Session MOB. There are 5 abstracts in this session.



Session: BIOMOLECULAR 1 - LIQUIDS/DYNAMICS, time: 10:45 - 11:10 am

Characterising Interactions and Dynamics of Side Chains in Medium-to-Large Proteins by Protonless 13C-detected NMR Spectroscopy


D. Flemming Hansen
ISMB, Univ. College London, London, United Kingdom

Side chains cover protein surfaces and are fundamental to processes as diverse as substrate recognition, protein folding and enzyme catalysis. However, characterisation of side-chain motions has so far been restricted to small proteins and methyl-bearing side-chains. Several new methods with focus on protein side chains and anchored in 13C-detected NMR spectroscopy are presented. One set of methods is aimed at elucidating the interactions and dynamics of the guanidinium group of arginine side chains, whereas a second set of methods allows a characterisation of the structure and dynamics of six different side chains in proteins with sizes up to 80 kDa. The presented methods promise characterisations of side-chain behaviour at a level that has hithereto been reserved for the protein backbone.


Session: BIOMOLECULAR 1 - LIQUIDS/DYNAMICS, time: 11:10 - 11:35 am

Probing the structure and dynamics of large protein complexes using segmental isotopic labeling and solid state NMR


Yuuki Wittmer; Rong Hu; Rachelle Stowell; Rebecca Rafique; Natalie Boulos; Dylan Murray
Univ. of California, Davis, Davis, 0

In theory, solid state NMR allows for the characterization of protein structure and dynamics of large protein assemblies with no upper limit in size. Limited spectral space for the signals from the protein molecules places a practical upper limit on the size of the assembly that can be probed. We implement segmental isotopic labeling using intein chemistry to selectively incorporate NMR-visible isotopes into subdomains of protein molecules. We present results for proteins involved in the formation of intermediate filament networks in the cell cytoskeleton and RNA-binding proteins that form both membraneless organelles and pathological inclusions in neurodegenerative disease. Our data show that segmental isotopic labeling is a key and viable technology for probing the molecular conformations of these proteins.


Session: BIOMOLECULAR 1 - LIQUIDS/DYNAMICS, time: 11:35 - 11:50 am

Allosteric Motions of the CRISPR-Cas9 Endonuclease Probed by NMR and Molecular Dynamics


Kyle East; George Lisi
Brown University, Providence, RI

CRISPR-Cas9 is an RNA-guided DNA endonuclease with applications to genome editing. However, its susceptibility to off-target effects and poor temporal control hamper its potential for therapeutic bioengineering. To circumvent these risks, new studies have examined the recently proposed allosteric mechanism of Cas9, which synchronizes binding and double-stranded cleavage of DNA at three spatially distinct sites. Understanding this crosstalk can enhance spatial and temporal control of the enzyme. We used NMR spin relaxation and computational approaches to prove picosecond-to-millisecond dynamics in a critical Cas9 nuclease, HNH, that is suggested to regulate allostery. We found a contiguous ms dynamic pathway spanning a novel engineered construct of HNH to its interfaces with adjacent domains, suggesting it may be relevant to allosteric signal transduction.


Session: BIOMOLECULAR 1 - LIQUIDS/DYNAMICS, time: 11:50 - 12:05

Site-specific detection and characterization of Ubiquitin carbamylation by NMR spectroscopy


Wes Pawloski1; Teppei Komiyama1; Christos Kougentakis2; Ananya Majumdar3; George Lorimer1; David Fushman1
1University of Maryland, College Park, MD; 2Department of Biophysics, Johns Hopkins University, Baltimore, MD; 3Bio. Mol. NMR Facility, Johns Hopkins University, Baltimore, MD

The post-translational modification of lysine sidechain amines has been demonstrated to alter protein structure and function by modifying steric or electrostatic interactions. These modifications are often difficult to detect or put into a functional context by standard biochemical methodologies, however NMR spectroscopy is uniquely situated to answer these questions. This work will highlight the use of H2CN triple resonance experiments to probe the reactivity of ubiquitin amines to carbomylation by the cyanate ion and correlate these reactivities to pKas. These results share similarities to the process of ubiquitin acetylation and overall highlight an evolutionary mechanism for regulating ubiquitin chain forming reactions by lysine pKas and environmental conditions.


Session: BIOMOLECULAR 1 - LIQUIDS/DYNAMICS, time: 12:05 - 12:20 pm

Functionally relevant rapid protein motions captured with high power Relaxation Dispersion in dedicated solution-NMR probe


Supriya Pratihar1; G J Reddy1; S Becker1; B. L. de Groot1; D Lee2; Christian Griesinger1
1MPI for Biophysical Chemistry, Gottingen, Germany; 2University of Louisville, Louisville, KY

Conformational flexibility governs protein-function. We aim to close the supra-τc (4ns-40µs) gap in the dynamics timescale-spectrum. In conventional cryo-probes, with 40 kHz RF-power, we found functionally relevant supra-τc motion in GB3. This method also revealed fast-folding kinetics (4µs at 263K) in the binding domain of tumor-suppressor protein-p53 (IDP). Supra-τc gap can be closed with 300 kHz power in solenoid-coil, in a 0.7mm MAS probe which accommodates only 0.3µl of sample. To perform high-power-RD on challenging proteins at low concentration, we are currently developing a solution-NMR probe which allows 300 kHz power to 50µl sample volume, with lock and gradient. 300 kHz RD data leading to the direct detection of a pincer-mode motion in ubiquitin will be presented.