Conclusions

 An overexpression system has been developed for CNI fused to a HALO7- 6His which yields an amount of fusion protein that can be used for further study of the tagged version or CNI by itself after protease cleavage.

 From the detergents and buffers screened, DM in a Tris buffer was the most successful in solubilising and stabilising the protein fusion.

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Figure 38: LC-MS analysis of Full length CNI-HALO7-His fusion protein. The mass spectrometry measurement matches the calculated size of 55 541.6 Da based on the amino acid sequence very closely, proving that the purified protein is the full length protein. A second protein species is detected at 56240. 55 Da, 700.49 Da higher than the full length protein. All measured values are accurate to ± 1 Da.

Figure 39: LC-MS analysis of CNI-HALO7-His cleavage products: The HALO7-6HIS-tag. One product is the Halo-6His-tag with a calculated size of 34270.2 Da which is matched closely by this LC-MS analysis confirming that cleavage was successful and was carried out at the correct site in the protein fusion. All measured values are accurate to ± 1 Da.

Figure 40: LC-MS analysis of CNI-HALO7-His cleavage products: CNI. The calculated size of CNI is 20431.4 Da, but including the amino acids left over after cleavage at the 3C cleavage site the calculated size is 21289.4 Da, which is matched closely by the lower peak detected by LC-MS. The higher peak is 702.5 Da larger than the full length cleavage product. Allowing for ±1 Da measuring error this matches the difference observed in Figure 38.

Chapter 4. In vivo and in vitro Colicin N immunity protein

interaction with Colicin N

4.1 Introduction

Due to their hydrophobicity immunity proteins of pore-forming colicins are difficult to study in vitro. Hence, most published research (Chapter 1: Introduction) has focused on mutagenesis to define an interaction site in vivo, assuming a direct interaction between Colicin N and its immunity protein. While some immunity proteins are highly specific and only protect against one colicin, others provide immunity to several (Chapter 1: Introduction). The Colicin N immunity protein (CNI) is only known to provide immunity to Colicin N (ColN), while the Colicin A immunity protein (CAI) is only known to provide immunity to Colicin A (ColA). Inspired by mutagenesis studies of other Colicin immunity proteins (see Chapter 1: Introduction), this chapter focuses on the determination of an ColN-CNI interaction site using mutagenesis as well as attempts to recreate the interaction in vitro using pull-down assays and a surface plasmon resonance assay.

Presumably, if residues crucial for protein-protein interaction are mutated, CNI will no longer protect against ColN. However, mutagenesis always involves the risk of changing structurally important residues, therefore abolishing function through structural collapse rather than the change of functionally important sidechains. Therefore, usually, mutated proteins must be shown to be structurally intact, using biophysical techniques to assess stability such as CD, DSC, fluorescence spectroscopy and others. Since it is very difficult to purify stable CNI (chapter 3: CNI overexpression), here, structural integrity must be shown in vivo. Therefore, instead of just abolishing activity against ColN, it was attempted to create activity against ColA. Residues involved in specificity are not necessarily the same as residues involved in activity as seen in previous mutagenesis studies (chapter 1: introduction). A mutated immunity protein may still be able to perform its function of providing immunity against a colicin, even though it can change its specificity from Colicin N to Colicin A, for example. However, residues responsible for specificity and activity are likely to be located in the same region and given that structural analysis is difficult, analysing specificity rather than activity it is a good alternative. Hence, the following mutagenesis study aimed to establish the residues responsible for CNI specific

interaction with ColN, which may or may not be the same residues which are responsible for its activity.

Predominantly based on the hypothesis proposed by (Song and Cramer, 1991), this chapter tests if individual helices of CNI are involved in its interaction with Colicin N. It was planned to mutate whole helices first and then narrow down the interaction site to individual residues once the general area was known. The predicted helices of CNI and its closest homologue Colicin A immunity protein (CAI) have been swapped (Figure 41). CNI may cope well with mutagenesis of functional domains if the tertiary structure is flexible, but the secondary structure is fixed. The secondary structure is predicted to be predominantly α-helical. A diagram of the predicted topology is shown in Figure 42.

Figure 41: Helix swap between Colicin N and Colicin A immunity proteins. Helices are numbered 1-4 from N- to C-terminus. Each helix of CNI is replaced by the homologous helix of CAI. Ligation sites were chosen in regions of high sequence identity.

Figure 42: Predicted topology for CNI (A) and CAI (B). The TMHMM tool predicts 4 transmembrane helices spanning the E. coli inner membrane. N- and C- termini are located in the cytoplasm.

The sequence alignment of Colicin A immunity protein (NCBI Reference Sequence: WP_008323617.1) and Colicin N immunity protein (Stroukova and Lakey, 2015, in press, see appendix) are shown in Figure 43. Underlined amino acids indicate the predicted helical structure in both proteins (Krogh et al. (2001), Sonnhammer et al. (1998), online prediction tool10), while coloured regions mark swapped regions (yellow: helix 1, green: Helix 2, blue: helix 3, magenta: helix 4). The number indicates which helix of CNI was swapped for a helix from CAI. Overall, the proteins share a high nucleotide and amino acid sequence similarity. There is no region which stands out with special features, like a particularly high or low sequence identity, although, the region around helix 3 shows the least similarity.

Swapping a whole helix rather than individual residues not only increases the chance of swapping specificity but may be advantageous because a whole functional unit is

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swapped, increasing the likelihood of maintaining structural integrity. In homologues several residues must be changed in the same region to see a change in functionality and specificity and no single residue seems to be more important than the others (Lindeberg and Cramer, 2001; Smajs et al., 2006; Smajs et al., 2008).

