Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B.

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties

Design of novel metalloenzymes and investigation of protein-metal interactions

Metalloproteins are the proteins that utilize metals or metal complexes as cofactors and exhibit a wide range of functions from oxygen transport and regulation of transcription to hydroxylation of alkanes and water splitting, even though they employ a limited set of metals and metal-binding ligands. The investigation of metalloprotein amino acid sequence, structure and function relationships can uncover the principles behind natural metalloprotein design and opens the possibility of their implication as scaffolds for the design of novel catalysts and biomaterials. Protein engineering is a set of techniques that allows to install new functionalities into existing proteins or to design new proteins from scratch (de novo design) for various applications.Chapter 1 of this dissertation will summarize the principles behind protein-metal interactions and metalloprotein design by discussing current methods in protein engineering and the successful examples of engineered metalloproteins. The work presented in Chapter 2 provides insight into the extraordinary metal affinity to UFsc, a single-metal-binding de novo designed protein. The introduction of a single mutation in a two-metal-binding site of DFsc, a de novo designed protein, resulted in the elimination of one coordination site. The rearrangement of the active site altered protein’s metal affinity to selected transition metals and resulted in the high affinity to zinc (II) ion with the dissociation constant in the picomolar range. We have shown that the fine tuning of the protein scaffold is important for effective metal binding in designed proteins. Chapter 3 will discuss the development of a new protein engineering method, NMR-guided directed evolution. Directed evolution is a protein engineering technique used to identify proteins with improved functions by generating large libraries of mutants with further high-throughput screening in a functional assay. Our work was focused on establishing a method that will help to identify the positions for potential productive mutations to improve the output of directed evolution. We have found a strong correlation between the HSQC NMR chemical shift perturbations of backbone amide resonances of amino acid residues interacting with the substrate transition state analog and the probability of finding a beneficial mutation in the vicinity of those residues. The combination of three mutations identified by NMR-guided directed evolution resulted in the design of FerrElCat, the most effective designed Kemp eliminase reported to date. The design of a metalloprotein-based PET tracer, presented in Chapter 4, will provide an example of the application of metalloproteins for theranostic purposes. The introduction of one mutation in the calcium-binding site of AlleyCat7, a calmodulin-derived Kemp eliminase, completely eliminated protein’s affinity to calcium (II) but preserved affinity to yttrium (III). This protein, fused to a liver-cancer-specific antibody, can be used to deliver metals to the cancer cells for PET imaging and radiotherapy. Finally, the application of a minimalist approach to the design of amyloid-binding peptides that can reduce the infectivity of HIV virions will be discussed in Chapter 5. The amyloid fibrils, formed by the peptides derived from PAP and SEM proteins, have been found in human semen and shown to enhance HIV infection. We have designed a set of peptides to bind to these fibrils specifically and identified the promising candidates that differentiate between fibrillated and monomeric forms of amyloidogenic peptides. The identified peptides also showed sequence specificity. The developed peptides can potentially be used as microbicides for the prevention of HIV infection

Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties

Peptide hydrogel with self-healing and redox-responsive properties

We have rationally designed a peptide that assembles into a redox-responsive, antimicrobial metallohydrogel. The resulting self-healing material can be rapidly reduced by ascorbate under physiological conditions and demonstrates a remarkable 160-fold change in hydrogel stiffness upon reduction. We provide a computational model of the hydrogel, explaining why position of nitrogen in non-natural amino acid pyridyl-alanine results in drastically different gelation properties of peptides with metal ions. Given its antimicrobial and rheological properties, the newly designed hydrogel can be used for removable wound dressing application, addressing a major unmet need in clinical care