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Crystallization of Hemagglutinin Receptor Binding Domain to Determine the Binding Mode of a Novel Inhibitor

Anindya, A.L. (2015) Crystallization of Hemagglutinin Receptor Binding Domain to Determine the Binding Mode of a Novel Inhibitor. Master's Thesis / Essay, Biology.

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Abstract

Hemagglutinin (HA) is one of the major surface protein in influenza virus, along with neuraminidase and M2 proteins. It plays a major role at the beginning of the viral cycle, allowing the influenza virus to enter host cells by binding to sialic acid on the host cell’s surface. While drugs targeting neuraminidase and M2 proteins have been marketed widely, such as amantadines, rimantadines, zantamivir, and oseltamivir, hemagglutinin-directed drugs still remain to be developed. In addition to its major role in the initial stage of viral attachment to host cells, hemagglutinin is not prone to mutation, unlike neuraminidase and M2. The conformation of its receptor-binding domain is relatively conserved due to the binding specificity to sialic acid. Mutation in this region may risk the ability of the virus to bind to the host cell in the first place. The sialic acid binding pocket in hemagglutinin protein is therefore an interesting starting place to develop antiviral drugs. Using in silico modelling ANCHOR.QUERY, HA-directed small molecules have been synthesized and screened for activity. Crystallization is needed to determine the binding mode of several small molecules to the sialic acid binding pocket. HA-receptor binding domain (HA-RBD) was known to be successfully expressed from E. coli bacteria as insoluble inclusion bodies, which need to be denatured by a strong chaotropic agent, for example urea. Denatured protein can be refolded into its native state in optimal conditions, otherwise it can be misfolded into aggregates. To estimate this optimal condition, 4 refolding methods were screened: fast dialysis, slow dialysis, Immobilized Metal Affinity Chromatography (IMAC), and shock dilution. Arginine and ßME were also used in the experiment to help promote correct refolding. HA-RBD protein was found to be refolded in all refolding methods to various degrees, with IMAC and shock dilution methods having the best results for producing monomeric protein in solution. HA-RBD protein crystals were obtained from scaling up expression and refolding protocols (using the shock dilution method), at a protein concentration of 5.6 mg/mL. HA-RBD protein was shown to have a better thermal stability in our dialysis buffer than the buffer used in a previous study by DuBois et al. (2011). Diffraction of HA-RBD crystals shows the protein has crystallised and we have performed crystal-compound soaking experiments.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Biology
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 08:23
Last Modified: 15 Feb 2018 08:23
URI: http://fse.studenttheses.ub.rug.nl/id/eprint/14334

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