The regions for swapping were chosen based on the predicted helix structure in both proteins (underlined in Figure 43).Maintaining the same residues at the ends of each region allowed a neat transition from one protein to the other. Furthermore, it was aimed to make the transition point in more flexible regions, like loops and turns, to allow tertiary structure folding and adjustment in case of steric hindrance. The predicted loops and turns connecting the helices were partially excluded from mutagenesis where possible based on previous research, which suggested that their role in protein – protein interaction is unlikely (Song and Cramer, 1991; Smajs et al., 2006; Smajs et al., 2008). The effect of mutagenesis was tested using two killing assays, a spot test assay on agar plates and a survival assay in liquid culture.

A CAI ATGATGAATGAACACTCAATAGATACGGACAACAGAAAGGCCAATAACGCATTGTATTTA 60 CNI ---ATGGAT---ATAAAAGACAGAAATAAGATAT-CAAAAAA---AATATCATTC 45 ***.** *::*.* **.**.*:.***:* *.**:** *:*.*.:**. CAI TTTATAATAATCGGATTAATACCATTATTGTGCATTTTTGTTGTTTACTACAAAACGCCA 120 CNI AGTCTTCTGCTCTTACTTTCCCCATTCGCATTAATATTTTTCAGTTATAATAATGCACCA 105 : *.*:.*..** * *:: .*****. .* .**:*** * . *** :* **:.*.*** CAI GACGCTTTACTTTTACGTAAAATTGCTACAAGCACTGAGAATCTCCCGTCAATAACATCC 180 CNI ATACCACTCCT----CGAAAAAAT-CATCGCATACCTA-TCCCTACCAGGATTTCATTCA 159 .:. *: *.** **:****:* *::*... ** * :. **.**. *:*:..:**. CAI TCCTACAACCCATTAATGACAAAGGTTATGGATATTTATTGTAAAACAGCGCCTTTCCTT 240 CNI TTAAACAACCCGCCCCTAAGCGAAGCATTCAATCTCTATGTTCATACAGCCCCTTTAGCT 219 * .:*******. ..*.* ..*.* ::* .**.* *** *.*:***** *****. * CAI GCCTTAATACTATACATCCTAACCTTTAAAATCAGAAAATTAATCAACAACACCGACAGG 300 CNI GCAATCAGCTTATTCATATTCACACACAAAGAATTAGAGTTAA-AACCAAAGTCGTCACC 278 **.:*.* . ***:***. *.**. : ***.:.: *.*.**** .*.***.. **:** CAI AACACTG-TACTTAG-ATCTTGTTTATTAAGTCCATTGGTCTATGCAGCAATTGTTTATC 358 CNI TCTGCGGGCACTAAAGATATTAACTCCTTTCACTATT-CTTTATATATCCATGATATACT 337 :. .* * ***:*. **.**.: *. *:: :* *** * ***. * *.** .*:** CAI TATTCT-GCTTCCGAAATTTTGAGTTAACAACAGCCGGAAGGCCTGTCAGATTAATGGCC 417 CNI GTTTCTTGCTAAC-TGACACAGAACTAACCTTGTCATCAAAAACATTTGTATTAATGTCA 396 :**** ***:.* :.* : :**. ****.: . *. **...*: * . ******* *. CAI ACCAATGACGCAACACTATTGTTATTTTATATTGGTCTGTACTCAATAATTTTCTTTACA 477 CNI AAAAACGATCTGTTTTTGTCTTTTTTCTATATAACACTATATATTGGGATATATATATTC 456 *..** ** .: : *.* **:** *****:. :**.** : :. .**:*: :*:: . CAI ACCTATATCACGCTATTCACACCAGTCACTGCATTTAAATTATTAAAAAAAAGG--- 531 CNI ACATATTTGTACTTTTGGTTCCTTATAGGAACATATAAGCTATTTACCAGGGGAGGAATA 516 **.***:* :. *:* : .* :.*.. :.***:***. ****:*..*...*. CAI --CAGTAA---- 537 CNI CTCCGTCGACAC 528 *.**.. B CAI MMNEHSIDTDNRKANNALYLFIIIGLIPLLCIFVVYYKTPDALLLRKIATSTENLPSITS 60 CNI -MDIK----DRNKISKKISFSLLLLLSPFALIFFSYNNAPIPLLEKIIAY--LSLPGFHS 53 *: : *..* .: : : ::: * *: **. * ::* .** : ** .**.: * CAI SYNPLMTKVMDIYCKTAPFLALILYILTFKIRKLINNTDRNTVLRSCLLSPLVYAAIVYL 120 CNI LNNPPLSEAFNLYVHTAPLAAISLFIFTHKELELKPKSSPLRALKILTPFTILYISMIYC 113 ** :::.:::* :***: *: *:*:*.* :* ::. .*: .::* :::* CAI FCFRNFELTTAGRPVRLMATNDATLLLFYIGLYSIIFFTTYITLFTPVTAFKLLKKRQ-- 178 CNI FLLTDTELTLSSKTFVLMSKNDLFLSFFYITLYIGIYIFTYLYFWFLIGTYKLFTRGGIL 173 * : : *** :.:.. **:.** * :*** ** *:: **: :: : ::**:.: CAI --- CNI RRH 176

Figure 43: CAI and CNI alignment using ClustalW. (A) Nucleotide sequence. (B) amino acid sequence. Highlighted text marks swapped parts; helix 1: yellow, helix 2: green and red, helix 3: blue and red, helix 4: magenta. Nucleotides and amino acid highlighted red are the endpoint of Helix 2 in the Helix 2 mutant as well as the beginning of Helix 3 in the Helix 3 mutant. Underlined text indicates predicted α- helical structure based on TMHMM (Krogh, 2001).