Related ArticlesSite-specific protonation kinetics of acidic side chains in proteins determined by pH-dependent carboxyl 13C NMR relaxation.
J Am Chem Soc. 2015 Feb 10;
Authors: Wallerstein J, Weininger U, Khan MA, Linse S, Akke M
Abstract
Proton transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on 13C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3)×106 s-1 and kon in the range (0.6-300)×109 M-1s-1, which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between logkon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.
PMID: 25665463 [PubMed - as supplied by publisher]
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection
Abstract
Protein dynamics on the microsecondâ??millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the...
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06-19-2014 10:21 PM
[NMR paper] The Involvement of Amino Acid Side Chains in Shielding the Nickel Coordination Site: An NMR Study.
The Involvement of Amino Acid Side Chains in Shielding the Nickel Coordination Site: An NMR Study.
The Involvement of Amino Acid Side Chains in Shielding the Nickel Coordination Site: An NMR Study.
Molecules. 2013;18(10):12396-12414
Authors: Medici S, Peana M, Nurchi VM, Zoroddu MA
Abstract
Coordination of proteins and peptides to metal ions is known to affect their properties, often by a change in their structural organization. Side chains of the residues directly involved in metal binding or very close to the coordination centre...
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10-11-2013 10:43 AM
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected 13C CPMG relaxation dispersion
Abstract Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding...
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07-30-2012 07:42 AM
13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy
13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy
Abstract Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of 13C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of...
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07-05-2012 04:13 AM
On the calculation of 3Jαβ-coupling constants for side chains in proteins
On the calculation of 3Jαβ-coupling constants for side chains in proteins
Abstract Structural knowledge about proteins is mainly derived from values of observables, measurable in NMR spectroscopic or X-ray diffraction experiments, i.e. absorbed or scattered intensities, through theoretically derived relationships between structural quantities such as atom positions or torsional angles on the one hand and observable quantities such as squared structure factor amplitudes, NOE intensities or 3 J-coupling constants on the other. The standardly used relation connecting 3 J-couplings...
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06-25-2012 04:41 AM
[NMR paper] Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of
Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of HIV-I protease tethered dimer by real time NMR spectroscopy.
Related Articles Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of HIV-I protease tethered dimer by real time NMR spectroscopy.
Biochem Biophys Res Commun. 2000 Mar 16;269(2):387-92
Authors: Panchal SC, Hosur RV
HIV I protease has been the target of extensive and variety of investigations in recent years because of its importance in the AIDS viral life cycle. We...
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11-18-2010 09:15 PM
Site-specific (19)F NMR chemical shift and side chain relaxation analysis of a membra
Site-specific (19)F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid.
Related Articles Site-specific (19)F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid.
Protein Sci. 2010 Nov 15;
Authors: Shi P, Wang H, Xi Z, Shi C, Xiong Y, Tian C
Site-specific (19)F chemical shift and side chain relaxation analysis can be applied on large size proteins. Here, one dimensional (19)F spectra and T(1), T(2) relaxation data were acquired...
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11-17-2010 05:49 PM
Site-Specific Protein Backbone and Side-Chain NMR Chemical Shift and Relaxation Analy
Site-Specific Protein Backbone and Side-Chain NMR Chemical Shift and Relaxation Analysis of Human Vinexin SH3 Domain using a Genetically Encoded (15)N/(19)F-Labeled Unnatural Amino Acid.
Related Articles Site-Specific Protein Backbone and Side-Chain NMR Chemical Shift and Relaxation Analysis of Human Vinexin SH3 Domain using a Genetically Encoded (15)N/(19)F-Labeled Unnatural Amino Acid.
Biochem Biophys Res Commun. 2010 Oct 11;
Authors: Shi P, Xi Z, Wang H, Shi C, Xiong Y, Tian C
SH3 is a ubiquitous domain mediating protein-protein interactions....
Site-specific protonation kinetics of acidic side chains in proteins determined by pH-dependent carboxyl 13C NMR relaxation.
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