Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Marcin Wolny,Michelle Peckham,Peter J Knight,Matthew Batchelor,Marta Kurzawa,Emanuele Paci,Emily G Baker,Gail J Bartlett,Lorna Dougan,Derek N Woolfson

doi:10.1038/srep44341

Marcin Wolny, Michelle Peckham + Show 8 more

Open Access

https://doi.org/10.1038/srep44341

Copy DOI

Abstract

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu–Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized.

Highlights

Pairs are more helix-stabilizing than the reversed E →K(+4) orientation[17], resolving previous conflicting reports[16,18,19]
To test the relative contributions of Lys and Arg to SAHs, we designed de novo polypeptides that contained either E–K or E–R pairs termed EK3 and ER3 (Fig. 1), both of which were expected to behave as SAHs
To match the 3.6 residues per turn of the α-helix as closely as possible, the de novo polypeptides were based on the 7-residue repeats, AEEEKKK and AEEERRR, respectively (Fig. 1a)

Summary

Introduction

Pairs are more helix-stabilizing than the reversed E →K(+4) orientation[17], resolving previous conflicting reports[16,18,19]. To determine how Lys and Arg contribute to the stability of long SAHs, we first designed, expressed and characterized de novo SAHs. To determine how Lys and Arg contribute to the stability of long SAHs, we first designed, expressed and characterized de novo SAHs These long (98-residue) polypeptides contain 7-residue repeats, AEEEXXX, where X is K or R, and were designed to emulate the properties of SAHs using simpler constructs. Their length was chosen to be similar to that of many natural SAHs2 as a better test of the properties of a SAH compared to short peptides. Our findings provide clear design rules for generating or re-engineering these domains

Methods

Results

Conclusion