The design process described herein relies on a combination of optimization strategies utilizing directed evolution by phage display and synthetic pharmaceuticalization via α-helix stapling. Protein engineers are increasingly able to rely on structure-function insights, computational methods, and deeper understanding of natural biosynthesis processes, to streamline the design and applications of enzymes. The engineered conjugate is capable of assembling into nanostructures once dispersed in water, with average diameter of 80. We have previously shown that the incorporation of an 8-atom all-hydrocarbon 'staple' at positions i and i + 3 of a synthetic peptide results in substantial stabilization of the α-helical conformation. All-Hydrocarbon Stapled α-Helical Peptides 3. This quantity of Methods in Enzymology appears to be like at Protein Engineering for Therapeutics. Methods Generation of Dual-Variable-Domain Immunoglobulin Molecules for Dual-Specific Targeting 1.
We further report that alterations of single base-pairs or a single amino acid substitution R112A can induce strandwise translocation. Concluding Remarks and Perspectives Acknowledgments Engineering Cyclic Peptide Toxins 1. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. The Verdine laboratory is developing powerful new chemistry-based platform technologies to address undruggable targets, including transcription factors. The methods described are also applicable to screening directly on cells and can be ported to other in vitro directed evolution systems utilizing non-immunoglobulin scaffolds.
Lead Optimization for Next-Generation Proteases 6. Here we report high resolution structural analysis of such recognition variants. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Conclusions Acknowledgment Oral Enzyme Therapy for Celiac Sprue 1. Considerable interest has therefore arisen in next-generation targeting molecules that combine the broad target recognition capabilities of protein therapeutics with the robust cell-penetrating ability of small molecules.
The suite of currently used drugs can be divided into two categories - traditional 'small molecule' drugs with typical molecular weights of 5000 Da that are not orally bioavailable and need to be delivered via injection. Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. Cationic Cell Permeable Peptides 3. Furthermore, it is essential that researchers, educators, and students alike remain aware of the fundamental comparative differences in the reproductive biology of domestic species. In some embodiments, these chirally controlled oligonucleotides show patterns of cleavage of a target nucleotide sequence that differ from those seen with ref. Expression, Purification, and Applications of Soluble scTv Proteins 5.
This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. We demonstrate that hydrocarbon stapling confers cellular permeability to a p53 peptide that is then capable of modulating transcriptional activity. In these complexes, the protein is observed to translocate by one nucleotide on one strand while remaining untranslocated on the complementary strand. With an international board of authors, this volume is split into sections that cover subjects such as antibodies, protein conjugates, peptides, enzymes and scaffolds. The highly mutagenic A:oxoG 8-oxoguanine base-pair is generated mainly by misreplication of the C:oxoG base-pair, the oxidation product of the C:G base-pair. In some embodiments, a provided chirally controlled oligonucleotide compn. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs.
The prototype for a transcription factor controlled by posttranslational modification is E. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule binding pockets on the Ras surface. The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compns. But natural products still offer unmatched structural variety, especially as new environmental niches are explored, and their usefulness can be further extended by engineering the proteins that produce them and by using them to probe biological pathways. Part B Author: K Dane Wittrup; Gregory L Verdine Publisher: San Diego, Calif. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. Reactivation of the p53 tumor suppressor pathway by a stapled p53 peptide.
Such peptides, which are conformationally and proteolytically stabilized with all-hydrocarbon staples, are an emerging class of biologics that are capable of disrupting protein-protein interactions and thus have broad therapeutic potential. Physicochemical Characterization of Immunoliposomes 4. Mutation of this residue to asparagine, another helix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. Summary Engineering and Identifying Supercharged Proteins for Macromolecule Delivery into Mammalian Cells 1. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair.
Hydrocarbon-stapled α-helical peptides are a new class of targeting molecules capable of penetrating cells and engaging intracellular targets formerly considered intractable. UvrA also harbors two unique domains; we show that one of these is required for interaction with UvrB, its partner in lesion recognition. Inactivation of p53 is the most prevalent defect in human cancers. © 2014 Wiley Periodicals, Inc. Will Targeting Increase Nanoparticle Accumulation in a Tumor? Inhibitors of the Mdm2-p53 interaction that restore the functional p53 constitute potential nongenotoxic anticancer agents with a novel mode of action. As part of our ongoing effort to explore the scope and utility of all-hydrocarbon peptide stapling systems, we have investigated and report herein the properties of a new i, i + 3 stapling system that employs a 6-carbon cross-link. To determine the factors involved in the specific recognition function of MutM, free energy potential of mean force simulations and targeted molecular dynamics simulations are performed for a number of different systems.
In Vitro and In Vivo Characterization of Immunoliposomal Therapeutics Cell-Targeting Fusion Constructs Containing Recombinant Gelonin 1. Free energy difference calculations show that both attractive and repulsive interactions have an important role in the preferential binding of oxoG compared with G to the active site. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. Interestingly, the mutation of the catalytically important Glu3 E3 residue to Gln Q significantly changes the free-energy surface so that oxoG can bind stably in the active site of the enzyme.
As part of our ongoing effort to explore the scope and utility of all-hydrocarbon stapling systems, we have investigated and report herein the properties of a new i, i + 3 stapling system that employs a 6-carbon cross-link. The X-ray structure reveals that, upon irradiation, the oxoG lesion has transited from the exosite to the active site pocket, but has not undergone cleavage by the enzyme. Affinity Engineering and Selection of T Cell Receptors by T Cell Display 4. Stapling can greatly improve the pharmacologic performance of peptides, increasing their target affinity, proteolytic resistance, and serum half-life while conferring on them high levels of cell penetration through endocytic vesicle trafficking. Inspired by the structure—function relationships of antibodies immunoglobulins , such an alternative protein scaffold provides a robust, conserved structural framework that supports an interaction site which can be reshaped for the tight and specific recognition of a given bio molecular target. These findings highlight the need for a more thorough understanding of the forces that underlie helix stabilization by hydrocarbon staples.