:00

Designing Polarizing Agents for Sensitive and Selective DNP-enhanced NMR   

Ildefonso Marin-Montesinos1; Diego Gauto1; Frédéric Mentink-Vigier1, 2; David Goyard5; Thomas Halbriter3; Olivier Renaudet5; Anne Imberty4; Snorri Sigurdsson3; Daniel lee1; sabine hediger1; Gael De Paepe1
1Univ. Grenoble Alpes, CEA, CNRS, INAC-MEM, Grenoble, France; 2National High Magnetic Field Laboratory, Tallahassee, Florida; 3University of Iceland, Science Institute, Reykjavik, Iceland; 4Univ. Grenoble Alpes, CNRS, CERMAV, Grenoble, France; 5Univ. Grenoble Alpes, CNRS, DCM, Grenoble, France
I will discuss our recent work towards developing efficient polarizing agents (PAs) at high magnetic field and fast MAS. The approach relies on the development of MAS-DNP simulations that allow, once coupled with DFT and high-field EPR, to predict DNP efficiency of known PAs as well as to design new candidates, such as the AsymPol family. Furthering this approach, Sel-DNP (Selective Dynamic Nuclear Polarization) will be introduced, where we use ligand-functionalized PAs to selectively highlight and identify residues present in a protein binding site. Since this method produces clean and resolved difference spectra containing a limited number of residues, resonance assignment can be performed without any limitation with respect to the biomolecular system.