Protein mis-folding and human disease

Serum Amyloid P Component (SAP), a putative molecular chaperone, is a homopentameric<br/>plasma protein of 25kDa subunits. It binds to the amyloid fibrils of<br/>misfolded proteins, which cause amyloidosis in humans. SAP not only stabilizes<br/>amyloid fibrils but also protects them from proteolytic and cell mediated<br/>degradation. SAP has been co-crystallized with three different aminoalkyl<br/>phosphonates that bind at the amyloid recognition site of SAP, and the X-ray<br/>crystal structures were determined at atomic resolution.<br/><br/>A secondary aim of this work was to understand the enhanced amyloidogenic<br/>potential of L55P and V30M transthyretin (TTR) protein. TTR misfolding has been<br/>implicated in number of human diseases such as senile systemic amyloidosis,<br/>familial amyloid polyneuropathy and familial amyloid cardiopathy. TTR protein is a<br/>thyroxine binding protein (14kDa) existing as a tetramer in vivo. L55P and V30M<br/>mutant TTR are the most aggressive and most common mutants, respectively, in<br/>causing FAP. L55P and V30M mutant TTR protein were expressed in E.coli and<br/>purified using anion-exchange chromatography and gel filtration. L55P and V30M<br/>TTR were co-crystallised with MDS84, a compound that has been demonstrated to<br/>stabilize the tetramer in vitro. The X-ray structures of L55P and V30M, TTR mutant<br/>proteins have been determined at 1.5Å and 2.1Å resolution.<br/><br/>In addition, research was carried out on the bacterial protein Burkholderia invasion<br/>protein D (BipD). BipD (33kDa) belongs to type III secretion system of Burkholderia<br/>pseudomallei. It creates a pore in the host cell membrane to help the B.<br/>pseudomallei invasion. This bacterial infection causes melioidosis disease in<br/>humans. To determine the ligand recognition site of BipD, its high-resolution crystal<br/>structure has been determined at 1.5Å. This high resolution BipD structure is more<br/>complete than its previously solved structures and is in the new space group C2.<br/>The BipD structure presented in this research may help to design potential<br/>chemical inhibitors of BipD to prevent bacterial invasion into human body.<br/><br/>The molecular chaperones play an important role in the protein refolding and<br/>assembly. ATJ11 is of 14kDa protein belongs to DnaJ co-chaperone family. The<br/>protein has been expressed in E.coli and purified by affinity and cation exchange<br/>chromatography. The biophysical study of ATJ11 (CD spectrum) has been<br/>conducted showing its predominant ?-helical structure and attempts were made to<br/>crystallise it but no protein crystals have been obtained.<br/>

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2010

Author dc:creator

• Pal, Mohinder

Advisors dc:contributor.advisor

• Wood, Steve P.
• Coker, A.R.