The Stability, Reduction Potential and Ligand State of Two Conformations of a C-Type Cytochrome from the Diatom Thalassiosira Pseudonana

Abstract

region forms a rod-like structure that projects the N-terminal functional domain of the protein from the bacterial cell surface. We have used SHRImP-TIRFM, a super-resolution fluorescence microscopy technique, to measure the end-to-end distance of a 7 repeat protein construct, and SEC-MALS-QELS to measure its hydrodynamic radius. Combined with crystal structures of both single and double repeat structures, we will assess the elongation of the repetitive region. Neuropilin-1 (NRP1) is the cellular growth factor that interacts with semaphorin 3A, placenta growth factor-2 and vascular endothelial growth factor (VEGF165). The interactions of NRP1 with these proteins initiate signaling pathways that have implications on fundamental cellular processes. NRP1 have been designated as a potential target for the treatment of various types of cancers. It is composed of two CUB domains (a1 and a2) that are connected with F5/8 type C1 (b1) and C2 (b2) domains, followed by a flexible linker, a MAM domain, membrane-anchored region and a cytoplasmic tail. The high-resolution structures of various domains of human NRP1 (a2, b1 and b2 and MAM domains) are available. However, the information of how the a1 domain is connected to the a2, b1 and b2 domains and how the overall assembly behaves is unavailable. Recently, a crystal structure of mouse a1, a2, b1 and b2 (PDB:4Z9) domains suggested that the a1 domain is linked with the rest of the domains via a flexible linker. We characterized the wild-type NRP1 composed of a1, a2, b1 and b2 domains and a mutant version that lacks glycosylation using the dynamic light scattering, analytical ultracentrifugation, and small angle X-ray scattering techniques. The results from all three techniques suggest that the glycosylation is crucial for the stability and homogeneity of NRP1. Furthermore, all though no major difference was found between these two versions of NRP1 in terms of their low-resolution structures obtained using small angle X-ray scattering, their solution conformation differs significantly compared to the crystal structure of the deglycosylated version of mouse a1, a2, b1 and b2 structure. Based on our preliminary data, we hypothesize that the flexible linker between the a1 and a2 domains allow efficient interaction with ligands to initiate signaling pathways.