Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected (13)C CPMG relaxation dispersion.
Weininger, Ulrich LU ; Respondek, Michal LU and Akke, Mikael LU
(2012)
In Journal of Biomolecular NMR
54(1).
p.9-14
- 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 interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation... (More)
- 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 interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/2966622
- author
- Weininger, Ulrich
LU
; Respondek, Michal
LU
and Akke, Mikael
LU
- organization
- publishing date
- 2012
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Conformational exchange, Relaxation dispersion, Protein folding, Aromatic side chain, TROSY
- in
- Journal of Biomolecular NMR
- volume
- 54
- issue
- 1
- pages
- 9 - 14
- publisher
- Springer
- external identifiers
-
- wos:000307979100002
- pmid:22833056
- scopus:84865633476
- pmid:22833056
- ISSN
- 1573-5001
- DOI
- 10.1007/s10858-012-9656-z
- language
- English
- LU publication?
- yes
- id
- 116d1bc5-7e73-433e-a625-4a873dbde3e4 (old id 2966622)
- date added to LUP
- 2016-04-01 10:06:06
- date last changed
- 2022-03-12 02:05:33
@article{116d1bc5-7e73-433e-a625-4a873dbde3e4,
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 interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains.}},
author = {{Weininger, Ulrich and Respondek, Michal and Akke, Mikael}},
issn = {{1573-5001}},
keywords = {{Conformational exchange; Relaxation dispersion; Protein folding; Aromatic side chain; TROSY}},
language = {{eng}},
number = {{1}},
pages = {{9--14}},
publisher = {{Springer}},
series = {{Journal of Biomolecular NMR}},
title = {{Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected (13)C CPMG relaxation dispersion.}},
url = {{http://dx.doi.org/10.1007/s10858-012-9656-z}},
doi = {{10.1007/s10858-012-9656-z}},
volume = {{54}},
year = {{2012}},
}