<i>De novo</i> design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy

Despite efforts for over 25 years, de novo protein design has not succeeded in achieving the TIM-barrel fold. Here we describe the computational design of 4-fold symmetrical (β/α)(8)-barrels guided by geometrical and chemical principles. Experimental characterization of 33 designs revealed the importance of sidechain-backbone hydrogen bonding for defining the strand register between repeat units. The X-ray crystal structure of a designed thermostable 184-residue protein is nearly identical with the designed TIM-barrel model. PSI-BLAST searches do not identify sequence similarities to known TIM-barrel proteins, and sensitive profile-profile searches indicate that the design sequence is distant from other naturally occurring TIM-barrel superfamilies, suggesting that Nature has only sampled a subset of the sequence space available to the TIM-barrel fold. The ability to de novo design TIM-barrels opens new possibilities for custom-made enzymes

ICR ANNUAL REPORT 2020 (Volume 27)[All Pages]

This Annual Report covers from 1 January to 31 December 202

Designing peptide nanoparticles for efficient brain delivery

The targeted delivery of therapeutic compounds to the brain is arguably the most significant open problem in drug delivery today. Nanoparticles (NPs) based on peptides and designed using the emerging principles of molecular engineering show enormous promise in overcoming many of the barriers to brain delivery faced by NPs made of more traditional materials. However, shortcomings in our understanding of peptide self-assembly and blood–brain barrier (BBB) transport mechanisms pose significant obstacles to progress in this area. In this review, we discuss recent work in engineering peptide nanocarriers for the delivery of therapeutic compounds to the brain, from synthesis, to self-assembly, to in vivo studies, as well as discussing in detail the biological hurdles that a nanoparticle must overcome to reach the brain

Coordination Geometries in Metallobundles Enforcing Oxidative and Hydrolytic Catalysis and Designing Biomaterials for Use As Antimicrobials

De novo protein design permits discovery of intricate folds and functions emulating natural enzymes in simpler, yet robust model constructs. Helical bundles serve as premier scaffolds to incorporate diverse reactivities from oxidative, reductive, to hydrolytic transformations observed in much grander O2-utilizing metalloproteins such as radical-generating ribonucleotide reductases and catalases. One notable family of de novo proteins is the Due Ferri or DF series of four-helix bundles providing a dinuclear site for metal incorporation and are amenable for tailoring active site reactivity. As reactive oxygen species and radical-based intermediates are prevalent and necessary for steering life-essential processes through reactive radicals stabilized by metals, we were curious to understand how the nature and number of metal ions influence sequestration of such species. We investigated the influence metal has on semiquinone stabilization in DF bundles, a minimalist model representing active sites of more complex natural diiron and dimanganese proteins. Coordination sphere was modified in the original DF single chain version to incorporate a 2-His-1-carboxylate facial triad, a single metal binding motif, mirroring morphology of mononuclear non-heme enzymes. We discovered breaking symmetry of the metal coordination site leads to a stable construct with one site exhibiting tight affinity for metal, and this single metal binding construct Uno Ferro single chain stabilizes semiquinone radical anions equally efficient as a two-metal binding version. We envision a robust and easy to modify DFsc and UFsc family of proteins would be versatile tools for gaining mechanistic insights of metalloenzymes. One application of DF constructs we\u27ve pursued is to create a hydrolase bridging a heteronuclear metallosite. Phosphoester substrates were introduced to DFsc and UFsc to characterize hydrolase features. Defining the structural and functional properties of hydrolases in DF proteins would facilitate creation of catalysts for bioremediation purposes. We further apply minimalist design approaches to redesign natural proteins displaying no inherent catalytic properties (or showing marginal activity) for a chosen chemical transformation. Calmodulin and myoglobin were selected as model proteins to introduce enzymatic properties rationally designed using non-rigorous set of computational tools to modify the substrate pocket for evolution. We use HSQC-NMR to guide the directed evolution process to arrive at beneficial mutations which enhance reactivity of generated enzymes. Our minimalist design approach also demonstrates the practical application of our calmodulin designs to reveal an acid-base promoted catalysis in conversion of therapeutics to their metabolically active forms. Only a single precisely positioned amino acid is needed to promote conversion of an antirheumatic prodrug leflunomide bearing an isoxazole ring for proton abstraction to the active form teriflunomide. Furthermore, we dissected possible defense mechanisms microorganisms utilize to survive habitats under oxidative stress. A reported diiron catalase conserved exclusively in Mycobacterium tuberculosis (Mtb) was characterized to determine if the protein acts as a dimanganese catalase as we observed the coordination ligands are similar to nonheme catalases. Therefore, we sought to identify metal preference of this four-helix bundle and establish possible mechanisms utilized by Mtb to disproportionate toxic oxygen metabolites. Conversely, a possible defense strategy by humans to promote bacterial clearance in cases where invasive bacteria trigger human innate immune responses was examined. Hemoglobin is found to be antimicrobial generating cytotoxic radicals to sustain microbicidal action. We considered myoglobin involved in similar peroxidatic processes could be reactive in presence of pathogen associated molecular patterns, specifically lipopolysaccharides (LPS), to produce radical cations. Hence, myoglobin was characterized in a mixture of different proteases and LPS for peroxidase activation. Lastly, design of antimicrobial hydrogels is described. Our hydrogel design is based on self-assembly of peptides induced by silver or copper complexation and does not incorporate permanent crosslinking agents. Short peptides of alternating polar and nonpolar amino acids affixed by an unnatural amino acid pyridyl alanine which coordinate metal form a strong hydrogel. The hydrogel is self-healing and can be delivered in syringe format for application in wound healing therapy

