Protein networks in all organisms comprise homologous interacting pairs. In these networks, some proteins are specific, interacting with one or a few binding partners, whereas others are multispecific and bind a range of targets. We describe an algorithm that starts from an interacting pair and designs dozens of new pairs with diverse backbone conformations at the binding site as well as new binding orientations and sequences. Applied to a high-affinity bacterial pair, the algorithm results in 18 new ones, with cognate affinities from pico- to micromolar. Three pairs exhibit 3-5 orders of magnitude switch in specificity relative to the wild type, whereas others are multispecific, collectively forming a protein-interaction network. Crystallographic analysis confirms design accuracy, including in new backbones and polar interactions. Preorganized polar interaction networks are responsible for high specificity, thus defining design principles that can be applied to program synthetic cellular interaction networks of desired affinity and specificity.

The functional sites of many protein families are dominated by diverse backbone regions that lack secondary structure (loops) but fold stably into their functionally competent state. Nevertheless, the design of structured loop regions from scratch, especially in functional sites, has met with great difficulty. We therefore developed an approach, called AbDesign, to exploit the natural modularity of many protein families and computationally assemble a large number of new backbones by combining naturally occurring modular fragments. This strategy yielded large, atomically accurate, and highly efficient proteins, including antibodies and enzymes exhibiting dozens of mutations from any natural protein. The combinatorial backbone‐conformation space that can be accessed by AbDesign even for a modestly sized family of homologs may exceed the diversity in the entire PDB, providing the sub‐Ångstrom level of control over the positioning of active‐site groups that is necessary for obtaining highly active proteins. This manuscript describes how to implement the pipeline using code that is freely available at https://github.com/Fleishman‐Lab/AbDesign_for_enzymes.

Recent advances in protein‐design methodology have led to a dramatic increase in reliability and scale. With these advances, dozens and even thousands of designed proteins are automatically generated and screened. Nevertheless, the success rate, particularly in design of functional proteins, is low and fundamental goals such as reliable de novo design of efficient enzymes remain beyond reach. Experimental analyses have consistently indicated that a major reason for design failure is inaccuracy and misfolding relative to the design conception. To address this challenge, we describe complementary methods to diagnose and ameliorate suboptimal regions in designed proteins: first, we develop a Rosetta atomistic computational mutation scanning approach to detect energetically suboptimal positions in designs (available on a web server https://pSUFER.weizmann.ac.il ); second, we demonstrate that AlphaFold2 ab initio structure prediction flags regions that may misfold in designed enzymes and binders; and third, we focus FuncLib design calculations on suboptimal positions in a previously designed low‐efficiency enzyme, improving its catalytic efficiency by 330‐fold. Furthermore, applied to a de novo designed protein that exhibited limited stability, the same approach markedly improved stability and expressibility. Thus, foldability analysis and enhancement may dramatically increase the success rate in design of functional proteins.

Recent advances in protein-design methodology have led to a dramatic increase in its reliability and scale. With these advances, dozens and even thousands of designed proteins are automatically generated and screened. Nevertheless, the success rate, particularly in design of functional proteins, is low and fundamental goals such as reliable de novo design of efficient enzymes remain beyond reach. Experimental analyses have consistently indicated that a major cause of design failure is inaccuracy and misfolding relative to the design model. To address this challenge, we describe complementary methods to diagnose and ameliorate suboptimal regions in designed proteins: first, we develop a Rosetta atomistic computational mutation scanning approach to detect energetically suboptimal positions in designs; second, we demonstrate that the AlphaFold2 ab initio structure prediction method flags regions that may misfold in designed enzymes and binders; and third, we focus FuncLib design calculations on suboptimal positions in a previously designed low-efficiency enzyme, thereby improving its catalytic efficiency by 330 fold. Thus, foldability analysis and enhancement may dramatically increase the success rate in design of functional proteins.

Over half the proteins form homo or hetero-oligomeric structures. Experimentally determined structures are often considered in determining a protein's oligomeric state, but static structures miss the dynamic equilibrium between different quaternary forms. The problem is exacerbated in homo-oligomers, where the oligomeric states are challenging to characterize. Here, we re-evaluated the oligomeric state of 17 different bacterial proteins across a broad range of protein concentrations and solutions by native mass-spectrometry (MS), mass photometry (MP), size exclusion chromatography (SEC), and small-angle x-ray scattering (SAXS), finding that most exhibit several oligomeric states. Surprisingly, many proteins did not show mass-action driven equilibrium between the oligomeric states. For approximately half the proteins, the predicted oligomeric forms described in publicly available databases underestimated the complexity of protein quaternary structures in solution. Conversely, AlphaFold Multimer provided an accurate description of the potential multimeric states for most proteins, suggesting that it could help resolve uncertainties on the solution state of many proteins.