Session ThOD. There are 6 abstracts in this session.

Session: Bioliquids, time: 11:00am-11:20am

Unraveling Cooperativity in Molecular Machines Using Methyl-TROSY

Rui Huang1, 2; Felipe Pérez1; Zev Ripstein1, 2; John Rubinstein1, 2; Lewis Kay1, 2
1University of Toronto, Toronto, Canada; 2Hospital for Sick Children, Toronto, Canada
Molecular machines are usually comprised of multiple subunits which often work in a concerted manner to achieve their biological functions. We have developed an approach to investigate cooperative structural changes and dynamics in homo-oligomeric molecular machines by constructing complexes of distinctively isotopic labeled subunits and using methyl-TROSY-based solution NMR experiments. We present here two examples of such an approach on (i) heptameric a7-ring of the proteasome core particle from Thermoplasma acidophilum (180 kDa) and (ii) hexameric ND1 domain of human AAA+ ATPase p97 (320 kDa). Our studies established noncooperative conformational changes of the proteasome gates and positive cooperativity in dynamics between neighboring N-terminal domains of p97.

Session: Bioliquids, time: 11:20am-11:40am

Development of Heteronuclear-Detected NMR-Experiments for Characterization of NH- and NH2-Groups in RNA

Robbin Schnieders1; Christian Richter1; Antje C. Wolter1; Jens Wöhnert1; Harald Schwalbe1; Boris Fürtig2
1Goethe University Frankfurt, Frankfurt Am Main, Germany; 2Graduate School CLiC- Goethe Univesity, Frankfurt am Main, Germany
Heteronuclear-detected NMR-experiments are especially beneficial in nucleic acids due to the inherent low proton-density and limited 1H-chemical shift dispersion. Here, we present 15N- and 13C-detected NMR-experiments that enable characterization of imino- and amino-groups in RNA. For imino-groups, we apply 15N-detected H-N correlation experiments and investigate the effect of molecular size on resolution, sensitivity and relaxation. Further, amino-groups are difficult to detect as their proton-resonances usually exhibit large line-widths due to a restricted rotation around the C-NH2 bond. We here introduce a 13C-detected C(N)H-HDQC experiment which enables the detection of sharp resonances for all amino-groups independent of their bond-rotation. Further, we develop a 13C-detected ‘amino’-NOESY experiment where amino-NOE contacts can be observed, which are not detectable in conventional 1H-based NOESY experiments.

Session: Bioliquids, time: 11:40am-12:00

Applications of Weak Field Cross Polarization in Solution Biomolecular NMR   

Luke Arbogast1; Frank Delaglio1; Joel R Tolman2; John P. Marino1
1NIST-IBBR, Rockville, MD; 2Johns Hopkins University, Baltimore, MD
The application of weak-field cross-polarization (WFCP) to biomolecules in solution enables the exquisite manipulation of spin-coupled pairs to achieve a number of spectroscopic outcomes ranging from selective coherence transfer to selective removal of target spin pairs.   Such versatility results from the ability to manipulate specific cross-polarization parameters, such as RF field strength, contact time and frequency offsets, and in combination with traditional pulse/delay elements, to design specific sequences that guide spin-trajectories into desired states.  Furthermore, as a double resonance approach, WFCP can take advantage of the signal dispersal afforded in multidimensional experiments to achieve selectivity, where traditional band-selective approaches fail.  We detail novel applications of WFCP to biomolecular systems and the theoretical framework that has allowed for development these methods.

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

A New NMR Technique to Measure Very Fast Hydrogen Exchange in Proteins:  Do Protection Factors for Alpha-Synuclein Signal Structuration?

Frans Mulder1; Enrico Corlianò2; Rupashree Dass1
1Aarhus University, Aarhus, Denmark; 2University of Florence, Sesto Fiorentino, Italy
We describe a novel approach to measure peptide hydrogen exchange rates in the range 10 – 10,000 s-1. Although several methods exist to measure chemical exchange, these are limited to the determination of very slow to slow processes. The new application relies on the principle of decorrelation of spin order reported by Skrynnikov and Ernst, applied to anti-phase operators, as elegantly described by Pelupessy and colleagues. Briefly, the technique uses spin-echoes to generate different admixtures of in-phase and anti-phase operators, where only the latter are subject to decorrelation by intermolecular hydrogen exchange. Applying this technique to the intrinsically disordered protein alpha-synuclein revealed surprisingly high protection factors at the C-terminus. Co-solute Paramagnetic Relaxation Enhancement shows that these effects are electrostatic in origin.

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

Looped, PROjected Concatenated Cross-Polarization (L-PRO CCP): An improved transfer scheme for tackling H->N->C transfers in solvent-exchanging systems

Mihajlo Novakovic; Michael J. Jaroszewicz; Lucio Frydman
Weizmann Institute of Science, Rehovot, Israel
INEPT- and HMQC-based pulse sequences are widely used to transfer polarization between heteronuclei, yet these methods are limited when fast solvent chemical exchanges are present –including many biomolecular NMR scenarios. Alternatives employing J-driven cross polarization (J-CP) proved to be more immune against exchange and relaxation effects. Although significantly improved vis-à-vis INEPT counterparts, H->N->C transfers using combination of triple- and double- resonance cross polarization (Concatenated CP, CCP) remain inefficient in presence of rapid chemical exchange. We present a method based on repeated Looped, PROjected (L-PRO) CCP operations, capable of improving H->N->C transfers and bring them close to a theoretical maximum. Simulation-predicted enhancements were corroborated on urea at fast exchange rates; implications of these triple resonance concepts to IDPs will be examined.

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

Chemical Shift Prediction of RNA Imino Group and the Application in Characterizing RNA Transient States

Yanjiao Wang1; Ge Han1; Tairan Yuwen2; Hashim Al-Hashimi3, 4; Yi Xue1
1School of Life Sciences, Tsinghua University, Beijing, China; 2University of Toronto, Toronto, Canada; 3Duke University Medical Center, Durham, NC; 4Department of Chemistry, Duke University, Durham, NC
NH group in proteins and nucleic acids is the most challenging target for chemical shift prediction. In this work we prepared and measured dozens of RNA hairpins containing all combinations of base pair triplet composed of GC, UA, and GU base pairs, and established a lookup table which relates experimental imino chemical shifts of the central base pair to the corresponding base pair triplet. This table can be used to predict imino chemical shifts of RNAs with unprecedented accuracy. More strikingly, we found that ring-current shift is the dominating factor for chemical shift variations among all base pair triplets. At the end we demonstrated an application of this predictor in characterizing secondary structures of RNA transient states.