Self-assembling Peptide for HIV-1 Vaccine Design

Human immunodeficiency virus-1 (HIV-1) is a worldwide epidemic, which cannot be eliminated by any current therapeutics, even with highly active antiretroviral therapy, which only can control virus replication. A safe and effective vaccine against HIV-1 that can elicit both potent humoral and cellular responses has been considered a best solution to prevent the infection or to reduce the viral load. However, despite the fact that over 250 clinical trials have been conducted based on different concepts, no vaccine has been successfully developed. The extraordinary diversity of HIV-1, the capability of the virus to escape from the adaptive immunity, the difficulty in inducing broadly neutralizing antibodies, and the lack of clear immune correlates of protection represent the major challenges obstructing the development of HIV-1 vaccines. Designing a peptide-based vaccine that stimulates cytotoxic T lymphocytes (CTLs) specifically against the highly conserved epitopes in HIV-1 has been considered a promising strategy. This can provide two theoretical advantages: maximizing the immunological coverage and minimizing the viral escape from recognition of T cells. However, owning to the short sequence (normally 8-10 amino acids for CTL epitopes), these conserved epitopes are weakly immunogenic, requiring potent adjuvants to boost the efficiency. Some novel strategies have been reported to achieve an efficient adjuvant without causing any side effect. Among them, nanoparticle based delivery systems that can provide targeted delivery to immune cells and/or self-adjuvant effect, are emerging as a promising approach. In this thesis, we present a self-assembling peptide based delivery platform efficiently integrating antigenic peptides and immune potentiators in the formulation of a nanoparticle vaccine, and evaluate the immunogenicity in vitro and in vivo. Three parts are involved in this work: (1) feasibility study of the delivery of HIV-1 CTL epitope with the self-assembling peptide EAK16-II (sequence: AEAEAKAKAEAEAKAK) and the cross-presentation efficiency by dendritic cells (DCs); (2) co-delivery of an antigenic peptide and a toll like receptor (TLR) agonist within one nanoparticle to target and maturate DCs, leading to enhanced CTL response; (3) formulating a prophylactic peptide vaccine against HIV-1 by the combination of CD4 epitope-conjugated EAK16-II, CD8 epitope-conjugated EAK16-II, and a TLR agonist R848, which was subsequently assessed the immunogenicity in the transgenic mice. The peptide EAK16-II could self-assemble into nanofibers, which were stable in the acidic environment and in the presence of proteases. We hypothesized that by directly conjugating HIV-1 CD8 epitope with EAK16-II, the fibrillar structures of the conjugate would enhance the stability of epitope and thus improve the immunogenicity. To verify this, the CD8 epitope SL9 was conjugated with EAK16-II to obtain the epitope-loading peptide SL9-EAK16-II. Physicochemical characterizations revealed SL9-EAK16-II spontaneously assembled to short nanofibers in PBS, which were more stable in serum or oligopeptidase than unstructured SL9. Ex-vivo generated DCs that were pulsed with SL9-EAK16-II and activated by maturation cytokines, stimulated more poly-functional CD8+ T cells. This augment was explained by the evidence that SL9-EAK16-II was degraded more slowly than SL9 within DCs, therefore prolonging the stimulation to CD8 T cells. Moreover, the results from confocal microscopy suggested the cytosolic pathway for the cross-presentation of SL9-EAK16-II. However, SL9-EAK16-II itself failed to maturate DCs after internalization, which might cause antigen tolerance. To avoid the induction of tolerance and further enhance the antigenicity of epitope SL9, TLR agonist R837 or R848 was incorporated into the nanofiber formulation. The data from fluorescence spectra and calorimetric titration suggested the co-assembly between SL9-loaded nanofibers and TLR agonist was mainly driven by hydrogen bonding and hydrophobic interactions. The SL9-EAK16-II/R848 co-assemblies strongly facilitated the activation of DCs, and stimulated significantly more epitope specific CTLs when assessed in the form of DC based vaccine. The in vitro studies implied the potential of the self-assembling peptide EAK16-II as a nanocarrier in the formulation of vaccine. We further determined the applicability of this formulation in vivo. Since the activation of CD4+T cells plays a critical role in the generation of functional memory CTLs, we incorporated an additional CD4 epitope TL13 into the vaccine formulation, via conjugating with EAK16-II. The new formulation of antigen was characterized as nanofibers with average size of approximately 220 nm. The transgenic mice that were subcutaneously injected with these nanofibers produced as much as 1 fold increase in frequencies of SL9 specific CTLs, when compared with the mice vaccinated with either the mixture of epitopes and R848, or R848 alone. Moreover, almost 90% of the SL9 specific CTLs primed by the nanofibers were central memory CD8+ T cells (CD44+, CD62L+), which was the hallmark of the acquired immune response. The in-vivo study suggested not only enhanced magnitude, but also higher quality of T cell response was induced by the nanoparticle-based vaccine. Our findings demonstrated the self-assembling peptide had considerable promise as a delivery platform to integrate the principal components for cellular response-focusing vaccines

