Difference between revisions of "Part:BBa K1151000"
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<partinfo>BBa_K1151000 short</partinfo> | <partinfo>BBa_K1151000 short</partinfo> | ||
| − | The HpNikR protein is a pleiotropic regulator from Helicobacter pylori. In presence of nickel it can acts as an activator or a repressor depending of the specific promoter that contains its operator site. It consists of two dimeric DNA binding domains separated by a tetrameric regulatory domain that binds nickel. This domain corresponds to the C-terminal regulatory domain which contains four nickel binding sites at the tetramer interface. Binding nickel, then a conformational change allows it to activate or repress trascription. | + | The HpNikR protein is a pleiotropic regulator from ''Helicobacter pylori''. In presence of nickel it can acts as an activator or a repressor depending of the specific promoter that contains its operator site. It consists of two dimeric DNA binding domains separated by a tetrameric regulatory domain that binds nickel. This domain corresponds to the C-terminal regulatory domain which contains four nickel binding sites at the tetramer interface. Binding nickel, then a conformational change allows it to activate or repress trascription. |
| Line 8: | Line 8: | ||
===Biochemistry=== | ===Biochemistry=== | ||
| − | [[File: | + | [[File:Cattura122Unisalento.jpg]] [[File:Cattura7Unisalento.jpg]] |
| − | '''Figure 1:''' Representation of | + | '''Figure 1:''' Representation of Apo-NikR (from '''Dian et al.''', see the section References); HpNikR nickel-binding domain (tetramer) (from [[PDB Data Bank]]). |
| Line 17: | Line 17: | ||
[[File:Cattura1.jpg]] | [[File:Cattura1.jpg]] | ||
| − | '''Figure 2:''' Proteic sequence analysis (from | + | '''Figure 2:''' Proteic sequence analysis (from [[UniProt]]). |
===HpNikR expression using BL21 (DE3) cells=== | ===HpNikR expression using BL21 (DE3) cells=== | ||
| Line 23: | Line 23: | ||
First we made competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours. | First we made competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours. | ||
| − | [[File: | + | [[File:RitUnisalento.jpg]] |
'''Figure 3:''' Induction results. | '''Figure 3:''' Induction results. | ||
| Line 31: | Line 31: | ||
To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells). | To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells). | ||
| − | [[File: | + | [[File:42Unisalento.jpg]] |
'''Figure 4:''' PAGE of the separation. | '''Figure 4:''' PAGE of the separation. | ||
| Line 39: | Line 39: | ||
NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues. | NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues. | ||
| − | [[File: | + | [[File:41Unisalento.jpg]] |
'''Figure 5:''' Purification result (Eluate 1: 0,82 ug/ul). | '''Figure 5:''' Purification result (Eluate 1: 0,82 ug/ul). | ||
| − | + | As marker of the three gel seen so far we have used ColorBurst Electrophoresis Marker (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/c1992bul.pdf) by Sigma-Aldrich. | |
===HpNikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography=== | ===HpNikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography=== | ||
| Line 60: | Line 60: | ||
In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin. | In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin. | ||
| − | [[File: | + | [[File:resinaUnisalento.jpg]] |
'''Figure 6:''' Sephadex columns. | '''Figure 6:''' Sephadex columns. | ||
| Line 84: | Line 84: | ||
6. Sephadex resin after incubation with the sample n.4 | 6. Sephadex resin after incubation with the sample n.4 | ||
| − | [[File: | + | |
| + | (Protocol adapted from '''Abraham et al.''', see the section References). | ||
| + | |||
| + | [[File:machineryUnisalento.jpg]] | ||
'''Figure 7:''' Samples analysis. | '''Figure 7:''' Samples analysis. | ||
| Line 90: | Line 93: | ||
''Experimental data and results'' | ''Experimental data and results'' | ||
| − | [[File: | + | [[File:tabella1Unisalento.jpg]] |
| − | [[File: | + | [[File:icp3Unisalento.jpg]] [[File:icp2Unisalento.jpg]] |
| Line 101: | Line 104: | ||
