Protein Backbone Alteration in Non‐Hairpin β‐Turns: Impacts on Tertiary Folded Structure and Folded Stability

The importance of β‐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 β‐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 β‐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. Backbone modification in β‐turns is vital in the development of protein mimetics. Prior work has focused on the subset of turns that join strands of a β‐sheet. Here we report the evaluation of complementary strategies that stabilize β‐turns found in other structural contexts. Optimal artificial residue placement in a helix‐loop‐helix domain yields variants with folded structure identical to the prototype and improved thermal stability (Tm).