1H R1ρ relaxation dispersion experiments in aromatic side chains
Dreydoppel, Matthias ; Lichtenecker, Roman J. ; Akke, Mikael LU
and Weininger, Ulrich
LU
(2021)
In Journal of Biomolecular NMR
75(44846).
p.383-392
- Abstract
Aromatic 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... (More)
Aromatic 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.
(Less)
- author
- Dreydoppel, Matthias
; Lichtenecker, Roman J.
; Akke, Mikael
LU
and Weininger, Ulrich
LU
- organization
- publishing date
- 2021
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Aromatic ring-flip, Aromatic side chains, Conformational exchange, Protein dynamics, Rotating-frame relaxation
- in
- Journal of Biomolecular NMR
- volume
- 75
- issue
- 44846
- pages
- 383 - 392
- publisher
- Springer
- external identifiers
-
- scopus:85114955369
- pmid:34510298
- ISSN
- 0925-2738
- DOI
- 10.1007/s10858-021-00382-w
- language
- English
- LU publication?
- yes
- id
- 586f818b-680f-4423-9d3c-08570a3b0ed1
- date added to LUP
- 2021-10-11 14:20:59
- date last changed
- 2024-04-06 10:27:16
@article{586f818b-680f-4423-9d3c-08570a3b0ed1,
abstract = {{<p>Aromatic 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 <sup>1</sup>H R<sub>1ρ</sub> 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 <sup>1</sup>H–<sup>13</sup>C spin pairs with vicinal <sup>2</sup>H–<sup>12</sup>C moieties are necessary to avoid anomalous relaxation dispersion profiles caused by Hartmann–Hahn matching due to the <sup>3</sup>J<sub>HH</sub> couplings and limited chemical shift differences among <sup>1</sup>H 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, k<sub>flip</sub>, agree well with previous results from <sup>13</sup>C R<sub>1ρ</sub> relaxation dispersion experiments, and yield <sup>1</sup>H chemical shift differences between the two sides of the ring in good agreement with values measured under slow-exchange conditions. The aromatic<sup>1</sup>H R<sub>1ρ</sub> relaxation dispersion experiment in combination with the site-selective <sup>1</sup>H–<sup>13</sup>C/<sup>2</sup>H–<sup>12</sup>C labeling scheme enable measurement of exchange rates up to k<sub>ex</sub> = 2k<sub>flip</sub> = 80,000 s<sup>–1</sup>, and serve as a useful complement to previously developed <sup>13</sup>C-based methods.</p>}},
author = {{Dreydoppel, Matthias and Lichtenecker, Roman J. and Akke, Mikael and Weininger, Ulrich}},
issn = {{0925-2738}},
keywords = {{Aromatic ring-flip; Aromatic side chains; Conformational exchange; Protein dynamics; Rotating-frame relaxation}},
language = {{eng}},
number = {{44846}},
pages = {{383--392}},
publisher = {{Springer}},
series = {{Journal of Biomolecular NMR}},
title = {{<sup>1</sup>H R<sub>1ρ</sub> relaxation dispersion experiments in aromatic side chains}},
url = {{http://dx.doi.org/10.1007/s10858-021-00382-w}},
doi = {{10.1007/s10858-021-00382-w}},
volume = {{75}},
year = {{2021}},
}