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Review
. 2003 Jan;12(1):1-16.
doi: 10.1110/ps.0233303.

Weak alignment offers new NMR opportunities to study protein structure and dynamics

Ad Bax  1
Affiliations
Review

Weak alignment offers new NMR opportunities to study protein structure and dynamics

Ad Bax. Protein Sci. 2003 Jan.

Abstract

Protein solution nuclear magnetic resonance (NMR) can be conducted in a slightly anisotropic environment, where the orientational distribution of the proteins is no longer random. In such an environment, the large one-bond internuclear dipolar interactions no longer average to zero and report on the average orientation of the corresponding vectors relative to the magnetic field. The desired very weak ordering, on the order of 10(-3), can be induced conveniently by the use of aqueous nematic liquid crystalline suspensions or by anisotropically compressed hydrogels. The resulting residual dipolar interactions are scaled down by three orders of magnitude relative to their static values, but nevertheless can be measured at high accuracy. They are very precise reporters on the average orientation of bonds relative to the molecular alignment frame, and they can be used in a variety of ways to enrich our understanding of protein structure and function. Applications to date have focused primarily on validation of structures, determined by NMR, X-ray crystallography, or homology modeling, and on refinement of structures determined by conventional NMR approaches. Although de novo structure determination on the basis of dipolar couplings suffers from a severe multiple minimum problem, related to the degeneracy of dipolar coupling relative to inversion of the internuclear vector, a number of approaches can address this problem and potentially can accelerate the NMR structure determination process considerably. In favorable cases, where large numbers of dipolar couplings can be measured, inconsistency between measured values can report on internal motions.

