Hyper-Stable β-Sheets ‚ Amphipathic Peptides Made Easy
Analyzing folding of micro domains is useful for increasing knowledge of folding patterns for entire proteins. Toward these efforts, β-hairpin motifs, which incorporate two β-strands oriented in an antiparallel direction have proven useful. However, β-hairpins have historically required stabilizing features to maintain a highly populated folded state. This limitation has demanded optimized β-strands incorporating cross strand Trp/Trp pairs and/or a tight nucleating turn. Disulfide bonding can also join antiparallel β-strands, but is only energetically favorable if the cysteine residues are placed at non-hydrogen bonded (NHB) cross-strand positions.
Recently, Dr. Brandon Kier and co-workers published a new finding in the Journal of the American Chemical Society related to the ability of disulfide bonding to form hyperstable β-sheets without inclusion of tertiary interactions and turns. They synthesized a variety of natively unfolded peptides which formed double stranded sheets upon oxidative dimerization. In one example a seven residue peptide (KWRCIWD)2 was synthesized and dimerized with a centrally located cystine. Remarkably, this peptide, with only seven natural amino acids, was essentially 100% folded at 280 K with a melting point of 70 °C. Their research showed that a single disulfide can impart greater stability for a β-sheet than the best known hairpin-nucleating turn sequences.
Further insights into disulfide bonding allowed Dr. Kier and colleagues to tune the location and number of cystines for various effects. It was found that paired disulfides were even better β-sheet nucleators, and that heterodimers and diagonally linked sheets could be selectively formed. More recently, Dr. Kier has been able to develop very complex turnless β-sheets. This includes the rapid self-assembly of a triangle structure upon oxidation of an 11 residue N-terminal-dimerized strand. The folded triangle was found to have significantly different properties than the unfolded dimer.
Highly charged amphipathic β-sheets can be constructed due to the ability of disulfide bonding to overcome repulsion of cross-stranded charged arginine and lysine residues providing an easier route to amphipathicity. This route toward amphipathicity has many useful applications related to catalysis, metal binding, and selective detergents. Antimicrobial peptides function as amphipiles containing a high charge and lipophilic faces allowing disruption of bacterial membranes. Therefore, this technology has the potential to significantly aid in the synthesis of many potential peptides of interest.
Dr. Kier began using the Liberty Blue Peptide Synthesizer in 2014. He indicated a key benefit of the system is its ability to synthesize peptides quickly which has allowed many different users to make peptides on the system in a single day. Additionally, the use of higher temperature couplings at 90°C has been helpful for making their difficult sequences quickly. Dr. Kier commented, “The Liberty Blue has been very useful for our research and I would highly recommend the system for making lots of peptides quickly.”
Dr. Brandon Kier’s research is published:
Disulfide-Mediated β-Strand Dimers: Hyperstable β-sheets Lacking Tertiary Interactions and Turns
JACS, 2015, 137, 5363–5371