Due to the enhanced labeling capability of maleimide-based fluorescent probes in in vitro experiments, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. Here we show that, although no noticeable changes were detected from in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK-tag substantially altered DNA compaction rates by Bacillus subtilis ParB protein in in vitro single-molecule DNA flow-stretching experiments. Furthermore, our measurements and statistical analyses demonstrate that the KCK-tags also altered the ParB protein’s response to nucleotide (cytidine triphosphate CTP or its nonhydrolyzable analog CTPγS) binding and the presence of the specific DNA binding sequence (parS). Remarkably, the appended KCK-tags are capable of even reversing the trends of DNA compaction rates upon different experimental conditions. DNA flow-stretching experiments for both fluorescently-labeled ParB proteins and ParB proteins with an N-terminal glutamic acid-cysteine-glutamic acid (ECE) tag support the notion that electrostatic interactions between charges on the tags and the DNA backbone are an underlying cause of the protein’s property changes. While it is typically assumed that the short KCK-tag minimally perturbs protein function, our results demonstrate that this assumption must be carefully tested when using tags for protein labeling.
Molina, M., Way, L. E., Ren, Z., Liao, Q., Wang, X., & Kim, H. (2023). Lysine-Cysteine-Lysine (KCK) tag changes ParB action in vitro but not in vivo. bioRxiv : the preprint server for biology, 2023.03.19.533373. https://doi.org/10.1101/2023.03.19.533373