Materials and Methods

studies, POTRA 1 was shown to be irrelevant for the interaction with BamB. However, the glycine at amino acid position 70 located at the end of the second α-helix of POTRA domain 1 being in close proximity to POTRA 2 was substituted to a cysteine (G70C).

MGSSHHHHHHSSGLVPRGSHMM 21 AEGFVVKDIHFEGLQRVAVGAALLSMPVRTG 51 52 DTVNDEDISNTIRALFATGNFEDVRVLRDGDT 83 84 LLVQVKERPTIASITFSGNKSVKDDMLKQNLE 115 116 ASGVRVGESLDRTTIADIEKGLEDFYYSVGKY 147 148 SASVKAVVTPLPRNRVDLKLVFQEGVSAEIQQ 179 180 INIVGNHAFTTDELISHFQLRDEVPFFNVVGDR 212 213 KYQKQKLAGDLETLRSYYLDRGYARFNIDST 243 244 QVSLTPDKKGIYVTVNITEGDQYKLSGVEVSG 275 276 NLAGHSAEIEQLTKIEPGELYNGTKVTKMEDD 307 308 IKKLLGRYGYAYPRVQSMPEINDADKTVKLR 338 339 VNVDAGNRFYVRKIRFEGNDTSKDAVLRREM 369 370 RQMEGAFLGSDLVDQGKERLNRLGFFETVDT 400 401 DTQRVPGSPDQVDVVYKVKERNTGSFNFGIGY 432 433 GTE*

Fig. 4.2 Position of constructed single cysteines in PD-BamA. (A) PD-BamA contains a His-tag for purification (blue). Amino acid residues substituted by a cysteine are given in red together with the number showing the position of the amino acid within the sequence, whereby the His-tag was not included. For interaction studies with the lipid membrane, the construct contains nine amino acids (green) located C-terminal to the POTRA domains forming the first β-strand of the β-barrel domain of BamA. (B) Crystal structure of PD-BamA showing the POTRA domains 1 to 5 and the positions of the single cysteines in red. The configurated figure is based on the superimposed PDB structures 3efc and 3q6b containing the POTRA domains 1 to 4 and 4 to 5, respectively (Gatzeva-Topalova et al. 2008; Zhang et al. 2011). The figure was created with PyMol 1.8.0.7 for macOS.

CD spectroscopy was used to confirm correct folding of the mutants. For fluorescence studies, the single cysteine mutants of BamB or PD-BamA were spectroscopically labeled at the SH groups of the single cysteines. The experiments were either performed in the absence or in the presence of lipid vesicles composed of various lipid compositions to examine the relevance of the lipid bilayer for the formation of the BamAB complex. In a more detailed study, distances between various regions of both proteins were determined by fluorescence energy transfer (FRET).

4.3.2 Construction and purification of single cysteine mutants of PD-BamA

To study interactions between BamB and PD-BamA, single cysteine mutants of PD-BamA were designed by using an already existing pET15b vector encoding for a mutant of PD-BamA in which all tryptophan residues were replaced by phenylalanine (Talmon, 2016). PD-BamA does not contain any cysteines. The final plasmid, named pET15-WaF-PD-BamA, was used as template for the replacement of one residue by a cysteine. To prepare various single cysteine mutants of PD-BamA, the amino acids at positions 70, 136, 158 and 331 were selected for replacement by a cysteine. For mutagenesis, a PCR reaction was performed as described in the protocol of the QuikChange Site-Directed Mutagenesis XL Kit (Agilent technologies, USA).

Sequences for forward and reverse primers containing the desired mutation were selected with SnapGene Version 2.3.2 and these primers were then synthesized by Eurofins MWG Operon (Ebersberg, Germany) (Table 4.1).

