Residual dipolar couplings in NMR structure determination

Bioc 585, Feb 26, 2008

Long-range information in NMR• a traditional weakness of NMR is that all the structural restraints are

short-range in nature (meaning short-range in terms of distance, not in terms of the sequence), i.e. nOe restraints are only between atoms <5 Å apart, dihedral angle restraints only restrict groups of atoms separated by three bonds or fewer

• over large distances, uncertainties in short-range restraints will add up--this means that NMR structures of large, elongated systems (such as B-form DNA, for instance) will be poor overall even though individual regions of the structure will be well-defined.

to illustrate this point, in the picture at left, simulatednOe restraints were generated from the red DNA structure and then used to calculate the ensemble of black structures

short-rangestructure OK

long-rangestructure bad

Zhou et al. Biopolymers (1999-2000) 52, 168.

best fitsuperpositiondone for this end

Residual dipolar couplings

•recall that the spin dipolar coupling depends on the distance between 2 spins, and also on their orientation with respect to the static magnetic field B0.

•In solution, the orientational term averages to zero as the molecule tumbles, so that splittings in resonance lines are not observed--i.e. we can’t measure dipolar couplings. This is too bad, in a way, because this orientational term carries structural info, as we’ll see

•In solids, on the other hand, the couplings don’t average to zero, but they are huge, on the order of the width of a whole protein spectrum. This is too big to be of practical use in high-resolution protein work

•compromise: it turns out that you can use various kinds of media, from liquid crystals to phage, to partially orient samples, so that the dipolar coupling no longer averages to zero but has some small residual value

Proteins tumbling isotropically in solutionNo orientational biasDipolar interaction averages to zero with tumblingNo observable dipolar coupling.Too small!

Proteins in a single crystalComplete orientational biasEnormous dipolar coupling.Too big!

(Dipolar couplings as big asentire proton spectral range)

B0B0

Residual dipolar couplings: A Goldilocks tale

Proteins dissolved in liquid but oriented mediumSome liquid crystals acquire macroscopic order in a magnetic fielde.g. bicelles, filamentous phage, cellulose crystallitesCollisions w/protein impart a slight orientational bias A small “residual dipolar coupling” resultsJust right! --> gives interpretable information

B0

...but the third bowl of porridge was just right.

filamentous phage,lipid bilayer fragment,cellulose crystallite

--regular HSQC--decoupled in both dimensions--15N-1H splittings not observed

--HSQC without decoupling in 15N dimension-- isotropic solution--15N-1H splittings observed, equal to 15N-1H one-bond scalar coupling (~92-95 Hz)

--HSQC without decoupling in 15N dimension--partly oriented--15N-1H splittings observed, equal to 15N-1H one-bond scalar coupling plus RDC!Some RDC -, some +

Measurement of Residual Dipolar Couplings

This picture illustrates measurement of 15N-1H residual dipolar couplings for a protein in a 7% bicelle (fragments of lipid bilayer) solution. The bicelle preparation is isotropic (not ordered) at 25 °C (left), allowing measurement of the scalar couplings. Upon heating to 35 °C, the bicelle preparation becomes anisotropic (ordered) such that the measured coupling now includes an RDC component. RDCs can therefore be measured by comparing spectra taken at the different temperatures.RDCs can often be “tuned” by adjusting the composition of the liquidcrystal mixture.

Prestegard et al. Biochemistry (2001) 40, 8677.

SAG: Strain induced alignment in a gel

Proteins can be incorporated into cylindrical polyacrylamide gels within NMR tubes. If the gel is stretched or compressed, the pores become anisotropic and can impart partial order to a protein just like a liquid crystal can.

axially compressed,radially stretchedoblate ellipsoid pores

radially compressed,axially stretchedprolate ellipsoid pores

pores in gelcontainprotein

regular polyacrylamidegel

• Recall that the spin dipolar interaction between two nuclei depends upon their relative position with respect to an external magnetic field. The residual dipolar coupling will therefore be related to the angle between the internuclear axis and the direction of the partial ordering of the protein. See Tjandra et al. Nat Struct Biol, 4, 732 (1997) for a more thorough treatment. Because the internuclear axis will have a different orientation for different bonds in the protein, the RDCs will exhibit a broad range of values.

internuclear axis (bond vector)

“axis of partial ordering”:principal axissystem of magnetic susceptibility tensor

15N-1H residual dipolar coupling will differ for these two residues.

Interpretation of RDCs--what do they mean?

15N15N

1H

1H 15N15N

1H

1H

Note that the relative values of 15N-1H RDCs for a set of amide nitrogenhydrogen pairs do not depend upon the distance between those pairs, only on their relative orientation with respect to a common axis system!

two NH bond vectorsclose together

two NH bond vectors far apart, but with same orientation

RDCs give information about long-range order in proteins

In other words, RDCs can in principle tell us the relative orientation of twobond vectors even if they are on opposite ends of the molecule. Contrast thiswith NOE distance restraints and dihedral angle restraints which define shortrange order.

Most measured RDCs are one-bond couplings

Recall that the spin dipolar interaction, and therefore the RDC, has both a steep distance dependence and an orientational dependence. If we are considering a particular type of RDC, say a one-bond coupling between amide hydrogens and amide nitrogens, the interatomic distances are all the same and equal to an NH bond length. The RDC depends only on the orientational component. This would also still be true for a two-bond RDC, but for a three-bond RDC the distance would vary with the dihedral angle, making interpretation less straightforward.

Most measured RDCs are “one-bond”, e.g. between an amide proton and its directly attached nitrogen, since these correspond to distances less than < 1.5 Å generally. However, you’ll notice in the Chou paper that they measure five dipolar couplings per residue, including HN, HC, CC and CN one-bond couplings, but also including the 1H-13C’ two-bond coupling (C’ means the carbonyl carbon). So two-bond RDCs are not unheard of.

Illustration of effect of using residual dipolar couplings on the quality of nucleic acid structuredetermination by NMR

a) without rdcb) with rdc

Zhou et al. Biopolymers (1999-2000) 52, 168.

A problem with dipolar couplings is that one cannot distinguish the direction of an internuclear vector from its inverse. Thus the two opposite orientations below give the same RDC value:

15N--1H 1H--15N

This ambiguity makes calculating a structure de novo (i.e. from a random starting model) using only residual dipolar couplings very computationally difficult. If there is a reasonable starting model, however, this is not a problem. Thus residual dipolar couplings are especially good for refining models/low resolution structures.

Refining initial models with RDCs