Probing the Rules of Protein Structure-Function Relationships through the De Novo Design of Functional Proteins - Project Summary The accurate design of de novo proteins with predetermined structural features that enable their function is the ultimate test of our understanding of protein structure-function relationships. Here, we propose to enhance our understanding of these relationships through the de novo design of stimuli-responsive protein masks that serve as protein delivery vehicles for cell penetrating peptides (CPPs), and antimicrobial peptides (AMPs). These membrane active peptides were chosen as while they have great promise as therapeutic agents, their promise is unrealized because they lack tissue specificity and suffer from inherent low half-life and stability. To overcome these obstacles, and in the process gain important insights into protein structure-function relationships, I propose to develop the Enfolding Pipeline which will mask target peptides in a de novo protein (an Enfold), which would “unmask” only in the presence of an environmental or disease-specific cue. The first “unmasking” mechanism will be the introduction of protease cleavable loops between the mask and target peptide, such that the target peptide will be conditionally released upon cleavage by extracellular proteases that are up-regulated in disease state. Importantly, we will incorporate three design approaches into this pipeline (ii) parametric protein design and (iii) newly developed diffusion-based generative models. Molecular dynamics simulations will be incorporated to tune the stability of the Enfolds, thus accounting for both stability and dynamics, a long-standing goal in the field. This will allow us to compare each design approach, an important benchmark in the field of protein design. In Aim 1 we will develop and test this pipeline towards the design of Enfolds for both AMPs and CPPs. We will characterize the folding, stability and structure of the Enfolds and carry out in vitro cleavage experiments. In Aim 2 we will characterize the biological activity of the Enfolds and released target peptides. This will include antimicrobial assays such as MIC and Live/Dead analyses for the AMP Enfolds and, together with our collaborator Prof. Xiaokun Shu, we will evaluate the ability of Enfolds to conditionally release the masked CPP, attached to various cargo molecules, via cell-based assays. The results of the first Aims will allow us to correlate protein structure to function, and will be utilized for the iterative design of the masks. In Aim 3 we will extend the functionality of the Enfolds to include new mechanisms of “unmasking”. To improve delivery specificity, we will incorporate receptor targeting peptides such as integrin-specific peptides or GPCR ligands. As receptor mediated endocytosis is common when targeting these receptors, we will incorporate endosomal escape mechanisms in the form of either (i) cathepsin cleavable loops between the mask and target peptide as these proteases are over expressed in endosomes, or (ii) packing of the bundles with pH sensitive residues (e.g. histidine) that will reduce the stability of the Enfolds in the low endosomal pH. This function-directed approach expands our fundamental understanding of protein structure-function relationships and our ability to accurately design protein-peptide interactions, and addresses critical pitfalls in the use of bioactive peptides as therapeutics.
