• Part 1: Theory of RDCs (Introduction)
• Part 2: Preparing Liquid Crystal Samples
• Part 3: Collecting ²H Spectra and Shimming

Overview

Residual dipolar couplings (RDCs) and residual anisotropic chemical shifts (RACS) offer high-resolution orientation information of bonds and nuclei in a molecular alignment frame. In contrast to nuclear Overhauser effect (NOE) distance constrains, which generally offer low-resolution distances between ¹H atoms in a molecule and are used to correctly pack a molecule, RDCs are useful in orienting secondary structure units in a refinement of a molecular structure. Stated another way, NOEs provide structures with a resolution of a molten globule, and RDCs and RACSs are used to refine NMR structures to high resolution.

The objective of this series of posts is to introduce researchers unfamiliar with RDCs and RACSs with their collection, analysis and refinement in structures. Excellent, comprehensive reviews can be found at the following references.^1,2

Definitions and Abbreviations

NOE

Nuclear Overhauser Effect. The cross-relaxation rate measured between nuclei, usually between ¹H nuclei. Ignoring the effects of spin diffusion, this rate is proportional to the r^-6 distance between nuclei. NOEs can be used to solve a molecular structure.

RACS

Residual Anisotropic Chemical Shift. The small contribution of the chemical shift anisotropy tensor measured from partially aligned samples. These are commonly described as residual chemical shift anisotropies (RCSAs). However, I believe this is a misnomer since the anisotropy of the chemical shift tensor is not directly measured in partially aligned samples.

RDC

Residual Dipolar Coupling. The small contribution of the magnetic dipole-dipole coupling measured from partially aligned samples.

Theory

Introduction

RDCs change the magnitude of measured J-couplings whereas RACSs change the frequency of peaks in a spectrum.

J-couplings are readily measured in NMR, and they are the basis of coherent magnetization transfers in solution-state NMR. J-couplings arise from the polarization induced in the electron cloud between NMR active nuclei, and they tend to cluster in a small range of values, depending on bond lengths, torsion angles, hydrogen bonding and other factors. In proteins, the backbone ¹H-¹⁵N J-coupling for backbone amides tend to adopt values between -90 and -95 Hz. Backbone ¹H^α-¹³C^α J-couplings are typically around 145 Hz. J-couplings are accurately measured from the spacing of split peaks in a spectrum or by using a quantitative J-coupling experiment.

For a heteronuclear spin pair, the J-coupling Hamiltonian is represented as follows.

\[\begin{equation} \label{eq:Hamiltonian_J} H_J = 2 I_z S_z \cdot J_{IS} \end{equation}\]

The \(I_z\) and \(S_z\) operators are the nuclear spin operators for spins ‘I’ and ‘S’, respectively, and \(J_{IS}\) is the J-coupling between these spins. Likewise, the heteronuclear dipolar coupling Hamiltonian can be written:

\[\begin{equation} \label{eq:Hamiltonian_D} H_D = 2 I_z S_z \cdot D_{IS} \end{equation}\]

The Hamiltonian is identical to the heteronuclear J-coupling Hamiltonian, with the exception of the dipolar coupling, \(D_{IS}\). The J-coupling above is orientation independent³, and it remains despite the rapid and isotropic tumbling⁴ of the molecule in solution. The dipolar coupling does not have an orientation-independent contribution, and it only has an orientation-dependent component. Consequently, it cannot be directly observed from the spectrum of a molecular that is tumbling (rotating in solution) isotropically.

For a simple dipolar coupling in a powder sample⁵, the orientation dependence is evaluated as follows.

\[\begin{equation} \label{eq:dipolar_PAS_to_lab} D_{IS} = D_{IS}^0 \frac{1}{2} \left(3 cos^2\theta - 1 \right) \end{equation}\]

The \(D_{IS}^0\) coupling is the static dipolar coupling constant, and it is the largest value the dipolar coupling can adopt. The static dipolar coupling depends on the distance between nuclei.

\[\begin{equation} \label{eq:static_dipole} D_{IS}^0 = -\frac{\mu_0 \hbar \gamma_i \gamma_j}{4 \pi r_{IS}^3} \end{equation}\]

See the following post for a more detailed discussion on the calculation of dipolar couplings.

