Coiled coils are one of most common protein quaternary structures and represent the best understood relationship between amino acid sequence and protein conformation. Whereas the roles of residues at the canonical heptad positions the a, d, e, and g are understood in precise detail, conventional approaches often assume that the solvent-exposed b-, c-, and f-positions can be varied broadly for application-specific purposes with minimal consequences. However, a growing body of evidence suggests that interactions among these b, c, and f residues can contribute substantially to coiled-coil conformational stability. In the trimeric coiled coil described here, we find that b-position Glu10 engages in a stabilizing long-range synergistic interaction with c-position Lys18 (ΔΔΔGf = -0.65 ± 0.02 kcal/mol). This favorable interaction depends strongly on the presence of two nearby f-position residues: Lys 7 and Tyr14. Extensive mutational analysis of these residues in the presence of added salt vs. denaturant suggests that this long-range synergistic interaction is primarily electrostatic in origin, but also depends on the precise location and acidity of a side-chain hydrogen-bond donor within f-position Tyr14.

Sunflower trypsin inhibitor-1 (SFTI-1) structure is used for designing grafted peptides as a possible therapeutic agent. The grafted peptide exhibits multiple conformations in solution due to the presence of proline in the structure of the peptide. To lock the grafted peptide into a major conformation in solution, a dibenzofuran moiety (DBF) was incorporated in the peptide backbone structure, replacing the Pro-Pro sequence. NMR studies indicated a major conformation of the grafted peptide in solution. Detailed structural studies suggested that SFTI-DBF adopts a twisted beta-strand structure in solution. The surface plasmon resonance analysis showed that SFTI-DBF binds to CD58 protein. A model for the protein-SFTI-DBF complex was proposed based on docking studies. These studies suggested that SFTI-1 grafted peptide can be used to design stable peptides for therapeutic purposes by grafting organic functional groups and amino acids. However, when a similar strategy was used with another grafted peptide, the resulting peptide did not produce a single major conformation, and its biological activity was lost. Thus, conformational constraints depend on the sequence of amino acids used for SFTI-1 grafting.

Simple and efficient total synthesis of homogeneous and chemically modified protein samples remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy for facile preparation of proteins. In this strategy, proteins are split into ~100-residue blocks, and each block is assembled on solid support from synthetically accessible peptide fragments before ligated together into full-length protein in solution. With the new method, we increase the yield of CENP-A synthesis by 2.5-fold compared to the previous hybrid phase ligation approach. We further extend the new strategy to the total chemical synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight peptide segments with only one single purification. We demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

Prion protein misfolding is associated with fatal neurodegenerative disorders such as kuru, Creutzfeldt-Jakob disease, and several animal encephalopathies. While the C-terminal 106-126 peptide has been well studied for its role in prion replication and toxicity, the octapeptide repeat (OPR) sequence found within the N-terminal domain has been relatively under explored. Recent findings that the OPR has both local and long-range effects on prion protein folding and assembly, as well as its ability to bind and regulate transition metal homeostasis, highlights the important role this understudied region may have in prion pathologies. This review attempts to collate this knowledge to advance a deeper understanding on the varied physiologic and pathologic roles the prion OPR plays, and connect these findings to potential therapeutic modalities focused on OPR-metal binding. Continued study of the OPR will not only elucidate a more complete mechanistic model of prion pathology, but may enhance knowledge on other neurodegenerative processes underlying Alzheimer's, Parkinson's, and Huntington's diseases.

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, β3-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.

Since its first detection in 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) has been the cause of millions of deaths worldwide. Despite the development and administration of different vaccines, the situation is still worrisome as the virus is constantly mutating to produce newer variants some of which are highly infectious. This raises an urgent requirement to understand the infection mechanism and thereby design therapeutic‐based treatment for COVID‐19. The gateway of the virus to the host cell is mediated by the binding of the receptor binding domain (RBD) of the virus spike protein to the angiotensin‐converting enzyme 2 (ACE2) of the human cell. Therefore, the RBD of SARS‐CoV‐2 can be used as a target to design therapeutics. The α1 helix of ACE2, which forms direct contact with the RBD surface, has been used as a template in the current study to design stapled peptide therapeutics. Using computer simulation, the mechanism and thermodynamics of the binding of six stapled peptides with RBD have been estimated. Among these, the one with two lactam stapling agents has shown binding affinity, sufficient to overcome RBD‐ACE2 binding. Analyses of the mechanistic detail reveal that a reorganization of amino acids at the RBD‐ACE2 interface produces favorable enthalpy of binding whereas conformational restriction of the free peptide reduces the loss in entropy to result higher binding affinity. The understanding of the relation of the nature of the stapling agent with their binding affinity opens up the avenue to explore stapled peptides as therapeutic against SARS‐CoV‐2.