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Figures

Figure 1.
Figure 1.
Magnetic dipole-dipole coupling, illustrated for a 15N-1H spin pair. 15N and 1H magnetic moments are aligned parallel (or antiparallel) to the static magnetic field, Bo. The total magnetic field in the Bo direction at the 15N position can increase or decrease relative to Bo, depending on the orientation of the 15N-1H vector and the spin state of the proton (parallel or antiparallel to Bo).
Figure 2.
Figure 2.
Bands of allowed vector orientations, corresponding to a given value of a dipolar coupling, in the frame of the diagonalized alignment tensor. The width of the bands corresponds to a typical measurement uncertainty that is 3% of the total range of the couplings. The bottom cone corresponds to the inverse vector orientations relative to the top cone. Cones are distorted due to the nonzero rhombicity, R, in Equation 2B. Polar angles θ and φ are defined for the vector shown.
Figure 3.
Figure 3.
Morphology of liquid crystalline bicelles. (A) Cross-section orthogonal to the NMR sample cell shows domains of parallel lipid bilayers. Domain size is approximately 10 μm. (B) Section of an individual, highly porous bilayer. The total surface area of the pores is comparable to that of the lipid bilayers. (C) Expansion of a pore, with the rim lined with detergent (DHPC), and the planar surface consisting of DMPC.
Figure 4.
Figure 4.
Small regions of the 600 MHz 15N-1H correlation spectra of ubiquitin, recorded in the absence of 1H decoupling in the 15N dimension, at three different levels of molecular alignment. (A) Isotropic spectrum, with the marked splitting corresponding to 1JNH. (B) Spectrum recorded in 4.5% (w/v) bicelles, consisting of a 30:10:1 molar ratio of DMPC, DHPC, and cetyl-trimethyl ammonium bromide (CTAB). (C) Spectrum recorded in 8% (w/v) bicelles. Marked splittings in panels B and C correspond to the sum of the 1JNH and dipolar coupling. The broadening in the 1H dimension, observed in panels B and C relative to A is caused by 1H-1H dipolar couplings.
Figure 5.
Figure 5.
Bands of allowed vector orientations for the Gln40 backbone amide 15N-1H bond vector in ubiquitin, in the frame of the crystal structure (1UBQ). Band A corresponds to the orientations compatible with the experimental dipolar coupling value measured in neutral bicelles. Band B corresponds to allowed orientations in bicelles charged with CTAB. The solid dot marks the orientation of the N-H vector when the proton is model-built into the crystal structure, assuming that HN is located on the line bisecting the C′-N-Cα angle. (Reprinted, with permission, from Ramirez and Bax 1998.)
Figure 6.
Figure 6.
Anisotropic environment created by strained gels. (A) 3-mm gel, contained in a 4.5-mm inner diameter Shigemi sample cell, prior to compression by the plunger. (B) After compression by the plunger. (C) Device for achieving radial compression, resulting in axial stretching of the gel.
Figure 6.
Figure 6.
Anisotropic environment created by strained gels. (A) 3-mm gel, contained in a 4.5-mm inner diameter Shigemi sample cell, prior to compression by the plunger. (B) After compression by the plunger. (C) Device for achieving radial compression, resulting in axial stretching of the gel.
Figure 7.
Figure 7.
Structures of gp41[282–304] in (A) q = 0.25 bicelles and (B) DHPC micelles, determined from dipolar couplings. Sidechain χ1 angles are derived from 3JNCγ and 3JC′Cγ couplings. Hydrophobic and hydrophilic sidechains are shown in yellow and in aqua, respectively. (Reprinted, with permission, from Chou et al. 2002.)
Figure 8.
Figure 8.
Correlation of experimental dipolar couplings, measured for ubiquitin in 5% (w/v) bicelles, and values predicted by the crystal and solution structures, after best fitting the alignment tensor to the 13Cα-1Hα couplings by singular value decomposition (Losonczi et al. 1999). (A) Plot of the 13Cα-1Hα couplings vs. values predicted by the X-ray structure (Vijay-Kumar et al. 1987). (B) The experimental sidechain dipolar couplings vs. the X-ray structure. (C) The experimental sidechain dipolar couplings vs. the lowest-energy NMR structure, calculated in the absence of sidechain dipolar couplings. (D) The sidechain dipolar couplings vs. the average sidechain dipolar couplings predicted for an ensemble of 50 NMR structures. Q factors for these correlations are (A) 25%, (B) 73%, (C) 67%, and (D) 47%.
Figure 9.
Figure 9.
Correlations between experimental 1DNH values and values calculated from the shape-predicted alignment tensor of the third immunoglobulin binding domain of protein G in (A) 5% w/v bicelle medium, and (B) 28 mg/mL fd medium. Dashed lines correspond to y = x. The poor correlation in panel B indicates that in phage medium the protein alignment is dominated by electrostatic interactions, which are ignored in the alignment tensor prediction, and not by steric interaction. (Adapted with permission from Zweckstetter and Bax 2000.)
Figure 10.
Figure 10.
Flow diagram of the approach used to refine a starting model and bring it in agreement with experimental dipolar couplings. The simulated annealing process is carried out at very low temperature (<200 K) in order to prevent backbone angles from jumping to false minima. Typically, two or three rounds of the annealing process suffice to obtain agreement with the dipolar couplings. (For a detailed description of the protocol, see Chou et al. 2000.)
Figure 11.
Figure 11.
Protein backbone fragment, with dipolar interactions that can readily be measured marked by dashed lines. Although 1DNCα can be measured experimentally, its relative accuracy tends to be lower, and for transpeptide bonds its normalized value is very similar to that of 1DC′Cα of the preceding residue. 1DCαCβ is most useful in perdeuterated proteins, where no value for 1DCαHα can be measured, and where the slow transverse relaxation of the deuterated 13Cα permits its accurate measurement.
Figure 12.
Figure 12.
Backbone ribbon diagrams of the Ca2+-CaM solution structure, shown in red, and the 1-Å crystal structure (1EXR), in blue. (A) For the N-terminal domain, the superposition is optimized for residues 29–54 (helices II and III), revealing the large difference in the orientation of helices I (27°) and IV (21°). (B) For the C-terminal domain, residues 102–127 (helices VI and VII) are superimposed, showing much smaller orientation differences of 15° and 6° for helices V and VIII, respectively. (Reprinted, with permission, from Chou et al. 2001.)
Figure 13.
Figure 13.
Assembly of the backbone of a protein by the molecular fragment replacement (MFR) approach, using a seven-residue fragment length. For each set of seven contiguous residues, the entire PDB is searched for fragments that are compatible with the experimental dipolar couplings and chemical shifts. The 20 best-fitting fragments are minimized in a short simulated annealing protocol, and if convergence is obtained, the cluster of lowest energy structures is averaged and subsequently minimized. If only a single liquid crystalline medium is used, the orientation of each fragment relative to the alignment tensor frame is fourfold degenerate and best fitting to the preceding, partially overlapping fragment is needed to resolve this ambiguity. If dipolar couplings have been measured in two or more media, the absolute orientation of the fragment is known. Assembly is illustrated for the backbone of the RecA-inactivating protein DinI (Voloshin et al. 2001).
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