# Multispin Cross-Correlated Transverse Dipolar NMR Relaxation in Solution

Authors: Erik R. P. Zuiderweg
Journal: Concepts in Magnetic Resonance Part A, Bridging Education and Research
 (2022)

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## Abstract

In this paper, we want to consider what would be involved in calculating theR2 relaxation of amide protons in a protein caused by dipolar interactions with nearby protons, for which there are many. NMR textbooks give analytical equations and sometimes derivations for solution NMR relaxation due to dipolar interactions between two spins. There are also closed equations for dipolar interactions between three spins, which include relaxation interference, also known as cross-correlated cross-relaxation. We here derive an expression for interference between four spins. For larger systems, such as amide protons in a protein, we develop a local-field methodology, from which solution relaxation interference can be computed for a basically limitless number of interacting spins.


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## Body

## 1. Introduction

Several researchers have been interested in investigating whether proton-proton dipolar interaction in proteins can be utilized to obtain structural [1, 2] or dynamical information [3]. Here, we want to consider what would be involved in calculating the R2 relaxation of amide protons in a protein caused by dipolar interactions with other protons and, ultimately, compare those with experiment [4]. NMR textbooks give closed equations and sometimes derivations for solution NMR relaxation due to dipole-dipole relaxation between two spins and for chemical shift anisotropy (CSA) relaxation. There are also closed equations for relaxation due to interactions between three dipoles [5] and two dipoles and the CSA [6]. In these cases, relaxation interference, also known as cross-correlated cross-relaxation, has been taken into account. Dipolar-dipolar relaxation interference has been described very early in the history of NMR [7, 8] and has been exploited to achieve line narrowing [9] and structural information [1, 2]. CSA-dipolar cross-correlated relaxation is at the core of the line narrowing in the TROSY experiment [10].Here, we derive an analytical expression for dipolar interference between four spins. For larger systems, such as amide protons in a protein, we develop a local-field methodology, from which solution relaxation interference can be computed for a basically limitless number of interacting spins.


