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dc.contributorScholtz, J. Martin
dc.creatorSyed Ali, Abbas Razvi
dc.description.abstractA thermodynamic study of five homologous HPr proteins derived from organisms inhabiting diverse environments has been undertaken. The aim of this study was to further our understanding of protein stabilization in extremes of environment. Two of the proteins were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus subtilus. Genes for three of these homologous HPr proteins were for the first time cloned from their respective organisms into expression vectors and they were over-expressed and purified in Escherichia coli. Stability measurements were performed on these proteins under a variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent induced denaturation experiments. Stability curves were determined for every homologue and these reveal very similar conformational stability for these homologues at their habitat temperatures. The BstHPr homologue is the most thermostable and also has the highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr. Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of these variants is presented in an attempt to understand the origins of the differences in stability of the HPr homologues.
dc.publisherTexas A&M University
dc.subjectProtein Folding
dc.subjectConformational Stability
dc.subjectExtremophilic proteins
dc.subjectThermophilic proteins
dc.subjectDomain swapping
dc.subjectCD spectroscopy
dc.titleA comparative study of HPr proteins from extremophilic organisms

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