Table 4.1: Oligonucleotide primers for site-directed mutagenesis in PD-BamA

substitution sequence of primers^*

G70C 5’-CGCTCTGTTTGCTACCTGCAACTTTGAGGATGTTC -3’

G136C 5’-CATTGCCGATATCGAGAAATGTCTGGAAGACTTCTAC-3’

L158C 5’-GCTGTCGTGACCCCGTGCCCGCGCAACCGTGTTG-3’

L223C^a 5’ -AAACTGGCGGGCGACTGTGAAACCCTGCGCAG-3’

A331C 5’-GATGCCCGAAATTAACGATTGCGACAAAACCGTTAAATTAC-3’

A363C^b 5’-GAAGGTAACGATACCTCGAAAGATTGTGTCCTGCGTCGCG-3’

N390C^c 5’-AGGGTAAGGAGCGTCTGTGTCGTCTGGGCTTCTTTG-3’

* Only the sequence of the forward primers is shown. Nucleotide substitutions are in boldface type. ^a,b,c Primers were designed by Talmon (PhD thesis, 2016).

After mutagenesis and sequencing (GATC Biotech AG, Germany) to confirm the mutation, each plasmid was transformed into the E. coli BL21 (DE3) strain. The additional plasmids containing the genes of the single cysteine mutants L223C, A363C and N390C of PD-BamA were created by Talmon (PhD thesis 2016) and have already been transformed into the BL21 (DE3) strain (Table 4.2). The corresponding glycerol stocks (E. coli cells of the strain BL21(DE3) harboring the plasmids pET223 (L223C), pET363 (A363C) or pET390 (N390C)) as well as the previously transformed mutants were used to inoculate LBAmp Medium (ampicillin concentration: 100 μg/ml). The protocol of expression and purification was comparable to that described in section 3.3.2 and Talmon (2016). All mutants were analyzed by CD spectroscopy to confirm wild-type-like structure.

Table 5.2: List of plasmids and proteins of single cysteine mutants of PD-BamA^* plasmid vector Cys position substitution/product source

pET15b 70 G70C this work

pET15b 136 G136C this work

pET15b 158 L158C this work

pET223 pET15b 223 L223C Talmon, PhD thesis 2016

pET15b 331 A331C this work

pET363 pET15b 363 A363C Talmon, PhD thesis 2016

pET390 pET15b 390 N390C Talmon, PhD thesis 2016

* All native tryptophans have been replaced by phenylalanine.

4.3.3 Expression and purification of single cysteine mutants of BamB

The protocol of construction, overexpression and purification of single cysteine mutants of BamB is described in section 2.3.3.

4.3.4 Preparation of lipid vesicles

1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phospho-ethanolamin (DLPE) and 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG) were purchased in powder form from Avanti Polar Lipids (Alabaster, USA). LUVs with a diameter of 100 nm were either prepared in pure form (DLPC, DLPG) or in molar ratios (DLPE/DLPG (4:1)) by extrusion as described in section 3.3.4 in glycine buffer (10 mM glycine, 2 mM EDTA, pH 8.0).

4.3.5 CD spectroscopy

Folding of all single cysteine mutants of PD-BamA was confirmed by CD spectroscopy using a Jasco J-815 CD spectrometer (Jasco, Gross-Ulmstadt, Germany). All samples were dialyzed to remove residual urea in Tris buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Folded mutants of PD-BamA (diluted to 12 μM in Tris buffer) were analyzed as described in section 2.3.5 with identical instrument settings. Six scans were accumulated to obtain a spectrum of a PD-BamA mutant. A background spectrum was subtracted that was recorded for the same buffer, but in the absence of any PD-BamA. The CD-spectra were normalized and then analyzed using the software and reference data sets of the DICHROWEB server as described in section 2.3.5.

4.3.6 Labeling of the cysteine residue

The single cysteine residues of the various mutants of PD-BamA were spectroscopically labeled with the SH-reactive fluorescent probe 5-IAF (Sigma Aldrich, St. Louis, USA) or IAEDANS (Invitrogen, Thermo Fisher Scientific, USA) whereas the cysteine residues of BamB were labeled with IAEDANS or IANBD as described in section 2.3.6. For the labeling procedure of PD-BamA, 100 μΜ of the single cysteine mutant was diluted in 1 ml Tris buffer (20 mM Tris,

1 mM EDTA, 7 M urea, pH 7.2) before a 10-fold molar excess of TCEP was added and the mixture was incubated for 30 min at room temperature. Afterwards, a 20-fold molar excess of the appropriate reactive dye (5-IAF or IAEDANS) was added to the reduced protein and the mixture was reacted over night at 4°C in dark to protect the sensitive fluorophores from light.

