KCK-tag-induced quantitative and qualitative property changes of DNA-binding protein ParB.

Abstract

Lysine-cysteine-lysine (KCK) tags are commonly employed to label proteins with maleimide-fluorescent dye conjugates to enhance dye-labeling efficiency. In our study, we systematically quantified the actions of a DNA-binding CTPase ParB protein and its KCK-tagged versions on flow-stretched lambda DNA using an in vitro single-molecule DNA flow-stretching assay. We measured DNA compaction rates in a series of experimental conditions, including the presence of the specific binding sequence (parS), CTP, or its non-hydrolyzable analog CTPγS. We found that KCK tag (either at N- or C-terminus) artificially increased the compaction rates of flow-stretched DNAs and even reversed ParB proteins’ response to these experimental conditions. DNA flow-stretching experiments with fluorescently-labeled ParB protein showed that the appended KCK-tag enhances protein loading onto a DNA. Furthermore, DNA compaction by ParB protein with a negatively-charged glutamate-cysteine-glutamate (ECE) tag was inefficient compared with its non-tagged ParB protein. These results suggest that the quantitative and qualitative changes of the KCK-tagged proteins were, at least in part, derived from electrostatic interactions between positively-charged KCK-tag and negatively-charged DNA backbone. Interestingly, we found that the KCK-tagged ParB proteins do not lead to any noticeable changes from in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays. It implies that a lack of perturbation in in vivo assays does not necessarily guarantee intact in vitro results. Despite the small size of KCK-tag, when it is used for fluorescent-dye labeling on a protein, rigorous test must be accompanied to ensure absence of any in vitro artifacts.