Modulation of the Alignment Tensor of Macromolecules Dissolved in a Dilute Liquid Crystalline Medium

Abstract

Dipolar couplings measured for pairs of nuclei constrain the possible orientations of internuclear vectors relative to the molecule’s alignment tensor.1-3 For macromolecules such as proteins and nucleic acids, the required small degree of alignment with the magnetic field can result from the molecules’ own magnetic susceptibility anisotropy2-4 or can be induced by the use of a liquid crystalline medium.1 Provided the alignment is sufficiently weak, only directly bonded pairs of atoms give rise to a measurable dipolar coupling, which manifests itself as a change in the corresponding scalar interaction. The spectral simplicity of the high-resolution NMR spectrum is therefore retained, and measurement of dipolar couplings is straightforward. The most commonly used liquid crystalline medium for inducing macromolecular alignment consists of large, disk-shaped phospholipid particles, often referred to as bicelles.5,6 The volume fraction of phospholipid particles can be adjusted to obtain the desired degree of alignment.7 For the case where the diagonalized alignment tensor is axially symmetric, and the internuclear distance is accurately known, each dipolar coupling restrains the position of the corresponding internuclear vector to a cone about the unique axis of the alignment tensor. For the general case, where the alignment tensor is asymmetric, the cone is distorted and “taco-shaped”.8 Because the direction of a second rank tensor interaction cannot be distinguished from its inverse, the dipolar coupling actually defines two cones of possible bond vector orientations, in opposing directions. Although such information is clearly very useful in determining macromolecular structures more accurately,4,6 the continuum of possible orientations makes it difficult to determine local geometry in the absence of other information. Here we demonstrate that the degeneracy can be lifted by recording a second set of dipolar couplings under conditions where the orientation and/or rhombicity of the alignment tensor is altered. Three different approaches are shown to be successful: (1) addition of an unstructured, so-called His-tag peptide at the C-terminus of the protein, (2) changing the sample pH, and (3) changing the net charge of the bicelles. For recombinant proteins, it is frequently possible to add a short unstructured peptide at either the Nor C-terminus. Although such an additional peptide fragment does not alter the structure of the globular domain, it adds a flexible “tail” to the protein which affects the molecule’s alignment tensor. For example, a so-called His-tag sequence frequently is added to a heterologously expressed protein to facilitate its purification. Histidine residues have a pK of ∼7, and their net charge can be altered by lowering the pH from 7.5 to 5. If there is a small amount of net charge on the surface of the bicelles, this will change the very weak electrostatic attraction/repulsion between the protein and the bicelle, thereby altering the alignment tensor. Finally, we demonstrate that deliberately adding a net charge to the bicelles, recently shown to increase the stability of the liquid crystalline phase at low volume fractions,9 also can be used to alter the alignment tensor, in either the presence or absence of the Histag sequence. Experiments are carried out for samples of uniformly 13C/15Nenriched ubiquitin, either with an additional SHHHHHH peptide at its C-terminus (Martek Biosciences, Columbia, MD) or without (VLI Research, Southeastern, PA). Samples were ∼0.7 mM, in 93% H2O, 7% D2O, containing 4-5% (w/v) bicelles (consisting of a 3:1 molar ratio of DMPC to DHPC).5,6 As a second test, measurements were conducted for a sample of uniformly 15Nlabeled aprotinin (often referred to as BPTI) (0.2 mM in 4-5% (w/v) bicelles). The dipolar coupling between two nuclei, A and B, in a solute macromolecule of fixed shape is related to the traceless alignment tensor according to