Structure determination of integral membrane proteins by solution NMR represents one

Structure determination of integral membrane proteins by solution NMR represents one of the most important difficulties of structural biology. for application as weak-alignment media in answer NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400nm-long six-helix bundles each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment towards collection of linearly impartial restraints can be launched by reducing the unfavorable charge of DNA nanotubes counter ions Tamoxifen Citrate and small DNA binding molecules. This detergent-resistant liquid-crystal media offers a number of properties conducive for membrane protein alignment including high-yield production thermal stability buffer compatibility and structural programmability. Tamoxifen Citrate Production of sufficient nanotubes for 4-5 NMR experiments can be completed in one week by a single individual. and typically known in advance (e.g. bond length for Tamoxifen Citrate covalently linked nuclei) and to the angle between the vector connecting the interacting nuclei and the static magnetic field by the relation <3cos2Θ?1> where the brackets indicate time-averaged sampling. These couplings can be a useful source of angular structural data for NMR studies of macromolecules. This is because direct information around the orientations of the corresponding bond vectors relative to the protein’s steric alignment vector is provided. However molecular tumbling averages these interactions to zero in standard isotropic solutions24. It has been shown that RDCs can be measured by utilizing some type of anisotropic media to allow for partial alignment and therefore non-vanishing dipole-dipole interactions22. Such incomplete directional averaging of macromolecules in liquid crystalline media would allow routine measurement of residual dipolar couplings while retaining conditions essential for high-resolution solution-state NMR (i.e. quick tumbling). A highly effective EIF4EBP1 method to induce weak-alignment of proteins is through mixing them with a dilute liquid-crystalline medium such that the conversation between the protein and the medium is poor and highly transient (< ns lifetime). A number of liquid-crystal alignment media have been developed to measure accurate RDCs including filamentous phage25 DMPC/DHPC bicelles22 C12E5 polyethylene glycol26 ternary mixtures of cetylpyridinium Cl/Br hexanol and sodium Cl/Br26 27 cellulose crystallites28 and a highly hydrated anisotropically compressed polyacrylamide gel29. Except the polyacrylamide gel in some cases30-33 and the fd bacteriophage in one case34 most of these media Tamoxifen Citrate have been demonstrated to be incompatible with the detergents and lipids needed to solubilize membrane proteins. With the compressed gel it has been generally hard to soak membrane proteins to higher than 0.1-0.2 mM because of the inhomogeneous pore size of randomly cross-linked gel matrices limiting accuracy and signal-to-noise for NMR measurements. Tamoxifen Citrate Because of this we have by no means succeeded Tamoxifen Citrate to measure accurate RDCs for any single-chain protein longer than 150 residues; thus far our only success has been with homomultimeric proteins31. In addition for large systems the polyacrylamide gel may not be practical due to strong interactions with the acrylamide mesh which reduce the molecular tumbling rate. This can be tuned by the gel concentration but acrylamide gels can't be used at lower concentration than about 4 % due to mechanical instability. More recently two new detergent-compatible liquid crystals have been reported one based on collagen35 and the other based on nucleic-acid G-tetrad structures36. For these two new media the reduction in molecular tumbling rates for large systems is much less problematic and they are easy to produce and non-expensive. However both methods still have issues with signal-to-noise and/or general detergent and buffer compatibility. The alignment induced by collagen gels is quite small when compared to other alignment media. The d(GpG)-based G-tetrad stacks require an excess of potassium which enables effective stacking of pyrene moieties around the uncovered guanine tetrads but some specific detergents can be incompatible with the presence of potassium. Thus measuring RDCs for membrane proteins has remained a difficult challenge. DNA nanotubes and NMR of Integral Membrane Proteins Inspired by the architecture of.