Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/ [1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIAChitobiose

Chun Tang; Junji Iwahara; G. Marius Clore

doi:10.1007/s10858-005-1206-5

Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIA^Chitobiose

Chun Tang, Junji Iwahara, G. Marius Clore

Research output: Contribution to journal › Article › peer-review

11 Scopus citations

Abstract

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of H_γ (i)-H_N(i) and H_γ(i)-H_N(i + 1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C-¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.

Original language	English (US)
Pages (from-to)	105-121
Number of pages	17
Journal	Journal of Biomolecular NMR
Volume	33
Issue number	2
DOIs	https://doi.org/10.1007/s10858-005-1206-5
State	Published - Oct 2005
Externally published	Yes

Keywords

Isotope labeling
Leu and Val conformation
NMR
NOE
Residual dipolar couplings
Solution structure

ASJC Scopus subject areas

Biochemistry
Spectroscopy

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10.1007/s10858-005-1206-5

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Tang, C., Iwahara, J., & Clore, G. M. (2005). Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIA^Chitobiose. Journal of Biomolecular NMR, 33(2), 105-121. https://doi.org/10.1007/s10858-005-1206-5

Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIA^Chitobiose. / Tang, Chun; Iwahara, Junji; Clore, G. Marius.
In: Journal of Biomolecular NMR, Vol. 33, No. 2, 10.2005, p. 105-121.

Research output: Contribution to journal › Article › peer-review

Tang, C, Iwahara, J & Clore, GM 2005, 'Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIA^Chitobiose', Journal of Biomolecular NMR, vol. 33, no. 2, pp. 105-121. https://doi.org/10.1007/s10858-005-1206-5

Tang C, Iwahara J, Clore GM. Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIA^Chitobiose. Journal of Biomolecular NMR. 2005 Oct;33(2):105-121. doi: 10.1007/s10858-005-1206-5

Tang, Chun ; Iwahara, Junji ; Clore, G. Marius. / Accurate determination of leucine and valine side-chain conformations using U-[¹⁵N/¹³C/²H]/ [¹H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings : Application to the 34-kDa enzyme IIA^Chitobiose. In: Journal of Biomolecular NMR. 2005 ; Vol. 33, No. 2. pp. 105-121.

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title = "Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/ [1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cγ-Hγ/Cβ-Hβ residual dipolar couplings: Application to the 34-kDa enzyme IIAChitobiose",

abstract = "An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15N/13C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ (i)-HN(i) and Hγ(i)-HN(i + 1) NOEs in a 3D 15N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13C-1H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIAChitobiose, a 34 kDa homotrimeric phosphotransferase protein.",

keywords = "Isotope labeling, Leu and Val conformation, NMR, NOE, Residual dipolar couplings, Solution structure",

author = "Chun Tang and Junji Iwahara and Clore, {G. Marius}",

note = "Funding Information: We thank Frank Delaglio for assistance with data analysis using NMRPipe/nmrDraw. This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov). This work was supported in part by the AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G.M.C.).",

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AU - Tang, Chun

AU - Iwahara, Junji

AU - Clore, G. Marius

N1 - Funding Information: We thank Frank Delaglio for assistance with data analysis using NMRPipe/nmrDraw. This study utilized the high-performance computational capabilities of the Biowulf PC/Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov). This work was supported in part by the AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G.M.C.).

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N2 - An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15N/13C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ (i)-HN(i) and Hγ(i)-HN(i + 1) NOEs in a 3D 15N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13C-1H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIAChitobiose, a 34 kDa homotrimeric phosphotransferase protein.

AB - An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15N/13C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ (i)-HN(i) and Hγ(i)-HN(i + 1) NOEs in a 3D 15N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13C-1H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIAChitobiose, a 34 kDa homotrimeric phosphotransferase protein.

KW - Isotope labeling

KW - Leu and Val conformation

KW - NMR

KW - NOE

KW - Residual dipolar couplings

KW - Solution structure

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