In vivo site-specific photo-crosslinking using DiZPK

We used the site-specific photo-crosslinking method described previously (Cho et al., 2014) with some modifications. To incorporate N⁶-((3-(3-methyl-3H-diazirin-3-yl)propyl)carbamoyl)-L-lysine (DiZPK) into RcsF, we used the pSup-Mb-DIZPK-RS plasmid encoding an evolved Methanosarcina barkeri pyrrolysyl-tRNA synthetase (PylRS) and an optimized [OmpA-RcsF]=1000[RcsF]KdOmpA-RcsF suppressor (Zhang et al., 2011). DH300 ΔrcsF (PL358) cells were co-transformed with pSup-Mb-DIZPK-RS and one of the plasmids containing an amber codon in rcsF in pSC253. Cells were grown in 3-(N-morpholino)propanesulfonic acid (MOPS) minimal medium supplemented with 0.2% glucose, 0.2% L-arabinose (MOPS-glucose/arabinose minimal medium), and 0.8 mM DiZPK (no supplement of other amino acids; see the reasons for using MOPS medium below) (Neidhardt, Bloch, and Smith 1974). Cell cultures were grown to an OD₆₀₀ of 1-1.2 and 1 mL of samples was irradiated with UV light at 365 nm or left unirradiated for 10 min. Cells were precipitated with trichloroacetic acid (TCA), and the pellets were washed with acetone and solubilized in 60 μL of SDS-sample buffer before further analysis. We used a similar method to incorporate DiZPK into OmpA with minor modifications; DH300 ΔompA (PR46) cells were co-transformed with pSup-Mb-DIZPK-RS and one of the plasmids containing an amber codon in ompA in pPR21. Cells were grown in LB medium supplemented with 0.2% L-arabinose, 200 µM IPTG and 1 mM DiZPK. Cell cultures were grown to an OD₆₀₀ of 1 and 0.75 mL of samples was irradiated with UV light at 365 nm or left unirradiated for 10 min. Cells were precipitated with TCA, and the pellets were washed with acetone and solubilized in 100 µL of SDS-sample buffer before further analysis.

In previous experiments incorporating p-benzoyl-L-phenylalanine into RcsF, we used LB as the growth medium (Cho et al., 2014). However, we found that the expression levels of the DiZPK-containing RcsF mutant proteins were substantially lower when cells were grown in LB (data not shown); in contrast, the expression levels of RcsF were greatly enhanced in MOPS-glucose/arabinose minimal medium (data not shown). Therefore, we used MOPS-glucose/arabinose minimal medium for all photo-crosslinking experiments involving DiZPK-containing RcsF variants.