Related ArticlesMeasurement of State-Specific Association Constants in Allosteric Sensors through Molecular Stapling and NMR.
J Am Chem Soc. 2015 Aug 6;
Authors: Moleschi KJ, Akimoto M, Melacini G
Abstract
Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs. active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e. state-selective molecular stapling through disulphide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cAMP-dependent protein kinase (PKA-RI?), for which the structures of the inactive and active states are available, as needed to design the state-selective disulphide bridges. Surprisingly, the PKA-RI? state-specific association constants are comparable to those of a structurally homologous domain with ~10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.
PMID: 26247242 [PubMed - as supplied by publisher]
Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes
From The DNP-NMR Blog:
Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes
Garimella, P.D., et al., Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes. J. Am. Chem. Soc., 2013. 136(1): p. 164-168.
http://pubs.acs.org/doi/abs/10.1021/ja406760r
[U. of Ottawa NMR Facility Blog] Measurement of Long Range C H Coupling Constants
Measurement of Long Range C H Coupling Constants
The stereochemistry of compounds is assigned very often with proton - proton NOE's by applying the 2D NOESY technique or the 1D selective gradient NOESY technique. These methods fail, however when the distance between protons is too large to measure an NOE. When faced with this situation, it may be possible to measure long range proton - carbon coupling constants which are able to provide the necessary information. Three-bond carbon - proton couplings follow a Karplus relationship where the magnitude of the coupling constant is related to...
[NMR paper] Residue-specific real-time NMR diffusion experiments define the association states of
Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding.
Related Articles Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding.
J Am Chem Soc. 2002 Jun 19;124(24):7156-62
Authors: Buevich AV, Baum J
Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the...
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[NMR paper] NMR exchange broadening arising from specific low affinity protein self-association:
NMR exchange broadening arising from specific low affinity protein self-association: analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1.
Related Articles NMR exchange broadening arising from specific low affinity protein self-association: analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1.
J Biomol NMR. 1999 Aug;14(4):307-20
Authors: Pfuhl M, Chen HA, Kristensen SM, Driscoll PC
Nuclear spin relaxation monitored by heteronuclear NMR provides a useful method to probe the overall and internal molecular motion for...