## 2. Theory

Let us start by considering theR2 relaxation of spin I in a rigid molecule with three protons, S-I-Q, subject to fluctuating dipolar interactions IS and IQ. We assume here that I, S, and Q have different chemical shifts and are thus similar, but “unlike.” The first step is to add the relaxation rates due to the IS and IQ dipolar interactions, as given by equation (A.24) of the Appendix.(1)R2Itotal=R2IS+R2IQ,(2)R2Itotal=18μ04π2γHγHℏrIS32+γHγHℏrIQ325J0+9JωH+6Jω2H.However, in a rigid molecule, the stochastic fluctuations of theIS and IQ dipolar interactions are 100% correlated and relaxation interference occurs. Relaxation interference is also called cross-correlated cross-relaxation or cross-correlated relaxation.Interference between two dipolar mechanisms can be qualitatively understood from Figure1. Here, we consider a linear three-spin system with spin I in the center and spin S and Q equidistant from I.Figure 1
Pictorial representation of dipolar cross-correlation in a linear S-I-Q system. The dipolar fields ofS and Q at the location of I for A, independent of orientation with respect to the magnetic field (vertical). In B, the dipolar fields of S and Q reinforce each other at the location of I and modulate strongly as a function of molecular orientation.In this case, there are two relaxation rates for spinI, one for molecule A, where the dipolar fields of S and Q at the location I cancel independent of orientation with respect to the magnetic field, and another for molecule B, where the dipoles reinforce each other and fluctuate strongly depending on orientation. This is expressed as(3)R2Itotal=R2IS+R2IQ±R2CCIS−IQ.Of course, the dipoles flip byR1 processes. For macromolecules, R1 rates are much slower than R2 rates. Thus, for R2 cross-correlations, one may assume that orientations of the dipoles remain set during the typical R2 relaxation time.If the different dipolar permutations also give rise to different resonances, by either scalar or residual static dipolar coupling, one observes four NMR lines forI. The two outer lines have equal linewidths, and the two inner lines also have equal, but different, linewidths. Which set will be the broader one depends on the molecular geometry and the sign of the (scalar) coupling. If no such coupling exists, one will observe a superposition of a broader and narrower line for I.In order to obtain quantitative values for the dipolar-dipolar cross-correlation, one starts with expanding the double commutator master equation forR2 relaxation, as shown in equation (A.16) of the Appendix. We mostly follow a formalism as introduced by [5, 11].For twoR2 relaxation mechanisms IS and IQ, modulated by the same motions, the relaxation master equation is(4)dI+dt=−12∑m=−2m=+2I+,TmIS+TmIQ,TmIS†+TmIQ†jmωm,where TmIS are the tensor operators of the perturbing Hamiltonian, which for dipolar interaction between spins I and S are given by(5a)T0=2IzSz−12I+S−+I−S+,(5b)T±1=∓32I±Sz+IzS±,(5c)T±2=32I±S±,with similar equations for TmIQ.jmωm are the spectral densities of the molecular motion at the frequencies of the bilinear spin operators including magnitude terms.In the expansion, the “diagonal” terms such as(6a)∑m=−2m=+2I+,TmIS,TmIS†jmωm,(6b)∑m=−2m=+2I+,TmIQ,TmIQ†jmωm,and their complex conjugates will give rise to the autocorrelation rates R2IS and R2IQ, as shown in equation (A.22) of the Appendix.In the cross terms,(7a)∑m=−2m=+2I+,TmIS,TmIQ†jmccωm,(7b)∑m=−2m=+2I+,TmIQ,TmIS†jmccωm,and their complex conjugates, only spin operators with exactly the same precession frequency will be relevant, i.e., only the pair (IzSz, IzQz) and the four terms I+Sz, I−Sz, I+Qz, and I−Qz.We obtain the following equation from equations (6a) and (6b):(8)−dI+dt=4I+,IzSz,IzQzj0cc0+32I+,I−Sz,I+Qzj1ccωI,where the ½ term is dropped because there are two equivalent cross terms, as given in equations (7a) and (7b).The cross-correlation spectral densities are(9)jmccωm=−18μ04π2γIγSℏrIS3γIγQℏrIQ3P2cosθIS−IQJωm,with, for isotropic motion,(10)Jωm=25τc1+ωm2τc2,where the P2cosθIS−IQ=1/23cos2θIS−IQ−1 term originates from transforming the IQ dipolar vector into the IS frame or vice versa and using the addition theorem of spherical harmonics. Here, θIS−IQ is the angle between vectors IS and IQ and τc is the (rotational) correlation time.In contrast to theR2 autocorrelation, the double commutators drive cross-relaxation(11a)−dI+dt=4I+SzQzj0cc0+344I+SzQzj1ccωI,and back(11b)−d4I+SzQzdt=I+j0cc0+I+j1ccωI.One ends up with a set of coupled differential equations:(12)ddtI+4I+SzQz=−R2IIPR2ICCR2ICCR2IAPI+4I+SzQz.Here,R2IIP and R2IAP are the in-phase (IP) R2 rate for the <I+> coherence and antiphase (AP) R2 rate for the 4 <I+SzIz> coherence. The antiphase terms relax faster because of the extra Sz and QzR1 relaxation. For macromolecules, the difference can be neglected because R1 is small as compared