title: Antibody variable domain interface and framework sequence requirements for stability and function by high-throughput experiments.
abstract: Protein structural stability and biological functionality are dictated by the formation of intradomain cores and interdomain interfaces, but the intricate sequence-structure-function interrelationships in the packing of protein cores and interfaces remain difficult to elucidate due to the intractability of enumerating all packing possibilities and assessing the consequences of all the variations. In this work, groups of β strand residues of model antibody variable domains were randomized with saturated mutagenesis and the functional variants were selected for high-throughput sequencing and high-throughput thermal stability measurements. The results show that the sequence preferences of the intradomain hydrophobic core residues are strikingly flexible among hydrophobic residues, implying that these residues are coupled indirectly with antigen binding through energetic stabilization of the protein structures. By contrast, the interdomain interface residues are directly coupled with antigen binding. The interdomain interface should be treated as an integral part of the antigen-binding site.
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan; Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Hung-Ju
authors.initials: HJ
authors.lastname: Hsu
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Kuo Hao
authors.initials: KH
authors.lastname: Lee
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan; Institute of Biomedical Informatics, National Yang-Ming University, Taipei 112, Taiwan; Bioinformatics Program, Taiwan International Graduate Program, Institute of Information Science, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Jhih-Wei
authors.initials: JW
authors.lastname: Jian
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan; Institute of Biochemical Science, National Taiwan University, Taipei 106, Taiwan; Chemical Biology and Molecular Biophysics program, Taiwan International Graduate Program at Academia Sinica, Taipei 115, Taiwan.
authors.forename: Hung-Ju
authors.initials: HJ
authors.lastname: Chang
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Chung-Ming
authors.initials: CM
authors.lastname: Yu
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yu-Ching
authors.initials: YC
authors.lastname: Lee
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Ing-Chien
authors.initials: IC
authors.lastname: Chen
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan; Institute of Biomedical Informatics, National Yang-Ming University, Taipei 112, Taiwan; Bioinformatics Program, Taiwan International Graduate Program, Institute of Information Science, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Hung-Pin
authors.initials: HP
authors.lastname: Peng
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Chih Yuan
authors.initials: CY
authors.lastname: Wu
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Huang
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan; Institute of Zoology, College of Life Sciences, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Chih-Yun
authors.initials: CY
authors.lastname: Shao
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Kuo Ping
authors.initials: KP
authors.lastname: Chiu
authors.affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan; Genomics Research Center, Academia Sinica, Taipei 115, Taiwan. Electronic address: yangas@gate.sinica.edu.tw.
authors.forename: An-Suei
authors.initials: AS
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D057166:High-Throughput Screening Assays; D006801:Humans; D006860:Hydrogen Bonding; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D057127:Single-Chain Antibodies; D013205:Staphylococcal Protein A; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A