Carson, Kenneth Harris (2006-08). Study and characterization of a novel small heat shock protein from Babesia. Doctoral Dissertation. Thesis uri icon

abstract

  • Many proteins can easily attain a non-native fold and be of no use or even a detriment to the host. The host cell has a myriad of molecules dedicated to assisting nascent and existing proteins in folding properly and maintaining the native fold. Of these molecular chaperones, the small Heat Shock Proteins (sHSP's) are an important group and worthy of study. The sHSP's are a diverse group of proteins that have in common an a-crystallin domain and generally display a chaperone activity. A sHSP (HSP20) isolated from the cattle parasite Babesia bovis has similar activities, and limited sequence homology to other a-crystallins. The gene encoding HSP20 was cloned into an expression system where the gene product was induced and purified for study. It was shown that HSP20 inhibits thermally induced aggregation of alcohol dehydrogenase at equimolar ratios. HSP20 was also used to significantly reduce amyloid formation of the b-Amyloid (1-40) Peptide in vitro at the sub-stoichiometric ratio of 1:10. A study of the oligomeric forms of HSP20 using size exclusion chromatography and gel electrophoresis revealed a broad range of multimers present in solution. The distribution of oligomers was affected by altering the solution conditions and concentration of the protein. The domains responsible for multimerization of HSP20 were mapped via sequence homology with known a-crystallins. These regions correspond to 12 carboxy-terminal amino acids and 50 amino-terminal amino acids. Truncated versions of HSP20 lacking these proposed oligomerization domains were created using PCR of the original gene and cloning into an expression vector as before. Using size exclusion chromatography, gel electrophoresis and analytical centrifugation, we show that the deleted domains alter the multimeric population of the protein in solution. The carboxy-terminal domain has a slight effect on multimerization while the amino-terminal deletion results in a drastic reduction in any multimers above a dimer under the conditions tested. Despite this drastic change in the multimerization of HSP20, there were no changes in the activities observed when compared to the full-length form. From this we conclude that the regions responsible for multimerization play little role in the observed activities of HSP20.
  • Many proteins can easily attain a non-native fold and be of no use or even a
    detriment to the host. The host cell has a myriad of molecules dedicated to assisting
    nascent and existing proteins in folding properly and maintaining the native fold. Of
    these molecular chaperones, the small Heat Shock Proteins (sHSP's) are an important
    group and worthy of study. The sHSP's are a diverse group of proteins that have in
    common an a-crystallin domain and generally display a chaperone activity. A sHSP
    (HSP20) isolated from the cattle parasite Babesia bovis has similar activities, and limited
    sequence homology to other a-crystallins. The gene encoding HSP20 was cloned into an
    expression system where the gene product was induced and purified for study. It was
    shown that HSP20 inhibits thermally induced aggregation of alcohol dehydrogenase at
    equimolar ratios. HSP20 was also used to significantly reduce amyloid formation of the
    b-Amyloid (1-40) Peptide in vitro at the sub-stoichiometric ratio of 1:10. A study of the
    oligomeric forms of HSP20 using size exclusion chromatography and gel electrophoresis
    revealed a broad range of multimers present in solution. The distribution of oligomers
    was affected by altering the solution conditions and concentration of the protein. The
    domains responsible for multimerization of HSP20 were mapped via sequence homology with known a-crystallins. These regions correspond to 12 carboxy-terminal
    amino acids and 50 amino-terminal amino acids. Truncated versions of HSP20 lacking
    these proposed oligomerization domains were created using PCR of the original gene
    and cloning into an expression vector as before. Using size exclusion chromatography,
    gel electrophoresis and analytical centrifugation, we show that the deleted domains alter
    the multimeric population of the protein in solution. The carboxy-terminal domain has a
    slight effect on multimerization while the amino-terminal deletion results in a drastic
    reduction in any multimers above a dimer under the conditions tested. Despite this
    drastic change in the multimerization of HSP20, there were no changes in the activities
    observed when compared to the full-length form. From this we conclude that the regions
    responsible for multimerization play little role in the observed activities of HSP20.

publication date

  • August 2006