Structural characterization of proteins to investigate their roles in diseases: Focus on MID & LTA4H

• Protein molecules are responsible for many biological functions in cells. In order to fulfill their various biological roles, these chain-like molecules must fold into precise three-dimensional shapes. The knowledge of accurate molecular structures is a prerequisite for rational drug design and for structure based functional studies. Getting structural information of proteins can be a very difficult task, especially when it comes to high resolution. Moraxella catarrhalis is widely recognized human-restricted gram-negative bacterium for which it has become clear that it is a true pathogen of both the upper and lower respiratory tract. After Haemophilus influenzae and Streptococcus pneumonia, it is the third most common cause of otitis media in children. The bacterium can directly stimulate B-cells without any recognition of T-cells and it can therefore be classified as a T-cell independent antigen. The mitogenic activity of Moraxella catarhallis is performed by a 2139 residue long outer membrane protein MID. An IgD binding domain (MID962-1200) has been described and the colonization to human respiratory tract cells is mediated by a 150-residue adhesin domain (MID764-913). SAXS studies on the IgD binding domain showed that this domain has an elongated 3-fold organization and that there is the presence of unordered/flexible structures. CD data and prediction of secondary structure for both of the domains indicated the presence of large amounts of (∼33%) ß-sheet and ∼10% α-helix content. Native datasets for MID962-1200 to 2.3 Å resolution and for MID764-913 to 2.7 Å resolution are collected and processed. Vertebrate leukotriene A4 hydrolases are zinc metalloenzymes with an epoxide hydrolase and aminopeptidase activity belonging to the M1 family of aminopeptidases. The human enzyme produces LTB4, a powerful mediator of inflammation and is implicated in a wide variety of rheumatoid diseases. The yeast homolog scLTA4H contains only a rudimentary epoxide hydrolase activity and was shown to undergo a large conformational change upon binding of the inhibitor bestatin. In SAXS studies XlLTA4H shows a more compact form upon bestatin binding, but humLTA4H did not. It was confirmed that the LTA4H from Xenopus is a dimer and that it seems to contract in size upon bestatin binding. In contrast the human enzyme does not show any major difference in SAX scattering patterns upon inhibitor binding and it seems therefore that this enzyme does not display larger conformational changes.

• Popular Abstract in English Living organisms are composed of mainly carbohydrate, protein and lipid molecules. Protein molecules are responsible for many biological functions in cells. In order to fulfill their various biological roles, these chain-like molecules must fold into precise three-dimensional shapes. The knowledge of accurate molecular structures is a prerequisite for rational drug design and for structure based functional studies. Getting structural information of proteins can be a very difficult task, especially when it comes to high resolution. Different cell biological studies help us to identify protein molecules involved in living process as well as any molecule that might be responsible for malfunction in cellular metabolism. These kinds of protein molecules may come from a disease causing bacteria which help them to create establishment on human and take over control of the normal local cellular process. On the other hand protein molecules present in human body might not function properly or their excessive production in a pathway may lead to over reacting cellular metabolism. As a result human can get development of diseases. Upon identification of a responsible protein, it is possible to clone sequence of a gene into a vector which produces the protein in large amount. This large amount of protein allows studying them by different technique ranging from high to low resolution. Crystallography is a technique where crystals are grown from protein molecule using different methods. If successful, the crystal can be exposed to high pulse of x-ray and a diffraction pattern can be recorded. These diffraction pattern which comes in a form of image can be further analyzed with the help of some software or algorithm and a three dimensional model of a protein can be built. Small angle x-ray scattering (SAXS) is another technique where protein molecule in high concentration is exposed to x-ray and a scattering pattern can be obtained. These data containing scattering pattern can be used to build envelope like three-dimensional shape of the protein molecule. Another technique, which is widely used today, is circular dichroism where proteins secondary structure can be studied in solution. Compared to crystallography and SAXS, this technique is rapid and required less amount of sample. Moraxella catarrhalis is widely recognized human-restricted gram-negative bacterium for which it has become clear that it is a true pathogen of both the upper and lower respiratory tract. After Haemophilus influenzae and Streptococcus pneumonia, it is the third most common cause of ear pain in children. The bacterium can directly stimulate immuneregulatory (B) cells. The mitogenic activity of Moraxella catarhallis is performed by a 2139 amino acid long outer membrane protein MID. An IgD binding domain (MID962-1200) has been described and the colonization to human respiratory tract cells is mediated by a 150 amino acid adhesin domain (MID764-913). Vertebrate leukotriene A4 hydrolases are zinc metalloenzymes with an epoxide hydrolase and aminopeptidase activity belonging to the M1 family of aminopeptidases. The human enzyme produces LTB4, a powerful mediator of inflammation and is implicated in a wide variety of rheumatoid diseases. The yeast homolog scLTA4H contains only a rudimentary epoxide hydrolase activity and was shown to undergo a large conformational change upon binding of the inhibitor bestatin. The structural information obtained and discussed in this thesis will help the scientific community to better understand their role in cellular metabolism. This will also lead to design of specific drug to prevent the diseases.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

Moraxella IgD binding protein

adhesin

leukotriene A4 hydrolase

small angle x-ray scattering

crystallography

circular dichroism

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