Difference between revisions of "Part:BBa K1151000"

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