Crystallization and preliminary X-ray crystallographic studies on Moxarella IgD-binding (MID) 764 mutant protein from Moraxella catarrhalis

Abstract

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My Experiments with Protein Crystallography
Visualization of proteins is very important to understand their structure – function relationship, it helps to identify and work with important drug targets. X-ray crystallography is basically a kind of high-resolution microscopy allowing us to realize such structures of proteins. In principle, the electron density of a macromolecule diffracts X-rays, which carries information regarding the respective electron density to a detector. Such diffraction data is used mathematically to backcalculate structural information on the macromolecule. A protein molecule individually is not great diffractor of X-Rays, but when arranged continuously in a 3-dimentional array the diffraction signal is amplified. The higher the order in such 3-dimentional arrays, better the diffraction data.

The sitting drop vapor diffusion method of crystallization uses a concentrated reservoir solution and a protein sample diluted with the reservoir solution in a sealed environment. Water diffuses from the diluted (protein) solution to a concentrated (reservoir) solution, increasing both protein and precipitant solutions in the sample drop. This will drive the system towards supersaturation where slow precipitation of the protein takes place and as water molecules become more scares, selforganization of the protein molecules through non-covalent bonds can eventually lead to nucleation and crystal formation.

Crystallization trials of MID764
The protein I choose for this work is from Moraxella catarrhalis. It is a gram-negative bacterium known to cause several respiratory tract infections, such as otitis media, sinusitis and it can elevate symptoms of COPD. The immunoglobulin D (IgD)-binding (MID) protein of M. catarrhalis is a 200-kDa outer membrane, capable of binding to IgD without inducing an immunological response and to human alveolar cells (MID764-913). MID protein helps M. catarrhalis in the first steps of infection and hence defines its virulence.

The aim of this study was to produce protein crystals of MID764 capable of diffracting to a resolution enough to solve its structure. The MID764 protein was successfully expressed and purified in large quantities. Initial trials failed but magnesium chloride was observed to enhance the crystallization. Crystal seeding and changed drop ratios let to formulation of MID764 crystals in 0.24M Lithium sulfate monohydrate 0.10M Tris hydrochloride pH 8.25, 21.00% w/v Polyethylene glycol 4,000 and 12mM Magnesium Chloride (shown), diffracting up to a resolution of 2.7Å, for which a native diffraction data set was collected. XDS software package showed that the crystal was in Space Group C2221. Attempts were made to collect anomalous data but with little or no success.

Abstract

Moraxella catarrhalis, a gram negative bacterium has the rare ability to bind to immunoglobulins without inducing an immunological response. M. catarrhalis is known to cause several respiratory tract infections, such as otitis media, sinusitis and it can elevate symptoms of COPD. The immunoglobulin D (IgD)-binding protein of M. catarrhalis is a 200 kDa outer membrane autotransporter capable of binding to IgD (MID962-1200) and to human alveolar cells (Adhesive / MID764-913). MID protein defines the virulence of M. catarrhalis.
Immunization studies showed effective clearance of M. catarrhalis in mice immunized with MID764-913, proving it to be an important candidate in M. catarrhalis vaccination.

The aim of this study was to structurally define MID764-913 using X-Ray crystallography. MID764-913was purified by Ni-NTA affinity chromatography followed by size exclusion chromatography. Initial crystallization trials failed, but the addition of magnesium chloride appeared to enhance the crystal quality. Successive trials proved crystal seeding and changing drop ratio to complement crystallization of MID764-913. Eventually crystals diffracting up to 2.7Å were grown in 0.24M lithium sulfate monohydrate 0.10M Tris hydrochloride pH 8.25, 21.00% w/v polyethylene glycol 4,000 and 12mM magnesium chloride with crystal seeding at MAX Lab, Lund, Sweden. A native data set of space group C2221 was collected, followed by several attempts to obtain an anomalous data set by MAD and MIR phasing by co-crystallization and soaking experiments, but failed to yield any significant results.

• author: Srinivas, Vivek
• supervisor: Marjolein Thunnissen
• organization: Department of Chemistry
• course: KEMT30 20131
• year: 2013
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Proteinvetenskap, Protein Science
• language: English
• id: 4286248
• date added to LUP: 2014-02-07 13:05:09
• date last changed: 2014-02-07 13:05:09