Molecular characterization and structural modeling of sialic acid transporter SiaPQM in Haemophilus influenzae

Autor: Shao-En Chang, 張韶恩
Rok vydání: 2016
Druh dokumentu: 學位論文 ; thesis
Popis: 105
Non-typeable Haemophilus influenzae is associated with invasive pneumonia and meningoencephalitis in infants and young children. Lipopolysaccharide (LPS) is a critical virulence determinant of non-typeable H. influenzae. Sialic acid (Neu5Ac) is one of crucial components on the outer part of non-typeable H. influenzae LPS and it provides protection from being killed by human immune system due to the abundance of Neu5Ac in human host. Because H. influenzae does not encode any enzymes to biosynthesize Neu5Ac, it can sialylate LPS only by uptaking the Neu5Ac from the extracellular environment. An active transporter SiaPQM complex on inner membrane of H. influenza is in charge of the up-hill transportation of Neu5Ac from the periplasm to the cytoplasm. SiaPQM complex belongs to a TRipartite ATP-independent Periplasmic (TRAP) transporter family. From previous studies, it has shown that periplamic siaP selectively binds to Neu5Ac and then brings the substrates to transmembrane SiaQM. The crystal structure of SiaP in complex with Neu5Ac has also been determined, demonstrating the key residues for substrate binding. However, the molecular mechanism of Neu5Ac transportation remains unclear. Currently, SiaP and SiaQM have been overexpressed and purified successfully in the study. We utilized Size Exclusion Chromatography to monitor if SiaP form complex with SiaQM in detergent buffer. ITC experiments have also been performed to study the binding affinity of Neu5Ac to SiaP and SiaQM. Protein crystals of SiaQM purified in αDDM buffer have been obtained but X-ray diffraction was up ~20Å resolution. In order to improve the diffraction quality, detergent screening for SiaQM was performed using CPM assay to seek alternative detergents. We characterized the affinity between SiaPQM and Neu5Ac by using the biophysical methods. Homology modeling of SiaQM suggested that four amino acids might be Na+-bindind site. The sdudy optimized the detergents for protein purification and provided insight into future functional characterization.
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: