Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences) by human H3s from another library. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3)) onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd) (m01s) with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn) (Gong et al, JBC, 20). However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Lastly, these methods for objectively and comprehensively comparing SAXS profiles for systems critically affected by solvent conditions and structural heterogeneity provide an enabling technology for advancing the design and bioengineering of nanoscale biological materials.Libraries based on an isolated human immunoglobulin G1 (IgG1) constant domain 2 (CH2) have been previously diversified by random mutagenesis. Force plots comparing SAXS data sets further reveal more complex and controllable behavior in solution than captured by our crystal structures. Specifically, our results probed the influence of solution conditions and symmetry on stability and structural adaptability, identifying the dimeric interface as the weak point in the assembly. These new tools, which provided effective feedback on experimental constructs relative to design, have general applicability in analyzing the solution behavior of heterogeneous nanosystems and have been made available as a web-based application. To generate a phase diagram associating structure and assembly, we developed force plots that more » measure dissimilarity among multiple SAXS data sets. Enhancing the crystallographic results, high-throughput small-angle x-ray scattering (SAXS) comprehensively contrasted our modifications under diverse solution conditions. The monomeric unit is composed of a trimerizing apex-forming domain genetically linked to an edge-forming dimerizing domain. Here, we created and validated an advanced design of a 600-kDa protein homododecamer that self-assembles into a symmetric tetrahedral cage. of California, Los Angeles, CA (United States) Sponsoring Org.: USDOE Office of Science (SC) OSTI Identifier: 1799610 DOE Contract Number: FC02-02ER63421 Resource Type: Journal Article Journal Name: ACS Synthetic Biology Additional Journal Information: Journal Volume: 9 Journal Issue: 2 Journal ID: ISSN 2161-5063 Publisher: American Chemical Society (ACS) Country of Publication: United States Language: English Subject: Biochemistry & Molecular BiologyĬentral challenges in the design of large and dynamic macromolecular assemblies for synthetic biology lie in developing effective methods for testing design strategies and their outcomes, including comprehensive assessments of solution behavior. Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States.Department of Ecology and Evolutionary Biology, University of California, Los Angeles, California 90095, United States.UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California 90095, United States Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States.UCLA-DOE Institute for Genomics and Proteomics, Los Angeles, California 90095, United States Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States Molecular Biology Institute, University of California, Los Angeles, California 90095, United States.
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