In the absence of motion, the dipolar coupling is maximum (and equal to its static dipolar coupling constant) when the dipole is colinear with the applied magnetic field, \(B_0\), such that \(\theta = 0^\circ\). Likewise, it is equal to zero at the magic angle (\(\theta = 54.7^\circ\)) and it is equal to -0.5 of the static dipolar coupling at \(\theta = 90^\circ\).

\[\begin{align} \label{eq:dipolar_at_multiple_angles} D_{IS}(\theta = 0^\circ) &= D_{IS}^0 \nonumber \\ D_{IS}(\theta \approx 54.7^\circ) &= 0 \nonumber \\ D_{IS}(\theta = 90^\circ) &= -\frac{1}{2} D_{IS}^0 \nonumber \end{align}\]

Integrating all of the dipolar couplings over a sphere produces a Pake doublet spectrum from solid-state NMR.

If the molecule, and indeed the dipole, rotate rapidly, only an average value of the dipolar coupling can be measured. If all angles \(\theta\) are sampled equally and isotropically, then the average is equal to zero.

\[\begin{equation} \label{eq:2nd_legendre_integral} \int_{0^\circ}^{180^\circ} \left< \frac{1}{2} (3 cos^2\theta -1) sin\theta d \theta \right> = 0 \end{equation}\]

The \(sin \theta\) factor comes from the Jacobian of integration. It’s role is to account for the fact that there are more vectors at the equator of a sphere than at the poles.

It follows that the dipolar coupling isn’t measured directly in the solution state spectrum because the time average is zero.⁶

Of course, if the time average of the dipolar coupling is not zero, it may be observed directly in the spectrum. This is easily achieved by making the molecule tumble slightly non-isotropically (anisotropically) with partial alignment. In this case, the above integral is not equal to zero, and the dipolar coupling adopts a single, average value.

Since the J-coupling and dipolar Hamiltonians share the same spin operator terms, they combine in the measured Hamiltonian.

\[\begin{equation} \label{eq:Hamiltonian_J+D} H_J + H_D = 2 I_z S_z \left ( J_{IS} + D_{IS} \right) \end{equation}\]

Consequently, if partial alignment can be achieved in a sample, the \(\| J_{IS} + D_{IS}\|\) coupling can be measured, analogously to the J-coupling, and the J-coupling can be subtracted from this new coupling to collect the RDC, \(D_{IS}\).

In practice, the magnitude depends on how the molecule is aligned, and therefore its value depends on the orientation of the dipole vector with respect to this molecular (alignment) frame. This transformation requires at least two rotations, and the final result represents the RDC equation.²

\[\begin{equation} \label{eq:RDC} D_{IS} = D_{a} \left [ (3 cos^2 \theta -1 ) + \frac{3}{2} R sin^2\theta cos2\phi \right ] \end{equation}\]

This equation assumes the absence of motion and the same \(D_a\), for a given interaction type, throughout the molecule. Two rotational transformations are needed in describing the RDC, one from the dipole⁷ to the frame of the molecule or alignment frame and one from the molecular frame to the laboratory frame, due to uniaxial diffusion of the liquid crystal.

The \(D_{a}\) represents the static dipolar coupling constant, \(D_{IS}^0\), scaled by the degree of alignment. To produce high-resolution spectra with partial alignment, samples must only be weakly aligned–on the order of 10^-3. Altogether, the static dipolar coupling (-11.5 kHz for a ¹H-¹⁵N) and degree of alignment should produce a \(D_{a}\) of 5-15 Hz for an ¹H-¹⁵N coupling. If the \(D_{a}\) is larger than this value, other couplings may become larger and further split peaks, and the average tumbling time of molecules may become too large to produce high-resolution spectra. An example of the former effect is the splitting of amide peaks in the HSQC of a protein due to appreciable ¹H^N-¹H^α dipolar couplings.

The \(R\) represents the rhombicity of alignment. The rhombicity, \(D_{a}\) and the θ and φ angles are dictated by the nature of the interaction between the molecule and the alignment medium.

In the following post, we discuss general considerations in preparing liquid crystal samples for NMR.

References and Notes