Biophysical methods

2.4.1 Liquid chromatography–mass spectrometry (LC-MS)

The CNI-HALO7-6His samples were prepared for LC-MS using the final CNI-HALO7- 6His purification protocol with the Tris buffer outlined in 2.3.1. The final concentration of protein provided was 1.12 mg/ml. The LC-MS measurement and the data analysis was carried out by the LC-MS specialist Dr Joseph Gray, Newcastle University, as a service provided by Pinnacle Lab2.

2.4.2 Circular Dichroism

To open up the helix-bundle structure of ColN P-domain in preparation for the pull- down assays (2.4.3), ColN P-domain, purified using the procedure outlined in 2.3.1, was incubated in a Tris buffer (10 mM Tris, 300 mM NaCl, pH=7.4) with and without 0.1 % DM for 24 h at 4 °C. Near-UV Circular Dichroism was used to investigate whether the tertiary structure had changed due to the added DM (Dover et al., 2000). The spectra were measured on a CD Spectrometer Jasco J-810. Parameters: band width of 2 nm, response time: 4 sec, measurement range: 320-250 nm, data pitch: 0.5 nm, scanning speed 20 nm/min, cell length: 1 cm, temperature: 25 °C, protein concentration: 3.8453 x 10-5 mol/l. The traces are an average of 9 accumulations.

2.4.3 CNI pull-down assay

A pull-down assay was used to assess protein-protein interaction between CNI and Colicin N. CNI-HALO7-6His was bound via its Halo-tag to HaloLink resin (Promega) according to the manufacturer’s instructions by calibrating the HaloLink resin with a Tris buffer (50 mM Tris-HCl, pH = 7, 300 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 % DM, 10 % glycerol) and incubating the resin with CNI-HALO7-6His for 30 min at room temperature. Excess protein was washed off with Tris buffer and the resin was incubated with ColN, ColN P-domain, ColA and ColA P-domain in Tris buffers containing either 1.6 mM DM, 25 mM OG or 8 mM SDS at 4 °C overnight. Excess protein was washed off and to establish what remained bound to the beads due to protein-protein interaction, the beads were resuspended in protein loading buffer (0.125 M Tris HCl, pH = 6.8, 15 % glycerol, 5 mM NaEDTA. 2 % SDS, 0.1 %

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bromophenol blue, 1 % β-mercaptoethanol) and heat-denatured at 90 °C for 10 min. Washes and “pulled-down” proteins were analysed using SDS-PAGE.

2.4.4 Surface Plasmon Resonance (SPR)

In preparation for SPR, the proteins to be used here were purified with the protocols outlined in (2.3.1). For CNI, the CNI-Halo7-6His final purification protocol with Tris buffer was used. All proteins were buffer exchanged with a PD-10 column (GE Healthcare) against a HEPES buffer (10 mM HEPES pH = 7.5, 150 mM NaCl, 50 μM EDTA, different concentrations of detergents DM, OG or SDS) as used in Johnson et al. (2014) and incubated at 37 °C for 1 h to open up the P-domain structure with the detergent. The chip used for SPR was a GE Healthcare NTA Sensor Chip. The chip matrix consists of carboxymethylated dextran pre-immobilized with nitrilotriacetic acid (NTA). His-tagged proteins can be immobilised through nickel ions which bind the histidine imidazole rings as well as NTA.

Before the SPR binding experiments could be carried out, the polyhistidine-tag on ColN, ColA, ColN P-domain and ColA P-domain had to be modified with diethylpyrocarbonate (DEPC; Sigma-Aldrich) to prevent these proteins from binding to the NTA chip. The modification method was adapted from Lundblad and Noyes, 1984, chapter 9. A specific amount of DEPC was added to the protein solution and incubated at room temperature for 20 min, before quenching with an excess of 5 mM imidazole. It was unknown how much DEPC would be enough to modify the imidazole ring on the His-tag, but not disturb the protein structure and prevent potential protein-protein interaction. Therefore, different molar ratios were investigated and SPR was used to determine if the modified protein would still bind to the NTA-Ni+ chip.

Here, DEPC was added in molar ratios between DEPC and histidine molecules. All proteins were diluted to a final concentration of 15 µM. The molar concentration of histidine was calculated based on the protein concentration and the amount of histidines per molecule. For example, for a polyhistidine-tag with 6 histidines and a protein concentration of 15 µM, the solution had a histidine concentration of 6 x 15 = 90 µM. If DEPC was added in a 1:5 ratio, the final concentration of DEPC in the solution had to be 5 x 90 = 450 µM. ColN P-domain has 7 His, ColN has 13 His, ColA P-domain has 9 His and ColA has 13, all including the 6His-tag which is common to all these proteins. DEPC modifies the imidazole ring of histidine residues and cannot

discriminate between the histidine in the tag and the histidine in the main protein. The progress of modification was observed by measuring protein absorption at 240 nm and 280 nm with a UV spectrophotometer (UV-1800 Shimadzu). An optimisation experiment, testing if the modified protein still bound to the chip, was carried out with ColN P-domain, where DEPC was added in a ratio of 1:1 based on the 6 histidines in the tag only and based on all 7 histidines ratios of 1:1, 1:5, 1:10 and 1:25. To confirm 1:5 ratio as the optimal ratio, a further run was carried out with ratios 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 based on all 7 histidines.

The SPR was carried out on a BIAcore X100 at a flow rate of 5 μl/min. The NTA chip has two chambers, one for measuring and one for reference to detect any non- specific binding. The chip is cleaned with 0.5 % SDS, 2 M NaCl, HEPES regeneration buffer (10 mM HEPES, 150 mM NaCl2, 500 mM EDTA, 5 mM CaCl2, pH

= 8.3) and HEPES running buffer (10 mM HEPES, 150 mM NaCl2, 50 µM EDTA, 5

mM CaCl2, pH = 7, 0.1% DM). For each binding run, the HEPES Nickel solution (0.5

M NiCl2, 10 mM HEPES, 150 mM NaCl2, 50 µM EDTA, pH = 7.5) flows across the

measuring cell only. Subsequently, all proteins flow across both chambers. For experiments where OG and SDS were added to the P-domain to open up its structure even further than by 0.1 % DM addition only, P-domains were modified with DEPC first (as above) and then incubated with OG (0, 0.1 %, 0.25 %, 0.5 %, 0.731 % (=CMC), 1 %, 1.5 %.) or SDS (0.4 mM, 0.8 mM, 1.6 mM, 4 mM, 8 mM (=CMC), 16 mM) for 1 h at 37 °C. The procedure was adapted from Johnson et al. (2014).