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Peptides are a promising source of new therapeutics, but the biophysical characteristics of natural peptides, including their stability and propensity to aggregate, can limit their success. Protein engineering offers powerful tools to improve the properties of peptides for biological applications. In this review, we explain rational design, directed evolution, and computational methods and how these methods can be applied to improving the characteristics of peptides. We also provide a discussion of engineering the thermodynamic stability, self‐assembly, reduced aggregation, proteolytic stability, and binding affinity and specificity of peptides, along with a perspective on future directions in engineering therapeutic peptides.

Although the interactions of cell‐penetrating peptides (CPPs) with mammalian cells have been widely studied, much less is known about their interactions with fungal cells. To study how the properties of CPPs affect translocation into fungal cells, we designed variants of the peptides pVEC and SynB with altered levels of charge and hydrophobicity and evaluated the translocation of the variants into the important human fungal pathogenCandida albicans. Charge played a greater role in translocation efficacy of the peptides than hydrophobicity, with a higher net positive charge leading to higher level of translocation intoC. albicansand a higher level of cytosolic localization. Hydrophobicity had little effect on translocation efficacy, but a low level of hydrophobicity did lead to increased vacuolar localization and an energy‐dependent translocation mechanism. Our results suggest that CPPs can be designed for desired levels of cargo delivery into fungal cells and for desired translocation mechanisms.

Histatin 5 (Hst‐5) is an antimicrobial peptide with strong antifungal activity againstCandida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused byC. albicans. However, Hst‐5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced byC. albicans. Single‐residue substitutions can significantly affect the proteolytic resistance of Hst‐5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single‐residue modifications can be combined in a single peptide, K11R–K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single‐residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap‐dependent effects on degradation. Both single‐ and double‐substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.