Chemical Shifts of Artificial Monomers Used to Construct Heterogeneous-Backbone Protein Mimetics in Random Coil and Folded States.

The construction of protein-sized synthetic chains that blend natural amino acids with artificial monomers to create so-called heterogeneous-backbones is a powerful approach to generate complex folds and functions from bio-inspired agents. A variety of techniques from structural biology commonly used to study natural proteins have been adapted to investigate folding in these entities. In NMR characterization of proteins, proton chemical shift is a straightforward to acquire, information-rich metric that bears directly on a variety of properties related to folding. Leveraging chemical shift to gain insight into folding requires a set of reference chemical shift values corresponding to each building block type (i.e., the 20 canonical amino acids in the case of natural proteins) in a random coil state and knowledge of systematic changes in chemical shift associated with particular folded conformations. Although well documented for natural proteins, these issues remain unexplored in the context of protein mimetics. Here, we report random coil chemical shift values for a library of artificial amino acid monomers frequently used to construct heterogeneous-backbone protein analogues as well as a spectroscopic signature associated with one monomer class, ß3-residues bearing proteinogenic side chains, adopting a helical folded conformation. Collectively, these results will facilitate the continued utilization of NMR for the study of structure and dynamics in protein-like artificial backbones.

Protein Backbone Alteration in Non-Hairpin ß-Turns: Impacts on Tertiary Folded Structure and Folded Stability.

The importance of ß-turns to protein folding has motivated extensive efforts to stabilize the motif with non-canonical backbone connectivity. Prior work has focused almost exclusively on turns between strands in a ß-sheet (i. e., hairpins). Turns in other structural contexts are also common in nature and have distinct conformational preferences; however, design principles for their mimicry remain poorly understood. Here, we report strategies that stabilize non-hairpin ß-turns through systematic evaluation of the impacts of backbone alteration on the high-resolution folded structure and folded stability of a helix-loop-helix prototype protein. Several well-established hairpin turn mimetics are shown detrimental to folded stability and/or hydrophobic core packing, while less-explored modification schemes that reinforce alternate turn types lead to improved stability and more faithful structural mimicry. Collectively, these results have implications in control over protein folding through chemical modification as well as the design of protein mimetics.

Heterogeneous-Backbone Proteomimetic Analogues of Lasiocepsin, a Disulfide-Rich Antimicrobial Peptide with a Compact Tertiary Fold.

The emergence of resistance to clinically used antibiotics by bacteria presents a significant problem in public health. Natural antimicrobial peptides (AMPs) are a valuable source of antibiotics that act by a mechanism less prone to the evolutionary development of resistance. In an effort to realize the promise of AMPs while overcoming limitations such as poor biostability, researchers have sought sequence-defined oligomers with artificial amide-based backbones that show membrane-disrupting functions similar to natural agents. Most of this precedent has focused on short peptidomimetic analogues of unstructured chains or secondary folds; however, the natural antimicrobial arsenal includes a number of small- and medium-sized proteins that act via an ordered tertiary structure. Generating proteomimetic analogues of these scaffolds poses a challenge due to the increased complexity of the target for mimicry. Here, we report the development of heterogeneous-backbone variants of lasiocepsin, a 27-residue disulfide-rich AMP found in bee venom that adopts a compact tertiary fold. Iterative cycles of design, synthesis, and biological evaluation yielded analogues of the natural domain with ∼30 to 40% artificial backbone content, comparable antibacterial activity, reduced host cell toxicity, and improved stability to proteolytic degradation. High-resolution structures determined for several variants by NMR provide insights into the interplay among backbone composition, tertiary fold, and biological properties. Collectively, the results reported here broaden the scope of protein functional mimicry by artificial backbone analogues of tertiary folding patterns and suggest protein backbone engineering as a means to tune protein function by exerting site-specific control over protein folded structure.