Developing Computational Tools for the Study and Design of Amyloid Materials

The self-assembly of short peptides into amyloid structures is linked to several diseases but has also been exploited for the design of novel functional amyloid-based materials. Such materials are potentially biocompatible and biodegradable, while their unique molecular organization provides them with remarkable mechanical properties. Amyloid fibrils are among the stiffest biological materials and exhibit a high resistance to breakage. Apart from the aforementioned properties, they are particularly attractive due to their easy synthesis and the ability to be redesigned through mutations at sequence level, which can result in potential functionality. Previous studies have reported the rational based design of functional amyloid materials, designed through primarily scientists’ intuition, and their applications in several fields as agents for tissue-engineering, antimicrobial and antibacterial agents, drug carriers, materials for separation applications, etc. The current work starts from the use of previously reported protocols for the computational elucidation of the structure of amyloids, leading to the formation of amyloid materials, and the investigation of the functional properties of rationally designed self-assembling peptides, and introduces a new approach for the computational design of functional amyloid materials, based on engineering and biophysical principles. In summary, we developed a computational protocol according to which an optimization-based design model is used to introduce mutations at non-βsheet residue positions of an amyloid designable scaffold (amyloid with non-β-sheet forming residues at its termini). The designed amino acids are introduced to the scaffold in such a way so that they mimic how amino acids bind to particular ions/compounds of interest according to experimentally resolved structures (defined by us as materialphore models) and also aim at energetically stabilizing the bound conformation of the pockets. The optimum designs are computationally validated using a series of simulations and structural analysis techniques to select the top designed peptides, which are predicted to form fibrils with specific ion/compound binding properties for experimental testing. The computational protocol has been implemented first for the design of amyloid materials (i) binding to cesium ions, and in additional cases, for the design of amyloid materials (ii) serving as potential AD drug carriers, (iii) which could promote cell-penetration and possess DNA binding properties, and (iv) incorporating potential cell-adhesion, calcium and strontium binding properties. The computational protocol is also presented here as a step toward a generalized computational approach to design functional amyloid materials binding to an ion/compound of interest. This work can constitute a stepping stone for the functionalization of peptide/protein-based materials for several applications in the future