to R2, and one can easily diagonalize the rate matrix and obtain relaxation “eigenvectors”(13)ddtI++4I+SzQzI+−4I+SzQz=−R2IS+R2IQ+R2ICC00R2IS+R2IQ−R2ICCI++4I+SzQzI+−4I+SzQz,with(14)R2ICC=14μ04π2γIγSℏrIS3γIγQℏrIQ3P2cosθIS−IQ4J0+3JωI.The totalR2 relaxation for proton i, as stated before in equation (3), is then given by(15)R2Itotal=R2IS+R2IQ±R2CCIS−IQ.One notices that1H IS-autorelaxation contains a 5J(0) term (equation (2)), while the dipolar cross-correlation has a 4J(0) term (equation (14)). The extra J(0) in autorelaxation is due to the fact that J(0) and JωI−ωS are for two protons similar enough for the spectral densities to add (see Appendix), but ωI−ωS and ωI−ωQ are different enough for the spin operators to diphase and not cross-correlate. Thus, there will not be a complete cancelation of relaxation, as suggested in the situation of Figure 1. It can still happen, if the differences in distances IS and IQ would make up for the 4/5 term.If one cannot make the approximation thatR2IIP and R2IAP are equal, the rates become(16)R2Itotal=12R2IIP+12R2IAP±R2IIPxR2IIP4−R2IIPxR2IAP2+R2IAPxR2IAP4+R2CCIS−IQxR2CCIS−IQ.Let us now extend this formalism to a 4-spin system, with a central spinI and 3 other spins S, Q, and P in its vicinity.From the master equation, we arrive at three cross terms:(17)−dI+dt=4I+,IzSz,IzQzj0cc0+32I+,I−Sz,I+Qzj1ccωI+4I+,IzSz,IzPzj0cc0+32I+,I−Sz,I+Pzj1ccωI+4I+,IzQz,IzPzj0cc0+32I+,I−Qz,I+Pzj1ccωI,leading to terms such as 4I+SzQz, 4I+SzPz, and 4I+PzQz, driven by the rates(18a)R2IIS−IQ=14μ04π2γIγSℏrIS3γIγQℏrIQ3P2cosθIS−IQ4J0+3JωI,(18b)R2IIS−IP=14μ04π2γIγSℏrIS3γIγPℏrIP3P2cosθIS−IP4J0+3JωI,(18c)R2IIP−IQ=14μ04π2γIγQℏrIQ3γIγPℏrIP3P2cosθIQ−IP4J0+3JωI.This appears to lead to eight different relaxation ratesR2IS+R2IQ+R2QS±R2CCIS−IQ±R2CCIS−IP±R2CCIP−IQ. However, that cannot be right; adding one more dipole P to the S-I-Q situation should just give rise to two more rates, four in total. So, one should not stop here and construct the full relaxation matrix.Also, one must take cross-relaxation between the different four-spin terms into account (here only showing someJ(0) terms),(19a)−d4I+SzQzdt=44I+SzQz,IzSz,IzPz=4I+QzPz,(19b)−d4I+SzQzdt=44I+SzQz,IzQz,IzPz=4I+SzPz,(19c)−d4I+SzPzdt=44I+SzPz,IzPz,IzQz=4I+SzQz,and three reverse processes.The differential equations in matrix form will now become(20)ddtI+4I+SzQz4I+SzPz4I+QzPz=−R2IR2ccSQR2ccSPR2ccQPR2ccSQR2IR2ccQPR2ccSPR2ccSPR2ccQPR2ccQPR2ccSPR2IR2ccSQR2ccSQR2II+4I+SzQz4I+SzPz4I+QzPz.Here, we are neglecting the differences betweenR2 in-phase and antiphase relaxation. This matrix has four eigenvalues, which correspond to what must be physically correct. While the exact eigenvalues of this matrix could potentially be analytically obtained, it becomes harder when differences between R2 in-phase and antiphase relaxation have to be considered as well. However, because matters will quickly become more complicated when even more spins interact, we will not attempt to derive an analytical solution for the four-spin case here. For instance, in a protein, there are typically at least 20 protons around every amide proton in a 6 Å sphere which would all have to be taken into account. In such a case, one would need to diagonalize a 262144∗262144 matrix (220 − 2).We must take another approach. To arrive at an estimation for the effects in a multiproton spin system, we will start from a “solid-state NMR” point of view. We calculateBlociΩ, the net local magnetic field at center proton i due to the M surrounding protons j [12] for a certain orientation of the molecule in the external magnetic field:(21)BlociΩ=μ04π∑j≠ij=MDγiγjrij3P2cosθij.Here,θij is the angle between the internuclear vector ij and the magnetic field direction Ω in the molecular frame. D represents a particular distribution of the signs of the z-components of the dipoles j. If one varies the magnetic field direction according to a sphere distribution and adds the results, one obtains the powder pattern for that particular distribution D. Subsequently, one coadds all powder patterns for different values of D and normalizes to arrive at the “cross-correlated” dipolar powder pattern for the 1HN under consideration.It is the time dependence ofBloc, as caused by molecular motion, that drives the solution NMR dipolar relaxation. The R2 relaxation is then obtained as the second moment of the (cross-correlated) powder pattern [12]:(22)R2isolution=4τc∑ΩBloci−BlociΩ2,where the brackets indicate average overall orientations. In practice, we permute only the signs of the eight closest protons (256 different distributions D) and treat the spins further out with a single random distribution where spins up and down are on average equal. So, each distribution then gives rise to an individual resonance for i with its own R2 rate. The sum of all of those creates the inhomogeneous sum-line for resonance i.


