Recent Progress Using De Novo Design to Study Protein Structure, Design and Binding Interactions

De novo protein design is a powerful methodology used to study natural functions in an artificial-protein context. Since its inception, it has been used to reproduce a plethora of reactions and uncover biophysical principles that are often difficult to extract from direct studies of natural proteins. Natural proteins are capable of assuming a variety of different structures and subsequently binding ligands at impressively high levels of both specificity and affinity. Here, we will review recent examples of de novo design studies on binding reactions for small molecules, nucleic acids, and the formation of protein-protein interactions. We will then discuss some new structural advances in the field. Finally, we will discuss some advancements in computational modeling and design approaches and provide an overview of some modern algorithmic tools being used to design these proteins.

Structural resolution of switchable states of a de novo peptide assembly

De novo protein design is advancing rapidly. However, most designs are for single states. Here we report a de novo designed peptide that forms multiple α-helical-bundle states that are accessible and interconvertible under the same conditions. Usually in such designs amphipathic α helices associate to form compact structures with consolidated hydrophobic cores. However, recent rational and computational designs have delivered open α-helical barrels with functionalisable cavities. By placing glycine judiciously in the helical interfaces of an α-helical barrel, we obtain both open and compact states in a single protein crystal. Molecular dynamics simulations indicate a free-energy landscape with multiple and interconverting states. Together, these findings suggest a frustrated system in which steric interactions that maintain the open barrel and the hydrophobic effect that drives complete collapse are traded-off. Indeed, addition of a hydrophobic co-solvent that can bind within the barrel affects the switch between the states both in silico and experimentally.

Breakthroughs in computational design methods open up new frontiers for de novo protein engineering

Proteins catalyze the majority of chemical reactions in organisms, and harnessing this power has long been the focus of the protein engineering field. Computational protein design aims to create new proteins and functions in silico, and in doing so, accelerate the process, reduce costs and enable more sophisticated engineering goals to be accomplished. Challenges that very recently seemed impossible are now within reach thanks to several landmark advances in computational protein design methods. Here, we summarize these new methods, with a particular emphasis on de novo protein design advancements occurring within the past 5 years.

Design and engineering of artificial metalloproteins: from de novo metal coordination to catalysis

Metalloproteins are essential to sustain life. Natural evolution optimized them for intricate structural, regulatory and catalytic functions that cannot be fulfilled by either a protein or a metal ion alone. In order to understand this synergy and the complex design principles behind the natural systems, simpler mimics were engineered from the bottom up by installing de novo metal sites in either natural or fully designed, artificial protein scaffolds. This review focuses on key challenges associated with this approach. We discuss how proteins can be equipped with binding sites that provide an optimal coordination environment for a metal cofactor of choice, which can be a single metal ion or a complex multinuclear cluster. Furthermore, we highlight recent studies in which artificial metalloproteins were engineered towards new functions, including electron transfer and catalysis. In this context, the powerful combination of de novo protein design and directed evolution is emphasized for metalloenzyme development.