Session TOF. There are 6 abstracts in this session.

Session: Biomolecular 1, time: 4:00-4:20

Sortase Allows for Efficient Segmental Labeling of Large Intrinsically Disordered Regions.   

Kristina Boyko
Western Washington University, Bellingham, WA
NMR spectroscopy is a tool of choice for studying function and dynamics of proteins, especially those containing intrinsically disordered regions (IDRs). However, NMR spectra of large uniformly labeled IDRs can be obscured by severe spectral overlap. Segmental isotopic labeling allows simplification of the spectra by presenting only NMR signatures of smaller target portions within a large, unlabeled IDR context. Existing segmental labeling techniques have various limitations. To augment the repertoire of existing methods, we developed an application that utilizes sortase ligation enzymes for segmental labeling of IDRs. We employed, for the first time, and optimized this approach for segmental labeling of a plant villin, which contains a large IDR. We will also present solutions to the problems encountered.

Session: Biomolecular 1, time: 4:20-4:40

Studying multi-site kinetics of Cadherin-11 dimerization by pressure variation and by chemical exchange detection

Hans Koss; Arthur G Palmer
Columbia University, New York, NY
We are investigating the multi-site dimerization kinetics of the cell adhesion protein Cadherin-11 (type II). In particular, we are interested in the role of an intermediate strand-exposed conformation. Cadherin-11 wildtype is mostly dimerized at concentrations relevant for NMR; it is therefore challenging to assess whether a strand-exposed conformation is present in the monomer state. We gained experimental access to various conformational states using high pressure and temperature titrations. Mutants which disrupt strand-swapping, relaxation experiments and a customized NMR experiment to detect chemical exchange assisted in characterizing the various states. Having established methods to explore kinetic multi-site exchange processes in one-domain Cadherin-11 constructs, we are also addressing the role of strand-swapping in two-domain constructs.

Session: Biomolecular 1, time: 4:40-5:00

Online Flow-Based Numerical Simulations

Thomas Vosegaard
Aarhus University, Aarhus, Denmark
We present a flow-based programming solution, EasyNMR, for numerical simulations in NMR. EasyNMR is a web site that provides a highly flexible interface to allow users to setup any simulation with little effort. No software installation is required, and users may easily contribute their own models to EasyNMR and share their Flows with other researchers.The versatility of EasyNMR will be demonstrated by simulations of pulse sequences, lineshape patterns and rheo-NMR applications.

Session: Biomolecular 1, time: 5:00-5:20

Partitioning of Polarization Agents for Dynamic Nuclear Polarization in Living Biomass

Yiling Xiao; Jaka Kragelj; Whitney Costello; Carla Madrid; Kendra Frederick
UT Southwestern Medical Center, Dallas,
DNP-enhanced ssNMR allows the observation of a protein which adopts environmentally-sensitive conformations at its endogenous concentration inside cells. Optimizing sample preparation of intact cells for DNP MAS NMR is a critical step towards the goal of in vivo structural biology. To do so, we determined the effects of polarization agent AMUPol when incubated with live cells, by analyzing the representative NMR signals for the major biomass components: protein, lipid, RNA, and cell wall, for the DNP enhancement and the degree of partitioning to all the biomass components. We describe sample preparation methods for in-cell DNP-NMR that maximize enhancements and maintaining cell viability at low experimental temperature for both bacteria and yeast. 

Session: Biomolecular 1, time: 5:20-5:40

Water-Exchanging Hydrogens’ Positions in Biomolecules Detected via Long-Lived Coherences    and Hyperpolarized 2D COSY   

Aude Sadet1; Cristina Stavarache1; Mihaela Bacalum2; Mihai Radu2; Geoffrey Bodenhausen3; Dennis Kurzbach4; Paul Vasos1, 5
1Research Institute of the University of Bucharest, Bucharest, Romania; 2Horia Hulubei” National Institute for Physics, Bucharest-Magurele, Romania; 3École Normale Supérieure, Paris, France; 4University of Vienna, Faculty of Chemistry, Vienna, Austria; 5Extreme Light Infrastructure (ELI-NP) at IFIN-HH, Bucharest-Magurele, Romania
When absent from spectra correlations, water-exchanging amide protons impede the assignment of entire spin systems. We report herein 1D and 2D fast spectroscopic methods to elucidate biomolecular structure and interactions in the proximity of water-exchanging hydrogens in peptides and proteins. The 1D method relies on the effect of water-exchangeable protons of AlaGly dipeptide on the high-field decay rate constants of aliphatic Gly long-lived coherences. The 2D method transfers polarization-enhanced water prepared by dissolution-Dynamic Nuclear Polarisation to -HN protons in AlaGly to record hyperpolarized 2D COSY maps. These proton-only correlations acquired in one minute can identify solvent-exposed regions of peptides in the presence of liposomes, without recurring to heteronuclear isotope labels.


Session: Biomolecular 1, time: 5:40-6:00

Solution-State NMR Studies of Polystyrene Nanoparticle-Small Molecule Interactions

Hui Xu; Emma Mulry; Yunzhi Zhang; Leah Casabianca
Clemson University, Clemson, SC
Interactions between nanoparticles and small molecules in solution are important in a variety of fields. Here we report on recent work using solution-state NMR techniques, including Saturation-Transfer Difference (STD)-NMR, pulsed field gradient experiments to measure diffusion coefficients, relaxation time measurements, and NOESY experiments to characterize the binding between small molecules, such as amino acids and dyes, and the surface of polystyrene nanoparticles. This work may lead to methods for screening biologically-relevant molecules that interact with nano-scale plastic pollutants.