Examination of Surface Residues of Proteins using Cucurbit[6]uril and Tandem Mass Spectrometry




Srinivasa, Vishnu

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Cucurbit[6]uril (CB[6]) is a large macrocyclic molecule that binds electrostatically to lysine, and has previously showed promise as a probe to tag the most surface-accessible regions of proteins. In order to validate this, in conjunction with tandem mass spectrometry, as an effective method to study protein surface structure, 193 nm ultraviolet photodissociation (UVPD) mass spectrometry was used to characterize the binding interactions between CB[6] and lysine residues of ubiquitin, and a data analysis protocol for this data was developed to figure out the most effective way to locate the binding sites. UVPD was successfully able to produce fragment ions that retained the non-covalent interactions between CB[6] and ubiquitin, and analysis of the pattern of N-terminal and C-terminal fragments that retained CB[6] demonstrated that Lys48 was a possible binding site. This residue is strongly involved in polyubiquitination and other protein interactions, demonstrating that UVPD in conjunction with CB[6] was successful in identifying residues that are highly accessible for interactions. As well, observation of species with multiple CB[6] adducts showed that a common method for determination of binding sites, subtracting fragment abundances of the uncomplexed species from the complexed species, is not necessarily useful for analysis of CB[6] binding due to the bulk of the molecule causing suppressed fragmentation elsewhere. However, preliminary results from this method also suggest potential binding to lysines around residue 30, another surface-accessible region, although this binding region is not confirmed and will require further investigation to determine whether fragmentation suppression is due to binding or interference from elsewhere in the sequence. This study overall provides a foundation for the integration of supramolecular electrostatic tagging and tandem mass spectrometry to locate structural features of interest without relying on covalent tags or complex data analysis. As well, it demonstrates the utility of UVPD in opening up additional pathways for protein structural analysis.



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