Overproduction, Purification and Structural Studies
283
hydrochloride and the EDTA solutions, used for dialysis of TthSI4, contained
0.45 ppm (Table 20.1, Sample 7) and 0.35 ppm (Table 20.1, Sample 8) Zn, respectively. The addition of the two values gives a result (0.8 ppm Zn) that is extremely
close to the value obtained for the non-denatured protein (0.796 ppm, Table 20.1,
Sample 1). This allows us to infer that the zinc ion has been quantitatively
removed from the protein through the concerted action of a denaturing and a
chelating agent.
5.2.4
Zn Attachment - Protein Renaturation
The denatured protein was dialysed against a buffer of 20 mM Tris-HCI pH 7.5,
0.1 M NaCl, which contained ZnClz in various concentrations. In order to remove
absorbed zinc ions, the protein was dialysed twice against the same buffer as
above containing 50 mM EDTA and finally against water. In all cases zinc
remained bound on the protein and could not be removed even by the action of
EDTA, which is in accordance with our results for the native protein (Table 20.1,
Sample 3). The Zniprotein molar ratio was lower than one when the zinc concentrations that were used were not in large excess of the protein concentration. The
ratio approached unity, when a 10:1 excess of ZnCh concentration over TthS14
concentration was used (Table 20.1, Sample 9). Similar values were obtained in
the case of the zinc-finger peptides of the estrogen-receptors (Archer et ai. 1990).
Based on the failure of EDTA to remove zinc we conclude that TthS14 was renatured. The percentage of protein that was renatured was limiting for the amount
of Zn that was bound. The molar ratio exceeded unity only when an excess of
ZnClz over 1:20 was used and this could be attributed to non-specific absorption
of Zn ions on the protein.
Thus, the existence of a zinc ion bound to the TthS14 zinc-finger like motif is
supported by experimental evidence and we conclude that the protein bears tetrahedrally coordinated zinc in a molar ratio of one, which is typical for zincfinger motifs. The zinc ion can only be removed by denaturation and the protein
rebinds Zn upon renaturation.
5.3
The Possible Role of the Zinc-Finger Domain in TthS14
The existence of zinc-finger motifs in several ribosomal proteins (Wool et al.
1995) and the detection of zinc in rat ribosomes (Chan et al. 1993) have led to the
question: do ribosomal proteins and, more specifically, their zinc-finger motifs
bind Zn?
In this work it is proven, as explained above, that T. thermophilus S14, a protein with a zinc-finger like motif, contains Zn. The data are in agreement with the
participation of the zinc ion in the formation of a zinc-finger structure. The 1:1
stoicheiometry and the absolute requirement for denaturation in order to detach
the Zn from the protein are very strong evidence in support of the participation
of the motif and the Zn in the formation of the zinc-finger domain.
283
hydrochloride and the EDTA solutions, used for dialysis of TthSI4, contained
0.45 ppm (Table 20.1, Sample 7) and 0.35 ppm (Table 20.1, Sample 8) Zn, respectively. The addition of the two values gives a result (0.8 ppm Zn) that is extremely
close to the value obtained for the non-denatured protein (0.796 ppm, Table 20.1,
Sample 1). This allows us to infer that the zinc ion has been quantitatively
removed from the protein through the concerted action of a denaturing and a
chelating agent.
5.2.4
Zn Attachment - Protein Renaturation
The denatured protein was dialysed against a buffer of 20 mM Tris-HCI pH 7.5,
0.1 M NaCl, which contained ZnClz in various concentrations. In order to remove
absorbed zinc ions, the protein was dialysed twice against the same buffer as
above containing 50 mM EDTA and finally against water. In all cases zinc
remained bound on the protein and could not be removed even by the action of
EDTA, which is in accordance with our results for the native protein (Table 20.1,
Sample 3). The Zniprotein molar ratio was lower than one when the zinc concentrations that were used were not in large excess of the protein concentration. The
ratio approached unity, when a 10:1 excess of ZnCh concentration over TthS14
concentration was used (Table 20.1, Sample 9). Similar values were obtained in
the case of the zinc-finger peptides of the estrogen-receptors (Archer et ai. 1990).
Based on the failure of EDTA to remove zinc we conclude that TthS14 was renatured. The percentage of protein that was renatured was limiting for the amount
of Zn that was bound. The molar ratio exceeded unity only when an excess of
ZnClz over 1:20 was used and this could be attributed to non-specific absorption
of Zn ions on the protein.
Thus, the existence of a zinc ion bound to the TthS14 zinc-finger like motif is
supported by experimental evidence and we conclude that the protein bears tetrahedrally coordinated zinc in a molar ratio of one, which is typical for zincfinger motifs. The zinc ion can only be removed by denaturation and the protein
rebinds Zn upon renaturation.
5.3
The Possible Role of the Zinc-Finger Domain in TthS14
The existence of zinc-finger motifs in several ribosomal proteins (Wool et al.
1995) and the detection of zinc in rat ribosomes (Chan et al. 1993) have led to the
question: do ribosomal proteins and, more specifically, their zinc-finger motifs
bind Zn?
In this work it is proven, as explained above, that T. thermophilus S14, a protein with a zinc-finger like motif, contains Zn. The data are in agreement with the
participation of the zinc ion in the formation of a zinc-finger structure. The 1:1
stoicheiometry and the absolute requirement for denaturation in order to detach
the Zn from the protein are very strong evidence in support of the participation
of the motif and the Zn in the formation of the zinc-finger domain.