HYD1 is an all D‐amino acid linear 10‐mer peptide that was discovered by one‐bead‐one‐compound screening. HYD1 has five hydrophobic amino acids flanked by polar amino acids. Alanine scanning studies showed that alternating hydrophobic amino acid residues and N‐ and C‐terminal lysine side chains were contributors to the biological activity of the linear 10‐mer analogs. This observation led us to hypothesize that display of the hydrophobic pentapeptide sequence of HYD1 in a cyclic beta‐hairpin‐like scaffold could lead to better bioavailability and biological activity. An amphipathic pentapeptide sequence was used to form an antiparallel strand and those strands were linked via dipeptide‐like sequences selected to promote β‐turns. Early cyclic analogs were more active but otherwise mimicked the biological activity of the linear HYD1 peptide. The cyclic peptidomimetics were synthesized using standard Fmoc solid phase synthesis to form linear peptides, followed by solution phase or on‐resin cyclization. SAR studies were carried out with an aim to increase the potency of these drug candidates for the killing of multiple myeloma cells in vitro. The solution structures of 1, 5, and 10 were elucidated using NMR spectroscopy. 1H NMR and 2D TOCSY studies of these peptides revealed a downfield Hα proton chemical shift and 2D NOE spectral analysis consistent with a β‐hairpin‐like structure.

Naturally derived antimicrobial peptides have been an area of great interest because of high selectivity against bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. There are also a significant number of venom-derived peptides that exhibit antimicrobial activity in addition to activity against mammals or other organisms. Many venom peptides share the same net cationic, amphiphilic nature as host-defense peptides, making them an attractive target for development as potential antibacterial agents. The peptide ponericin L1 derived from Neoponera goeldii was used as a model to investigate the role of cationic residues and net charge on peptide activity. Using a combination of spectroscopic and microbiological approaches, the role of cationic residues and net charge on antibacterial activity, lipid bilayer interactions, and bilayer and membrane permeabilization were investigated. The L1 peptide and derivatives all showed enhanced binding to lipid vesicles containing anionic lipids, but still bound to zwitterionic vesicles. None of the derivatives were especially effective at permeabilizing lipid bilayers in model vesicles, in-tact Escherichia coli, or human red blood cells. Taken together the results indicate that the lack of facial amphiphilicity regarding charge segregation may impact the ability of the L1 peptides to effectively permeabilize bilayers despite effective binding. Additionally, increasing the net charge of the peptide by replacing the lone anionic residue with either Gln or Lys dramatically improved efficacy against several bacterial strains without increasing hemolytic activity.

Protein secondary and tertiary structure mimics have served as model systems to probe biophysical parameters that guide protein folding and as attractive reagents to modulate protein interactions. Here we review contemporary methods to reproduce loop, helix, sheet and coiled-coil conformations in short peptides.

Cell-penetrating peptides (CPPs) are useful tools for the delivery of a wide variety of cargo into cells. Our lab has developed two classes of CPPs based on β-hairpin sequences, one that folds at the surface of cell membranes and the other that is intrinsically disordered. Although these peptides can effectively deliver different types of cargo, their use in protein delivery has been hindered due to the presence of non-natural D-proline within the central turn region of both sequences, which prohibits functionalizing proteins with the CPPs via standard expression protocols. In this work, we describe new CPPs that replace the non-natural turn region with natural turn motifs amenable to protein expression. We first investigate how these changes within the turn affect various CPP-related properties in the absence of protein cargo, and then generate protein fusions for intracellular delivery.