## 3. Verification

We verified the algorithm with a three-atom arrangement (see Table1) with results in Figure 2. In this figure, the green points were calculated from equations (14) and (15) for dipolar-dipolar cross-correlation, while the drawn black line was calculated using the “solid-state” approach. The results are identical. In red is a relaxation curve calculated from the straight addition of R2 (IS) and R2 (IQ) (see equation (1)). It is clear that the cross-correlation cannot be neglected, except when one considers the first part of the curve. Fitting a single exponential against a complete cross-correlated relaxation curve (dashed line) yields an erroneous rate.Table 1
Coordinates of an arbitrary three-spin arrangement (Å).dIS = 2 Å, dIQ = 2.236 Å, and P2 cos(θIS−IQ) = 0.70.
xyzCenter spin (I)000Neighbor (S)020Neighbor (Q)0−21Figure 2
The green points were calculated from equations (14) and (15) for dipolar-dipolar cross-correlation, while the solid black line was calculated using the “solid-state” approach (equations (21) and (22)). Fitting a single exponential against a complete cross-correlated relaxation curve (dashed line) yields an erroneous rate. In red is a relaxation curve calculated from the straight addition of R2 (IS) and R2 (IQ) (equation (1)). A (isotropic) rotational correlation time τc of 10 ns was used in all calculations.In Figure3(a), we show calculated R2 relaxation rates for the HN of Asp45 of the protein GB1. This amide has 21 proton neighbors within a sphere of 6 Å. Here, the difference between relaxation curves with and without cross-correlations is smaller than the example case in Figure 2. From this, one would tend to conclude that many protons cancel each other’s cross-correlations. However, that is not necessarily true; in Figure 3(b), we show calculated R2 relaxation rates for the HN of Asn35 of the protein GB1. This amide has more (36) proton neighbors within a sphere of 6 Å, yet the difference between relaxation curve with and without cross-correlations is larger than for Asp45. This happens because the R2 relaxation of Asn35 is dominated by two close-by protons.Figure 3
The calculatedR2 relaxation rates (a) for the HN of Asp45 of the protein GB1 which has 21 proton neighbors within a sphere of 6 Å and (b) for the HN of Asn35 with 37 proton neighbors within a sphere of 6 Å. The black curves are with cross-correlations (equations (21) and (22)), while the red curves are computed as the sum of the R2 rates (equation (1)). A (isotropic) rotational correlation time τc of 10 ns was used in all calculations.
(a)(b)This manuscript was written in anticipation of the interpretation of experimentalT1rho data for protein amide protons, which we hope may contribute to more complete understanding of protein molecular dynamics (E.R.P. Zuiderweg and D.A. Case, in preparation). We conclude from the current analysis that one can still fit a single exponential to the beginning of the T1rho relaxation curves, even when many protons interact. This result is the same as for a 3-spin network, but that was not previously demonstrated. However, how far down a relaxation curve can go varies from proton to proton environment. Judging from Figure 3(b), one should limit the recording of T1rho relaxation curves to values larger than 0.8xI0, where I0 is the initial value of the decay curve.


## 4. Description of the Codes

The computer program requires as input a “protonated” PDB file, the radius of the sphere of protons around the amide protons one is interested in, the rotational correlation time, and the spectrometer frequency. Basically, the program consists of four nested loops: amides, protons around amides, permutation of dipole signs of these surrounding protons, and rotation of the magnetic field vector in the molecular frame.A set of 10 nested loops permutes the dipolar signs of the closest 10 hydrogens (1024 distributions). The more remote hydrogens in the sphere (if any) have their dipolar signs assigned according to a 50% random chance. The local dipolar field at a certain1HN due to the surrounding protons in a certain permutation D of surrounding dipoles is calculated according to equation (20). Here, the program takes the differences between “like” and “unlike” spins (see Appendix) into account. Then, the program calculates, according to equation (22), the R2 relaxation rate due to that permutation, by rotating the external field direction (the z-axis) through the molecular frame using an isotropic spherical distribution (5000 orientations) (http://corysimon.github.io/articles/uniformdistn-on-sphere/).The relaxation rate for that permutationD is then used to compute a R2 relaxation curve. The computed R2 relaxation curves for all different permutations D are then added to obtain the complete R2 relaxation curve, as shown in Figures 2 and 3.The program is written in Fortran 90 and contains no references to outside libraries. The source code is available from the author.



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*Source: 1617848-2022-04-12.xml*