Background: A small heat shock protein AgsA was originally isolated from Salmonella enterica serovar Typhimurium. We previously demonstrated that AgsA was an effective chaperone that could reduce the amount of heat-aggregated proteins in an Escherichia coli rpoH mutant. AgsA appeared to promote survival at lethal temperatures by cooperating with other chaperones in vivo. To investigate the aggregation prevention mechanisms of AgsA, we constructed N- or C-terminal truncated mutants and compared their properties with wild type AgsA.
Results: AgsA showed significant overall homology to wheat sHsp16.9 allowing its three-dimensional structure to be predicted. Truncations of AgsA until the N-terminal 23rd and C-terminal 11th amino acid (AA) from both termini preserved its in vivo chaperone activity. Temperature-controlled gel filtration chromatography showed that purified AgsA could maintain large oligomeric complexes up to 50°C. Destabilization of oligomeric complexes was observed for N-terminal 11- and 17-AA truncated AgsA; C-terminal 11-AA truncated AgsA could not form large oligomeric complexes. AgsA prevented the aggregation of denatured lysozyme, malate dehydrogenase (MDH) and citrate synthase (CS) but did not prevent the aggregation of insulin at 25°C. N-terminal 17-AA truncated AgsA showed no chaperone activity towards MDH. C-terminal 11-AA truncated AgsA showed weak or no chaperone activity towards lysozyme, MDH and CS although it prevented the aggregation of insulin at 25°C. When the same amount of AgsA and C-terminal 11-AA truncated AgsA were mixed (half of respective amount required for efficient chaperone activities), good chaperone activity for all substrates and temperatures was observed. Detectable intermolecular exchanges between AgsA oligomers at 25°C were not observed using fluorescence resonance energy transfer analysis; however, significant exchanges between AgsA oligomers and C-terminal truncated AgsA were observed at 25°C.
Conclusions: Our data demonstrate that AgsA has several regions necessary for efficient chaperone activity: region (s) important for lysozyme chaperone activity are located outer surface of the oligomeric complex while those region(s) important for insulin are located inside the oligomeric complex and those for MDH are located within the N-terminal arm. In addition, the equilibrium between the oligomer and the dimer structures appears to be important for its efficient chaperone activity.
Springer Nature|BioMed Central
© 2010 Tomoyasu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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