===ATR-FTIR assay=== | ===ATR-FTIR assay=== | ||
| − | To complete our analysis on the protein, we studied the conformational changes which HpNikR goes | + | To complete our analysis on the protein, we studied the conformational changes which HpNikR goes under in the bond with Ni2+ ions through Attentuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). With this technique it is possible to acquire the infrared absorption spectra of our control (Apo-HpNikR) and of our protein treated with NiSO4 (complex HpNikR:Ni), so as to obtain the IR differential spectrum. By means of these it will be possible to highlight how some spectrum peaks suffer changes in infrared absorption due to changes in protein conformation induced by the nickel binding. |
| − | [[File: | + | [[File:ATRUnisalento.jpg]] |
'''Figure 8:''' Results. | '''Figure 8:''' Results. | ||
| Line 109: | Line 112: | ||
''Our hypothesis'' | ''Our hypothesis'' | ||
| − | The analysis of the spectrum of the native protein shows two peaks at values of wave number of 1261 cm-1 and 800 cm-1; these peaks are absent in the protein in the presence of nickel and everything is confirmed by the differential spectrum.These peaks could be assigned to a tyrosine residue | + | The analysis of the spectrum of the native protein shows two peaks at values of wave number of 1261 cm-1 and 800 cm-1; these peaks are absent in the protein in the presence of nickel and everything is confirmed by the differential spectrum.These peaks could be assigned to a tyrosine residue, which due to the interaction with nickel undergoes a deprotonation of the hydroxyl functional group. So it can be assumed that the bond to the metal induces a change necessary to the regulator NikR for the interaction with DNA. |
| − | [[File: | + | [[File:y3Unisalento.jpg]] |
'''Figure 9:''' The 72 tyrosine residue. | '''Figure 9:''' The 72 tyrosine residue. | ||
| + | |||
| + | ===CryoTEM microscopy of purified HpNikR=== | ||
| + | |||
| + | |||
| + | [[File:cryobigUnisalento.jpg]] [[File:cryoUnisalento.jpg]] | ||
| + | |||
| + | '''Figure 10:''' CryoTEM analysis results. | ||
| + | |||
| + | |||
| + | The high quality of the CryoTEM image displays: | ||
| + | |||
| + | - We made a correct sample preparation | ||
| + | |||
| + | - We set up correct set of experimental and instrumental conditions | ||
| + | |||
| + | - There is high probability that the image is relative to purified HpNikR protein. Assuming that ribosomal particles, whose molecular weight is around 200 kDa, have a diameter around 20 nm, the particle of diameter of around 1-2 nm which can be seen may coincide with a particle of MW around 20 kDa. HpNikR MW is around 17 kDa. | ||
| + | |||
| + | ===RAMAN spectroscopy analysis=== | ||
| + | |||
| + | Raman spectroscopy is a spectroscopic method based on the interaction between electromagnetic radiation used to observe vibrational, rotational, and other low-frequency modes in a system. | ||
| + | With Raman spectroscopy analysis it is possible to state that in the 1000-1600 cm-1 region the difference signal comes from amide groups (I, II e III). Instead, to high frequences, in the 2900-3200 cm-1 region the signal comes from the stretching of the CH2 and CH3 bonds. | ||
| + | Conformational deformations could be seen in the amide regions but it is necessary further investigation. | ||
| + | |||
| + | |||
| + | [[File:SenzaNiUnisalento.jpg]] [[File:ConNiUnisalento.jpg]] | ||
| + | |||
| + | '''Figure 11:''' RAMAN results. | ||
| + | |||
===Other=== | ===Other=== | ||
Latest revision as of 11:28, 4 October 2013
Nickel-responsive pleiotropic regulator (HpNikR)
The HpNikR protein is a pleiotropic regulator from Helicobacter pylori. In presence of nickel it can acts as an activator or a repressor depending of the specific promoter that contains its operator site. It consists of two dimeric DNA binding domains separated by a tetrameric regulatory domain that binds nickel. This domain corresponds to the C-terminal regulatory domain which contains four nickel binding sites at the tetramer interface. Binding nickel, then a conformational change allows it to activate or repress trascription.
