The non-covalent forces that stabilise protein structures are not fully understood. One way to address this is to study equilibria between unfolded states and α-helices in peptides. For these, electrostatic forces are believed to contribute, including interactions between: side chains; the backbone and side chains; and side chains and the helix macrodipole. Here we probe these experimentally using designed peptides. We find that both terminal backbone-side chain and certain side chain-side chain interactions (i.e., local effects between proximal charges, or interatomic contacts) contribute much more to helix stability than side chain-helix macrodipole electrostatics, which are believed to operate at larger distances. This has implications for current descriptions of helix stability, understanding protein folding, and the refinement of force fields for biomolecular modelling and simulations. In addition, it sheds light on the stability of rod-like structures formed by single α-helices that are common in natural proteins including non-muscle myosins.Folded proteins are stabilised by many weak intramolecular forces between thousands of atoms, including: hydrogen bonds, van der Waals' contacts, and Coulombic interactions between formal charges (i.e., salt bridges). 1 Because of the interdependence of non-covalent interactions, and the resulting cooperativity of protein folding, it is extremely difficult to disentangle contributions from these various components. For example, in terms of backbone interactions alone, CO i ➔NH i+4 hydrogen bonds are regarded as the major factor stabilising α-helices. 2 Whilst this remains unchallenged, recent studies have identified additional contributions from CO i ➔CO i+1 n➔π* interactions, 3 which can "compete" with * To whom correspondence should be addressed: D.N.Woolfson@bristol.ac.uk. Author contributions EGB, CFJF and DNW designed the research; EGB made the synthetic peptides and performed the CD spectroscopy; MPC and EGB collected and analysed the NMR data; GJB, EGB and DNW performed the bioinformatics; EGB and RBS constructed and analysed atomistic models for the peptides; NL performed and analysed the helix-dipole calculations; EGB, GJB and DNW wrote the paper. All authors reviewed and contributed to the manuscript.The authors declare no competing financial interests.Dedication: DNW would like to dedicate this paper to the memory of Prof. Dudley H. William FRS, an inspirational scientist, mentor and person. Europe PMC Funders GroupAuthor Manuscript Nat Chem Biol. Author manuscript; available in PMC 2015 December 03. Published in final edited form as:Nat Chem Biol. 2015 March ; 11(3): 221-228. doi:10.1038/nchembio.1739. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts traditional hydrogen bonding. 4 Thus, it is important to develop continually, and to challenge our understanding of the fundamental forces underpinning protein folding, and of biomolecular interactions in water more generally. Improved understanding will lead to better modellin...

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.

The design and study of miniproteins, that is, polypeptide chains <40 amino acids in length that adopt defined and stable 3D structures, is resurgent. Miniproteins offer possibilities for reducing the complexity of larger proteins and so present new routes to studying sequence-to-structure and sequence-to-stability relationships in proteins generally. They also provide modules for protein design by pieces and, with this, prospects for building more-complex or even entirely new protein structures. In addition, miniproteins are useful scaffolds for templating functional domains, for example, those involved in protein-protein interactions, catalysis, and biomolecular binding, leading to potential applications in biotechnology and medicine. Here we select examples from almost four decades of miniprotein design, development, and dissection. Simply because of the word limit for this Account, we focus on miniproteins that are cooperatively folded monomers in solution and not stabilized by cross-linking or metal binding. In these cases, the optimization of noncovalent interactions is even more critical for the maintenance of the folded states than in larger proteins. Our chronology and catalogue highlights themes in miniproteins, which we explore further and begin to put on a firmer footing through an analysis of the miniprotein structures that have been deposited in the Protein Data Bank (PDB) thus far. Specifically, and compared with larger proteins, miniproteins generally have a lower proportion of residues in regular secondary structure elements (α helices, β strands, and polyproline-II helices) and, concomitantly, more residues in well-structured loops. This allows distortions of the backbone enabling mini-hydrophobic cores to be made. This also contrasts with larger proteins, which can achieve hydrophobic cores through tertiary contacts between distant regions of sequence. On average, miniproteins have a higher proportion of aromatic residues than larger proteins, and specifically electron-rich Trp and Tyr, which are often found in combination with Pro and Arg to render networks of CH-π or cation-π interactions. Miniproteins also have a higher proportion of the long-chain charged amino acids (Arg, Glu, and Lys), which presumably reflects salt-bridge formation and their greater surface area-to-volume ratio. Together, these amino-acid preferences appear to support greater densities of noncovalent interactions in miniproteins compared with larger proteins. We anticipate that with recent developments such as parametric protein design, it will become increasingly routine to use computation to generate and evaluate models for miniproteins in silico ahead of experimental studies. This could include accessing new structures comprising secondary structure elements linked in previously unseen configurations. The improved understanding of the noncovalent interactions that stabilize the folded states of such miniproteins that we are witnessing through both in-depth bioinformatics analyses and experimental testing will feed the...

Miniproteins simplify the protein-folding problem, allowing the dissection of forces that stabilize protein structures. Here we describe PPα-Tyr, a designed peptide comprising an α-helix buttressed by a polyproline II helix. PPα-Tyr is water soluble and monomeric, and it unfolds cooperatively with a midpoint unfolding temperature (T) of 39 °C. NMR structures of PPα-Tyr reveal proline residues docked between tyrosine side chains, as designed. The stability of PPα is sensitive to modifications in the aromatic residues: replacing tyrosine with phenylalanine, i.e., changing three solvent-exposed hydroxyl groups to protons, reduces the T to 20 °C. We attribute this result to the loss of CH-π interactions between the aromatic and proline rings, which we probe by substituting the aromatic residues with nonproteinogenic side chains. In analyses of natural protein structures, we find a preference for proline-tyrosine interactions over other proline-containing pairs, and observe abundant CH-π interactions in biologically important complexes between proline-rich ligands and SH3 and similar domains.

In nature, co-assembly of polypeptides, nucleic acids, and polysaccharides is used to create functional supramolecular structures. Here, we show that DNA nanostructures can be used to template interactions between peptides and to enable the quantification of multivalent interactions that would otherwise not be observable. Our functional building blocks are peptide–oligonucleotide conjugates comprising de novo designed dimeric coiled-coil peptides covalently linked to oligonucleotide tags. These conjugates are incorporated in megadalton DNA origami nanostructures and direct nanostructure association through peptide–peptide interactions. Free and bound nanostructures can be counted directly from electron micrographs, allowing estimation of the dissociation constants of the peptides linking them. Results for a single peptide–peptide interaction are consistent with the measured solution-phase free energy; DNA nanostructures displaying multiple peptides allow the effects of polyvalency to be probed. This use of DNA nanostructures as identifiers allows the binding strengths of homo- and heterodimeric peptide combinations to be measured in a single experiment and gives access to dissociation constants that are too low to be quantified by conventional techniques. The work also demonstrates that hybrid biomolecules can be programmed to achieve spatial organization of complex synthetic biomolecular assemblies.