RESUMEN
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 ß 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.
Asunto(s)
Región Variable de Inmunoglobulina/química , Anticuerpos de Cadena Única/química , Factor A de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Anticuerpos de Cadena Única/inmunología , Proteína Estafilocócica A/química , Proteína Estafilocócica A/inmunología , Relación Estructura-Actividad , Termodinámica , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.