Biochemistry
Figure 1: Representation of Apo-NikR (from Dian et al., see the section References); HpNikR nickel-binding domain (tetramer) (from PDB Data Bank).
Figure 2: Proteic sequence analysis (from UniProt).
HpNikR expression using BL21 (DE3) cells
First we made competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG for a time of 1, 2 and 4 hours.
Figure 3: Induction results.
Cytosol/membrane separation by Zerial method
To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells).
Figure 4: PAGE of the separation.
HpNikR purification by Ni-NTA resin
NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues.
Figure 5: Purification result (Eluate 1: 0,82 ug/ul).
As marker of the three gel seen so far we have used ColorBurst Electrophoresis Marker (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/c1992bul.pdf) by Sigma-Aldrich.
HpNikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography
We therefore decided to study the NikR protein: first, we focused on its characteristic to bind nickel. We have developed a protocol of incubation of the protein with nickel sulfate (stock: 10 ug/ul), in presence of an Incubation buffer (20 mM Tris pH 7,6, 100 mM NaCl). These are the samples we tested (each has a final volume of 100 ul):
1. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer
2. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer
3. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer
The samples were put on wheel at 4°C overnight.
In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin.
Figure 6: Sephadex columns.
In this way the protein with the nickel-binding sites saturated will be released first from the column, collecting the eluate easily. The nickel in excess will remain trapped in the pores of the resin.
ICP-AES assay
Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample. These are the samples (also here the final volume of each is 100 ul) that we have analyzed:
1. 100 ul Incubation buffer
2. 1,5 ul Nickel sulfate + 98,5 ul Incubation buffer
3. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer
4. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer
5. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer
6. Sephadex resin after incubation with the sample n.4
(Protocol adapted from Abraham et al., see the section References).
Figure 7: Samples analysis.
Experimental data and results
Discussion
Although it remains difficult to identify accurately the binding NikR-nickel stoichiometry: we can say, however, that between the two variables exists a direct relationship.
ATR-FTIR assay
To complete our analysis on the protein, we studied the conformational changes which HpNikR goes under in the bond with Ni2+ ions through Attentuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). With this technique it is possible to acquire the infrared absorption spectra of our control (Apo-HpNikR) and of our protein treated with NiSO4 (complex HpNikR:Ni), so as to obtain the IR differential spectrum. By means of these it will be possible to highlight how some spectrum peaks suffer changes in infrared absorption due to changes in protein conformation induced by the nickel binding.
Figure 8: Results.
Our hypothesis
The analysis of the spectrum of the native protein shows two peaks at values of wave number of 1261 cm-1 and 800 cm-1; these peaks are absent in the protein in the presence of nickel and everything is confirmed by the differential spectrum.These peaks could be assigned to a tyrosine residue, which due to the interaction with nickel undergoes a deprotonation of the hydroxyl functional group. So it can be assumed that the bond to the metal induces a change necessary to the regulator NikR for the interaction with DNA.
Figure 9: The 72 tyrosine residue.
CryoTEM microscopy of purified HpNikR
Figure 10: CryoTEM analysis results.
The high quality of the CryoTEM image displays:
- We made a correct sample preparation
- We set up correct set of experimental and instrumental conditions
- There is high probability that the image is relative to purified HpNikR protein. Assuming that ribosomal particles, whose molecular weight is around 200 kDa, have a diameter around 20 nm, the particle of diameter of around 1-2 nm which can be seen may coincide with a particle of MW around 20 kDa. HpNikR MW is around 17 kDa.
RAMAN spectroscopy analysis
Raman spectroscopy is a spectroscopic method based on the interaction between electromagnetic radiation used to observe vibrational, rotational, and other low-frequency modes in a system. With Raman spectroscopy analysis it is possible to state that in the 1000-1600 cm-1 region the difference signal comes from amide groups (I, II e III). Instead, to high frequences, in the 2900-3200 cm-1 region the signal comes from the stretching of the CH2 and CH3 bonds. Conformational deformations could be seen in the amide regions but it is necessary further investigation.
Figure 11: RAMAN results.
Other
The characterization of HpNikR-bound promoters can be seen at the pages BBa_K1151036 and BBa_K1151038.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]