Solution Conformation of Extracellular Matrix Proteins

Abstract

The extracellular matrix (ECM) is composed of large, multi-domain proteins with varying degree of shapes and post-translational modifications. Therefore, it is challenging to study such proteins with a single technique to gain reliable information about their size, shape and conformation. We have employed a multidisciplinary approach using a variety of techniques to investigate the solution conformation of laminin gamma-1 short arm, netrin-4 and G3 domain of agrin fused with human IgG-Fc (G3Fc). Briefly, the hydrodynamic properties of entire multi-domain proteins were studied using analytical ultracentrifugation, dynamic light scattering, and small angle X-ray scattering (SAXS). Further, the information from SAXS for entire multi-domain protein was then either used to determine ab initio structure or combined with high-resolution data for individual domains to obtain a detailed solution structure of desired multi-domain protein. Additionally, the SAXS models were verified by comparing the experimentally determined hydrodynamic parameters with the parameters calculated from solution structures. The ab initio structures for laminin gamma-1 short arm revealed an extended and curved assembly. We could also combine small portion of high-resolution structure from X-ray Crystallography data to obtain rigid-body models. Further, we found that the netrin4 has an elongated shape in solution using ab initio methods. Remarkably, we found that the G3Fc chimera is T-shaped and not Y-shaped like an antibody in solution by rigid body modelling. Thus, we conclude that by combining various methods, it is possible to explore large muldi-domain protein and protein-protein assemblies that will help enable us to understand structure-function relationship of these proteins with various forms of diseases.