Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.ĭepartment of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. of C(sp3)-metalated pincer complexes based on dibenzobarrelene scaffold. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. of peptides and modified peptides Protein biochemistry: new methods for. to the logical conclusion that JIP was a natural inhibitor of JNK signaling. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Scaffold proteins facilitate signal transduction by tethering molecules. This development started with the notion that immunoglobulins owe their function to the composition of a conserved framework region and a spatially well-defined antigen-binding site made of peptide segments that are hypervariable both in sequence and in conformation. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The use of so-called protein scaffolds has recently attracted considerable attention in biochemistry in the context of generating novel types of ligand receptors for various applications in research and medicine. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. 2.3 Structural ultra-stability A protein scaffold with ultra high. Here, we describe the rapid and systematic development of high-affinity 10 FN3 domain inhibitors of the MDM2 and MDMX proteins. While there has been considerable progress in designing protein-protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. For example, loop 1 of the Arabidopsis thaliana trypsin inhibitor (ATTp) and loop 2 of. Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Biologically Interesting Molecule Reference Dictionary (BIRD).