pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants |
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Authors: | LAURA POLITI EMILIA CHIANCONE LAURA GIANGIACOMO LAURA CERVONI ANNA SCOTTO D’ABUSCO STEFANO SCORSINO ROBERTO SCANDURRA |
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Institution: | 1. Dipartimento di Scienze Biochimiche ’A.Rossi-Fanelli’, Sapienza, Università di Roma, Ple A. Moro 5, 00185, Roma, Italy |
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Abstract: | Recombinant amidase from Sulfolobus solfataricus
occurred as a dimer of 110 kDa comprising identical subunits.
Only dimers were present at pHs above 7.0, but with decreasing pH,
dimers associated into octamers, with complete oligomerization
occurring at pH 3.0. Oligomerization showed reversible
temperature-dependence, with octamer formation increasing with
temperature from 36 °C to between 70 and 80° C. Increasing
salt concentrations, favored dissociation of the octamers. Among the
three investigated factors affecting the dimer–octamer
equilibrium, the most important was pH. Among four mutants obtained by
site-specific mutagenesis and selection for pH and temperature
sensitivity, the T319I and D487N mutant amidases, like that of the
native Sulfolobus solfataricus, responded to changes
in pH and temperature with a conformational change affecting the
dimer–octamer equilibrium. The Y41C and L34P mutant amidases
were unaffected by pH and temperature, remaining always in the dimeric
state. The differences among mutants in protein conformation must be
related to the position of the introduced mutation. Although the L34P
and Y41C mutations are located in the helical region 33–48
(LLKLQLESYERLDSLP), which is close to the amino-terminal segment of
the protein, the T319I mutation is located in a strand on the surface
of the protein, which is far from, and opposite to, the amino-terminal
segment. The D487N mutation is located in the center of the protein,
far distant from the 33–48 segment. These observations suggest
that the segment of the protein closest to the amino-terminus plays a
key role in the association of dimers into octamers. |
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Keywords: | amidase archaea hyperthermophile oligomerization signature amidase |
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