Allostery is a fundamental regulatory mechanism of protein function. Despite notable advances, understanding the molecular determinants of allostery remains an elusive goal. Our current knowledge of allostery is principally shaped by a structure-centric view, which makes it difficult to understand the decentralized character of allostery. We present a function-centric approach using deep mutational scanning to elucidate the molecular basis and underlying functional landscape of allostery. We show that allosteric signaling exhibits a high degree of functional plasticity and redundancy through myriad mutational pathways. Residues critical for allosteric signaling are surprisingly poorly conserved while those required for structural integrity are highly conserved, suggesting evolutionary pressure to preserve fold over function. Our results suggest multiple solutions to the thermodynamic conditions of cooperativity, in contrast to the common view of a finely tuned allosteric residue network maintained under selection.

The interaction between a bacteriophage and its host is mediated by the phage's receptor binding protein (RBP). Despite its fundamental role in governing phage activity and host range, molecular rules of RBP function remain a mystery. Here, we systematically dissect the functional role of every residue in the tip domain of T7 phage RBP (1660 variants) by developing a high-throughput, locus-specific, phage engineering method. This rich dataset allowed us to cross compare functional profiles across hosts to precisely identify regions of functional importance, many which were previously unknown. Substitution patterns showed host-specific differences in position and physicochemical properties of mutations, revealing molecular adaptation to individual hosts. We discovered gain-of-function variants against resistant hosts and host-constricting variants that eliminated certain hosts. To demonstrate therapeutic utility, we engineered highly active T7 variants against urinary tract pathogen. Our approach presents a generalized framework for characterizing sequence-function relationships in many phage-bacterial systems.

Splitting bioactive proteins into conditionally reconstituting fragments is a powerful strategy for building tools to study and control biological systems. However, split proteins often exhibit a high propensity to reconstitute even without the conditional trigger, limiting their utility. Current approaches for tuning reconstitution propensity are laborious, context-specific, or often ineffective. Here, we report a computational design strategy grounded in fundamental protein biophysics to guide experimental evaluation of a sparse set of mutants to identify an optimal functional window. We hypothesized that testing a limited set of mutants would direct subsequent mutagenesis efforts by predicting desirable mutant combinations from a vast mutational landscape. This strategy varies the degree of interfacial destabilization while preserving stability and catalytic activity. We validate our method by solving two distinct split protein design challenges, generating both design and mechanistic insights. This new technology will streamline the generation and use of split protein systems for diverse applications.

Allostery is a fundamental regulatory mechanism of protein function. Despite notable advances, understanding the molecular determinants of allostery remains an elusive goal. Our current knowledge of allostery is principally shaped by a structure-centric view which makes it difficult to understand the decentralized character of allostery. We present a function-centric approach using deep mutational scanning to elucidate the molecular basis and underlying functional landscape of allostery. We show that allosteric signaling exhibits a high-degree of functional plasticity and redundancy through myriad mutational pathways. Residues critical for allosteric signaling are surprisingly poorly conserved while those required for structural integrity are highly conserved, suggesting evolutionary pressure to preserve fold over function. Our results suggest multiple solutions to the thermodynamic conditions of cooperativity, in contrast to the common view of a finely-tuned allosteric residue network maintained under selection.