Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains ( http://AbLIFT.weizmann.ac.il ). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.

<p>Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the <i>T. acidophilum</i> archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (<i>S. cerevisiae</i>) and mammals (rat - <i>R.</i> <i>norvegicus</i>, rabbit - <i>O. cuniculus</i> and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies. </p>

<p>Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the <i>T. acidophilum</i> archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (<i>S. cerevisiae</i>) and mammals (rat - <i>R.</i> <i>norvegicus</i>, rabbit - <i>O. cuniculus</i> and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the <a></a><a>PSMA7 </a>subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies. </p>

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