Elucidating Molecular Design Principles for Charge-Alternating Peptides

The therapeutic potential of protein drugs has been hindered by difficulties with long-term stability and rapid clearance from the body. Recombinant fusion proteins provide a scalable platform for engineered biologics, whereby a polypeptide domain is appended to alter the physical characteristics of a therapeutic protein and enhance its pharmaceutical viability. Two simple design principles for recombinant fusion proteins, based on the physical properties of the polypeptide domain, have been separately applied to address issues with the stability and delivery of biologics. "Conformationally disordered" peptides, exemplified by the homo amino acid peptide polyG, have been shown to increase the circulation half-life and bioactivity of protein therapeutics in vivo. Superhydrophilic peptides, exemplified by the alternating-charge peptide poly(EK), have been shown to increase the thermostability of proteins in vitro. The combination of superhydrophilicity and conformational disorder in a single fusion peptide could simultaneously address concerns regarding the stability and therapeutic lifetime of biologics. In the current work, we use enhanced sampling molecular dynamics (MD) simulations to investigate the conformational ensemble of poly(EK) and glycine-substituted poly(EK) variants and validate our structural predictions with circular dichroism (CD). We find the (EK)₁₅ peptide exhibits a high propensity for forming antiparallel β-strand secondary structures, which are stabilized by extensive salt bridging of the positive and negative side chains. MD simulations predict that limited glycine substitutions effectively disrupt the secondary structure and promote disordered conformations at physiologically relevant temperatures. We conclude that the conformational disorder of alternating-charge peptides should be taken into account to improve their suitability for drug delivery applications. We also contribute a computational approach to quantify conformational disorder in polypeptides, which should facilitate the de novo design of effective fusion proteins.