One-dimensional 1H-detected solid-state NMR experiment to determine amide-1H chemical shifts in peptides

doi:10.1016/S0009-2614(01)01359-8

Chemical Physics Letters

Volume 351, Issues 1–2, 4 January 2002, Pages 42-46

https://doi.org/10.1016/S0009-2614(01)01359-8 Get rights and content

Abstract

A simple one-dimensional solid-state NMR experiment is proposed to detect amide proton chemical shifts directly in uniaxially oriented molecules under static experimental condition and powder samples under MAS. The efficacy of this method depends on the selective transfer of ¹⁵N transverse magnetization to ¹H nuclei via the $^{1} H –^{15} N$ dipolar coupling and also on the procedure to acquire the ¹H magnetization under $^{1} H –^{1} H$ dipolar decoupling. Experimental results obtained from an N-acetyl-l–¹⁵N-valyl-l–¹⁵N-leucine single crystal labeled with ¹⁵N at both the amide sites and a powder sample of [¹⁵N–Phe-16]-magainin2 are presented.

Introduction

In recent years, solid-state NMR spectroscopy has made important contributions to investigate the structure, dynamics and function of membrane-associated polypeptides. In one of the approaches that utilizes uniaxially oriented samples, NMR parameters such as chemical shift, dipolar coupling and quadrupole coupling are determined to elucidate the backbone conformation of a membrane-associated polypeptide [1], [2], [3], [4], [5], [6], [7], [8]. A series of powerful multidimensional experiments has recently been reported to resolve and assign resonances from uniformly ¹⁵N-labeled membrane proteins in oriented bilayer samples [9], [10], [11], [12], [13], [14], [15], [16], [17]. Since direct detection of protons is complicated due to spin diffusion effects, chemical shifts of amide protons are usually determined indirectly even for peptides that are labeled with $^{15} N$ or $^{13} C$ at a single site [11], [12], [18]. Amide- $^{1} H$ chemical shifts are also important to understand the geometry of hydrogen bonding, secondary structure, and cross correlation studies in polypeptides and proteins [19], [20], [21], [22]; these applications are becoming more powerful due to the availability of higher magnetic fields for solid-state NMR experiments. Since a $^{1} H$ -CRAMPS experiment [23], [24] is non-selective in providing $^{1} H$ resonances, it cannot be used to selectively detect amide- $^{1} H$ chemical shifts. Recently reported indirect $^{15} N$ detection methods perform well under fast spinning (∼30 kHz) conditions and are not useful to samples that cannot be spun faster [25], [26]. In addition, a successful amide- $^{1} H$ detection can lead to the development of solid-state NMR methods to measure various other NMR parameters from proteins. Therefore, it is important to develop experimental procedures for the selective excitation of amide- $^{1} H$ resonances. In this study, a one-dimensional experiment is proposed for the direct detection of amide- $^{1} H$ chemical shift. Experimental results obtained from N-acetyl-l– $^{15} N$ -valyl-l– $^{15} N$ -leucine (NAVL) single crystal and magainin2 (GIGKFLHSAKKFGKAFVGEIMNS-amide) labeled with $^{15} N$ –Phe-16) powder samples are presented.

Section snippets

Experimental

The one-dimensional pulse sequence is shown in Fig. 1. After cross polarization (CP) from $^{1} H$ to $^{15} N$ , the transverse $^{15} N$ magnetization is spin-locked while the $^{1} H$ magnetization is allowed to dephase. During the second-CP, a magic angle spin-lock (SL) (or $^{1} H /^{15} N$ spin exchange at the magic angle [18], [27]) sequence is used in the $^{1} H$ RF channel to suppress $^{1} H –^{1} H$ dipolar interactions. This allows the transfer of $^{15} N$ magnetization to protons that are dipolar coupled to $^{15} N$ (i.e. mainly amide

Results and discussions

The proton spectrum of an NAVL single crystal at an arbitrary orientation relative to the external magnetic field of the spectrometer is given in Fig. 2A. Proton-CRAMPS spectrum of a powder sample of NAVL is given in Fig. 2B for comparison. There are two magnetically inequivalent molecules in a unit cell of the single crystal [32] and therefore a maximum of four amide- $^{1} H$ chemical shift peaks is expected. Two of the amide- $^{1} H$ sites may have the same chemical shift, as only three peaks are well

Conclusion

A one-dimensional $^{1} H$ -detected experiment was successfully demonstrated to record amide- $^{1} H$ signals selectively from peptides that are labeled with $^{15} N$ at a single site. Experimental conditions for the best results depend on the magnitudes of $^{1} H –^{15} N$ and $^{1} H –^{1} H$ dipolar couplings. We strongly believe that this new method will be highly valuable to develop several multidimensional solid-state NMR experiments to study biological molecules in high magnetic field spectrometers.

Acknowledgements

This research was supported by NSF through a grant MCB-9875756 (CAREER Development Award to AR).

References (35)

D.K. Lee et al.
Chem. Phys. Lett.
(1997)
D.K. Lee et al.
Chem. Phys. Lett.
(1998)
D.K. Lee et al.
Chem. Phys. Lett.
(1998)
C.H. Wu et al.
J. Magn. Reson. A
(1994)
A. Ramamoorthy et al.
Solid State NMR Spectrosc.
(1995)
A. Ramamoorthy et al.
J. Magn. Reson. B
(1995)
A. Ramamoorthy et al.
J. Magn. Reson. B
(1996)
Z. Gu et al.
J. Magn. Reson.
(1999)
F.M. Marassi et al.
J. Magn. Reson.
(2000)
F.M. Marassi et al.
J. Magn. Reson.
(2000)

A. Ramamoorthy et al.

J. Magn. Reson. B

(1995)

I. Schnell et al.

J. Magn. Reson.

(2001)

A. Ramamoorthy et al.

J. Magn. Reson.

(1999)

M. Wilhelm et al.

J. Magn. Reson.

(1998)

D.K. Lee et al.

Chem. Phys. Lett.

(1999)

D.K. Lee et al.

J. Magn. Reson.

(1998)

S. Hafner et al.

J. Magn. Reson. A

(1996)

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One-dimensional 1H-detected solid-state NMR experiment to determine amide-1H chemical shifts in peptides

Abstract

Introduction

Section snippets

Experimental

Results and discussions

Conclusion

Acknowledgements

Chem. Phys. Lett.

Chem. Phys. Lett.

Chem. Phys. Lett.

J. Magn. Reson. A

Solid State NMR Spectrosc.

J. Magn. Reson. B

J. Magn. Reson. B

J. Magn. Reson.

J. Magn. Reson.

J. Magn. Reson.

J. Magn. Reson. B

J. Magn. Reson.

J. Magn. Reson.

J. Magn. Reson.

Chem. Phys. Lett.

J. Magn. Reson.

J. Magn. Reson. A

One-dimensional ¹H-detected solid-state NMR experiment to determine amide-¹H chemical shifts in peptides