Session WOC. There are 5 abstracts in this session.

Session: Biomolecular Structure & Function 2, time: 10:45-11:10
Structural studies of telomerase: a marriage of NMR and electron microscopy

Juli Feigon
University of California Los Angeles, Los Angeles, CA
Telomerase maintains the DNA at the ends of linear chromosomes, thereby preventing genomic instability. It is an RNA-protein complex that contains a catalytic core comprised of a non-coding telomerase RNA (TER) and a unique telomerase reverse transcriptase (TERT), and other proteins involved in biogenesis, assembly, and recruitment of other proteins of the DNA synthesis machinery and to telomeres. We have been using an integrative structural biology approach combining NMR, crystallography, mass spectrometry, and electron microscopy to study the structure and function of telomerase from the ciliate Tetrahymena and from humans. I will discuss how our NMR studies of telomerase RNA structure and dynamics have been combined with cryoelectron microscopy to help elucidate the roles of TER in this remarkable enzyme.

Session: Biomolecular Structure & Function 2, time: 11:10-11:35
Conformational Dynamics in Biomolecular Recognition  & Optimization of NMR Experiments for Ultrahigh Magnetic Fields  
Michael Sattler1, 2
1Technical University of Munich, Garching, DE; 2Helmholtz Zentrum Muenchen, Neuherberg, DE
We study molecular mechanisms and dynamics of multidomain proteins and regulatory protein-RNA and protein-protein complexes by combining solution NMR with complementary techniques, such as SAXS, SANS or FRET. Recent progress and examples will be presented that highlight the role of conformational dynamics and population shifts in molecular recognition of single- and double-stranded RNA.

Current progress will also be presented on the development of radiofrequency pulses and pulse elements using optimal control methods to enable NMR spectroscopy at ultrahigh magnetic fields (>1 GHz) which we pursue in collaboration with the Glaser lab at TUM.

Session: Biomolecular Structure & Function 2, time: 11:35-11:50
Looped, PRojected Overhauser SpectroscopY (L-PROSY): A sensitivity-enhanced experiment for detecting backbone/sidechain NOE cross-peaks
Mihajlo Novakovic; Samuel Cousin; Lucio Frydman
Weizmann Institute of Science, Rehovot, Israel
Dipole-dipole cross-relaxation phenomena leading to NOESY spectra lie at the center of structural determinations by NMR. One of NOESY’s drawbacks is its relatively low sensitivity, as off-diagonal cross-peaks involve a small fraction of the total magnetization. This study explores a simple approach, looped PROSY, capable of enhancing amide/aliphatic NOE cross-peaks in proteins. L-PROSY is based on repeating the perturbation done by NOE’s Ramsey measurement, multiple times. Parameters such as correlation times and amide-water chemical exchange rate will dictate L-PROSY’s overall SNR improvements; these parametric dependencies were examined, and predict enhancements ≈2-5x for typical macromolecules. These predictions were corroborated by novel 15N-1H HMQC-PROSY and 15N-filtered 2D L-PROSY experiments, performed on folded and unfolded proteins. 

Session: Biomolecular Structure & Function 2, time: 11:50-12:05
Alternative Labeling with Pyruvate: Backbone Resonance Assignment of Large Proteins from a Single Experiment
Scott Robson1; Koh Takeuchi2; Andras Boeszoermenyi1, 3; Paul Coote1; Abhinav Dubey1, 3; Sven G. Hyberts1; Gerhard Wagner1; Haribabu Arthanari1, 3
1Harvard Medical School, Boston, MA; 2National Institute of Advanced Industrial Science, Tokyo, Japan; 3Dana-Farber Cancer Institute, Boston, MA
Most investigations of proteins by NMR, whether focused on structural, interaction or dynamic properties, require assignment of backbone nuclei. This procedure is difficult for proteins >30 kDa and requires multiple lengthy experiments. We present a strategy that uses amino acid specific labeling that result from metabolism of a mixture of pyruvate isotopomers and only a single high-resolution and sensitive HNCA experiment. Amino-acid pairs in spin systems can be identified by the level of 1JCaCb suppression that results. Further resolution of amino-acid type is achieved by selectively decoupling 1JCaCb with new Cβ decoupling pulses. Combined with high resolution through NUS acquisition, we can make extensive backbone assignments for proteins greater than 40 kDa with ~4 days of collection time.  

Session: Biomolecular Structure & Function 2, time: 12:05-12:20
Stability and Water Accessibility of the Trimeric Membrane Anchors of the HIV‑1 Envelope Spikes
Alessandro Piai; Jyoti Dev; Qingshan Fu; James J. Chou
Harvard Medical School, Boston, MA
The transmembrane domain (TMD) of the HIV-1 envelope spike has several peculiar features that remain difficult to explain, most notably the presence of an arginine R696 in the middle of the transmembrane helix and the opposite nature of its N- and C-terminal halves (hydrophobic and hydrophilic). We used NMR spectroscopy to determine the membrane partition and solvent accessibility of the TMD in bicelles that mimic a lipid bilayer. Our data show that R696 is positioned close to the center of the bilayer, but, surprisingly, exchanges rapidly with water. We provide a rationale for how the bipolar nature of the TMD allows the placement of the arginine in an otherwise unfavorable lipid environment while reporting the overall stability of the trimer.