A Hendecad Motif Is Preferred for Heterochiral Coiled-Coil Formation.

The structural principles that govern interactions between l- and d-peptides are not well understood. Among natural proteins, coiled-coil assemblies formed between or among α-helices are the most regular feature of tertiary and quaternary structures. We recently reported the first high-resolution structures for heterochiral coiled-coil dimers, which represent a starting point for understanding associations of l- and d-polypeptides. These structures were an unexpected outcome from crystallization of a racemic peptide corresponding to the transmembrane domain of the influenza A M2 protein (M2-TM). The reported structures raised the possibility that heterochiral coiled-coil dimers prefer an 11-residue (hendecad) sequence repeat, in contrast to the 7-residue (heptad) sequence repeat that is dominant among natural coiled coils. To gain insight on sequence repeat preferences of heterochiral coiled-coils, we have examined three M2-TM variants containing substitutions intended to minimize steric clashes between side chains at the coiled-coil interface. In each of the three new crystal structures, we observed heterochiral coiled-coil associations that closely match a hendecad sequence motif, which strengthens the conclusion that this motif is intrinsic to the pairing of α-helices with opposite handedness. In each case, the presence of a hendecad motif was established by comparing the observed helical frequency to that of an ideal hendecad. This comparison revealed that decreasing the size of the amino acid side chain at positions that project toward the superhelical axis produces tighter packing, as determined by the size of the coiled-coil radius. These results provide a basis for future design of heterochiral coiled-coil pairings.

Dodecyl-ß-melibioside Detergent Micelles as a Medium for Membrane Proteins.

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-ß-melibioside (ß-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the ß-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-ß-maltoside (ß-DDM). ß-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ß-DDMB yielded activities that were 40% higher than those of DAGK purified into ß-DDM. ß-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ß-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

High-resolution structures of a heterochiral coiled coil.

Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

CHOBIMALT: a cholesterol-based detergent.

Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3−4 µM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1.

Bolaamphiphile-class surfactants can stabilize and support the function of solubilized integral membrane proteins.

Bolaamphiphile-class surfactants composed of two hydrophilic (maltoside) headgroups connected by long saturated alkyl chains were tested for their ability to stabilize a solubilized membrane protein, Escherichia coli diacylglycerol kinase (DAGK), and to sustain its native function. Members of this "Bis-MALT-C(18-28)" series were poor solubilizers of DAGK in the absence of conventional detergent. However, mixed micelles of the bolaamphiphiles with either dodecylphosphocholine or beta-n-decyl maltoside were more effective and enhanced DAGK's thermal stability relative to corresponding detergent-only conditions. Moreover, certain bolaamphiphiles were seen to be lipidlike by providing partial activation of DAGK's catalytic activity. Finally, addition of bolaamphiphiles to micellar NMR samples of DAGK did not result in a degradation of spectral quality, indicating their compatibility with high-resolution structural studies. To the best of our knowledge, this work represents the first documentation of the potential of bolaamphiphile-class surfactants for use in biochemical and biophysical studies of MPs.