Knot proteins were regarded as structural impossibilities, until they were observed embedded in naturally occurring proteins. They are fascinating proteins whose backbones adopt knotted configurations with a wide range of topologically interesting features. However, little is known about the folding/unfolding pathways. In the present work, the kinetically trapped intermediates of two knot proteins, ubiquitin C-terminal hydrolase (UCH) and a methyltransferase YibK, were investigated using trapped ion mobility spectrometry - mass spectrometry (TIMS-MS). Experimental insights on the structural integrity of the two knot proteins were provided by varying the starting solvent conditions (i.e., organic content), collisional energy (i.e., CIU), and temperature. Our results suggest that the integrity of the knot core of YibK is more stable than the knot core of UCH and the loss of the knot core can cause a structural collapsing or unfolding event. The potential of the present approach is demonstrated to provide an invaluable insight necessary for the structural integrity elucidation of the knot proteins in the gas-phase.

Insights on the structural integrity of knot proteins using trapped ion mobility spectrometry – mass spectrometry

2019 Spring National Meeting

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