The unreacted excess of the fluorescent dye was removed by extensive dialysis against Tris buffer (10 mM Tris, 2 mM EDTA, 7 M urea, pH 8.0). The protein concentration and the degree of labeling was estimated as previously described in section 2.3.6.

4.3.7 S-Methylation of cysteine

For calculations of the Först r’s distance R0 and of the actual donor-acceptor distance r, it is necessary to record fluorescence emission spectra of IAEDANS-labeled single cysteine mutants of BamB also in the absence of the FRET acceptor 5-IAF. Unmodified SH groups of cysteines are able to quench the fluorescence emission of neighboring fluorophores when they are in close proximity (Cowgill, 1967). To prevent fluorescence quenching, the SH groups of the cysteine residues were reacted with Methyl 4-nitrobenzene sulfonate (MNB) and converted into S-methyl derivates (Heinrikson, 1971). The S-methylation reaction was performed by diluting 50 μM of the PD-BamA single cysteine mutant in 1 ml borate buffer (50 mM borate, 1 mM EDTA, 7 M urea, pH 9.0). Disulfide bonds of cysteines were reduced in a 10-fold molar excess of TCEP and the sample was incubated for 30 min at room temperature. A 20-fold molar excess of MNB was added and the reaction was incubated for 90 min at 37°C. The unreacted excess of MNB was removed from the sample by extensive dialysis against Tris buffer (10 mM Tris, 1 mM EDTA, 7 M Urea, pH 8.0) at 4°C. After concentrating the labeled PD-BamA to a smaller volume, its concentration was estimated and the complete labeling with MNB was confirmed as described in section 2.3.6.

4.3.8 Fluorescence spectroscopy

4.3.8.1 Fluorescence studies on single cysteine mutants of IAEDANS-labeled PD-BamA

The fluorescence emission of 1 μM IAEDANS-labeled single cysteine mutants of PD-BamA was excited at 336 nm and the spectra were recorded from 410 nm to 650 nm using a Spex Fluorolog-3 spectrofluorometer (Horiba/Jobin-Yvon, Germany). The bandwidths of the excitation and emission monochromators were set to 2.5 nm and 5 nm, respectively. The integration time was 0.05 s and the increment for the scan was 0.5 nm. All measurements on PD-BamA were performed in glycine buffer (10 mM glycine, 2 mM EDTA, pH 8.0) at 25°C.

For binding studies, background spectra of lipid membranes or of wt-BamB were recorded at a

concentration of 600 mM total lipid (DLPE/DLPG (4:1) or of 1 μM wt-BamB. For the background spectra, three scans were averaged. For all other fluorescence spectra, six scans were averaged and the corresponding background spectra were subtracted. The fluorescence emission spectra were analyzed using IGOR Pro 8 (Wavemetrics, Oregon).

4.3.8.2 Fluorescence studies on single cysteine mutants of IANBD-labeled BamB Fluorescence spectra were recorded as described in section 4.3.8.1 for IAEDANS-labeled PD-BamA, but exciting the fluorescence of IANBD-labeled BamB (0.5 μM) at 478 nm and recording the spectra from 490 nm to 700 nm under otherwise identical instrument settings. For interaction studies between BamB and PD-BamA or the lipid membrane, lipid vesicles (DLPC, DLPG or DLPE/DLPG (4:1), 300 mM) or PD-BamA/wt-BamA (0.5 μM) were added in the background sample. Wt-BamA was folded overnight into lipid vesicles whereas PD-BamA was added immediately. All samples were incubated for 5 min in the cuvette before recording the spectra. The averaged background spectrum of three measurements without BamB was subtracted from the final measurement of six averaged spectra. The fluorescence spectra were analyzed using IGOR Pro 8 (Wavemetrics Oregon).

