scholarly journals Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

2014 ◽  
Vol 59 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Ulrich Weininger ◽  
Ulrika Brath ◽  
Kristofer Modig ◽  
Kaare Teilum ◽  
Mikael Akke
Keyword(s):  
Relaxation Dispersion ◽  
Side Chains ◽  
Rotating Frame ◽  
Rotating Frame Relaxation ◽  
Aromatic Side Chains

scholarly journals 1H R1ρ relaxation dispersion experiments in aromatic side chains

2021 ◽  
Author(s):  
Matthias Dreydoppel ◽  
Roman J. Lichtenecker ◽  
Mikael Akke ◽  
Ulrich Weininger
Keyword(s):  
Chemical Shift ◽  
Active Sites ◽  
Isotope Labeling ◽  
Relaxation Dispersion ◽  
Conformational Exchange ◽  
Side Chains ◽  
Transverse Relaxation Rate ◽  
Spin Lock ◽  
Spin Pairs ◽  
Aromatic Side Chains

AbstractAromatic side chains are attractive probes of protein dynamic, since they are often key residues in enzyme active sites and protein binding sites. Dynamic processes on microsecond to millisecond timescales can be studied by relaxation dispersion experiments that attenuate conformational exchange contributions to the transverse relaxation rate by varying the refocusing frequency of applied radio-frequency fields implemented as either CPMG pulse trains or continuous spin-lock periods. Here we present an aromatic 1H R1ρ relaxation dispersion experiment enabling studies of two to three times faster exchange processes than achievable by existing experiments for aromatic side chains. We show that site-specific isotope labeling schemes generating isolated 1H–13C spin pairs with vicinal 2H–12C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the 3JHH couplings and limited chemical shift differences among 1H spins in phenylalanine, tyrosine and the six-ring moiety of tryptophan. This labeling pattern is sufficient in that remote protons do not cause additional complications. We validated the approach by measuring ring-flip kinetics in the small protein GB1. The determined rate constants, kflip, agree well with previous results from 13C R1ρ relaxation dispersion experiments, and yield 1H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic1H R1ρ relaxation dispersion experiment in combination with the site-selective 1H–13C/2H–12C labeling scheme enable measurement of exchange rates up to kex = 2kflip = 80,000 s–1, and serve as a useful complement to previously developed 13C-based methods.

scholarly journals Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

2019 ◽  
Vol 73 (10-11) ◽  
pp. 633-639
Author(s):  
Heiner N. Raum ◽  
Julia Schörghuber ◽  
Matthias Dreydoppel ◽  
Roman J. Lichtenecker ◽  
Ulrich Weininger
Keyword(s):  
Active Sites ◽  
Relaxation Dispersion ◽  
Side Chains ◽  
Anomalous Relaxation ◽  
Cpmg Relaxation Dispersion ◽  
Key Residues ◽  
Site Selective ◽  
Unfolding Kinetics ◽  
Kinetics Of ◽  
Aromatic Side Chains

Abstract Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic 13C or 1H CPMG relaxation dispersion experiments. Aromatic 1H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by 3J 1H–1H couplings which are causing anomalous relaxation dispersion profiles. Here we show that this problem can be addressed by site-selective 1H/2H labeling of the aromatic side chains and that artifact-free relaxation dispersion profiles can be acquired. The method has been further validated by measuring folding–unfolding kinetics of the small protein GB1. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. Furthermore, the CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained directly from the spectra. In summary, site-selective 1H/2H labeling enables artifact-free aromatic 1H CPMG relaxation dispersion experiments in phenylalanine and the six-ring moiety of tryptophan, thereby extending the available methods for studying millisecond dynamics in aromatic protein side chains.

Privileged hydration sites in aromatic side chains: effect on conformational equilibrium

2017 ◽  
Vol 19 (42) ◽  
pp. 28684-28695 ◽  
Author(s):  
Belén Hernández ◽  
Fernando Pflüger ◽  
Manuel Dauchez ◽  
Mahmoud Ghomi
Keyword(s):  
Conformational Equilibrium ◽  
Theoretical Calculations ◽  
Side Chains ◽  
Aromatic Side Chains

The most energetically favourable hydration sites of aromatic (Phe, Tyr, Trp and His) side chains revealed by DFT-based theoretical calculations.

scholarly journals Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

2021 ◽  
Author(s):  
Pär Söderhjelm ◽  
Mandar Kulkarni
Keyword(s):  
Free Energy ◽  
Trypsin Inhibitor ◽  
Side Chain ◽  
Side Chains ◽  
Aromatic Residues ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Energy Surfaces ◽  
Aromatic Side Chains

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cβ-Cγ axis (χ2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed χ1 (N−Cχα) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

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