4.3.8.3 Intermolecular FRET measurements between BamB and PD-BamA

The transfer of energy was measured between the IAEDANS-labeled single cysteine mutants of BamB and the 5-IAF or S-methylated single cysteine mutants of PD-BamA using a Spex Fluorolog-3 spectrofluorometer (Horiba/Jobin-Yvon, Germany). The excitation wavelength was 336 nm for IAEDANS and the fluorescence spectra were recorded in a range from 410 nm to 650 nm. The bandwidths of the excitation and emission monochromators were 2.5 nm and 5 nm, respectively. First, 0.5 μM of the acceptor (either 5-IAF-labeled or S-methylated single cysteine mutant of PD-BamA) was added to the cuvette and diluted in 1 ml glycine buffer (10 mM glycine, 1 mM EDTA, pH 8.0) in the presence or in the absence of lipid vesicles composed of DLPE/DLPG (4:1, 300 mM). Afterwards, 0.5 μM of the donor, the IAEDANS-labeled single cysteine mutant of BamB, was added and the sample was incubated for 10 min at 25°C before the final spectrum was recorded six times. The background spectrum containing only the acceptor either in the absence or in the presence of lipid vesicles was subtracted.

4.3.8.4 Calculation of the efficiencies of energy transfer and of distances between donors and acceptors in complexes of labeled BamB and PD-BamA

To determine the efficiency of energy transfer ET and to calculate the distances between the donor (IAEDANS in labeled mutants of BamB) and acceptor (5-IAF in labeled mutants of PD-BamA), fluorescence emission spectra were measured. For mobile dipoles the energy in dipole-dipole interactions is inversely proportional to the sixth power of distance (1/r⁶) between the donor (D) and acceptor (A) molecules. The FRET efficiency ET describes the amount of absorbed donor photons transferred to the acceptor and is given by equation 4.1:

ET = R06/ R06 + r⁶ or r = R0 (1/ET –1)^1/6 (Eq. 4.1) with r being the distance between the donor and acceptor pair and R0 being the critical or

“Först r” distance at which the FRET efficiency is 50 % (Lakowicz, 2006). The distance r between the donor and acceptor molecules can be calculated from Eq. 4.1 if ET and Förster’s critical distance R0 are known. ET is calculated from the relative fluorescence intensities of the donor in the presence (FDA) and in the absence (FD) of the acceptor by using equation 4.2:

ET = 1 – FDA/FD (Eq. 4.2) The critical Först r’s distance R0 depends upon κ², n, QD and J(λ) and can be calculated by

R0 = 9.78 · 10³ (κ²n⁻⁴ QDJ(λ))^(1/6) in Å (Eq. 4.3) if the wavelength is expressed in cm and the overlap integral J(λ) in the units of M^–1 cm³. The orientation factor κ ² describes the average of orientation of the freely rotated dyes of the donor and acceptor leading to a mean value of 2/3. n is the refractive index, taken as 1.4 and QD

describes the quantum yield of the donor IAEDANS in the absence of the acceptor and is determined by (Kronman et al., 1971; Liang and Chakrabarti, 1982):

QD = (F/FIAED) · (AIAED/A) · 0.63 (Eq. 4.4) with F as the fluorescence of the donor in the absence of the acceptor and FIAED as the fluorescence of free IAEDANS, both integrated between 410 nm and 580 nm. A and AIAED are referring to the absorbances of the donor and of free IAEDANS at 336 nm, respectively. 0.63 is used as quantum yield of free IAEDANS (Dalbey et al., 1983; Boey et al., 1994). J is the

overlap integral given in M^–1 cm³ representing the degree of overlap between the donor emission spectrum and the acceptor absorbance spectrum and is defined as follows:

J = ∫ FD (λ) εA (λ) λ ⁴ d λ / ∫ FD (λ) d λ (Eq. 5.5) FD (λ) describes the fluorescence intensity of the donor in the absence of the acceptor and εA is the excitation coefficient of the acceptor in units M^–1 cm^–1.