Probing the affinity, selectivity and inhibition of ubiquitin-ubiquitin binding domain complexes by electrospray ionization mass spectrometry
Sokratous, Kleitos (2013) Probing the affinity, selectivity and inhibition of ubiquitin-ubiquitin binding domain complexes by electrospray ionization mass spectrometry. PhD thesis, University of Nottingham.
This thesis describes the development and application of a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to study the weak hydrophobic interactions seen in many Ub-Ub-binding domain (UBD) complexes. A range of UBDs has been screened against mono-Ub, di-Ub (Ub2) and tetra-Ub (Ub4). Affinities in the 2-200 J.lM range were found to be in excellent agreement with data obtained from other biophysical techniques. Insights into the UBD's preference for poly-Ub chain linkage and length are also provided by this methodology. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multisite interactions. A simple, clean and effective method for reducing charge states observed in ESI-MS without the use of any solution additives or instrumental modifications is also reported; with the charge reduction method ultimately promoting the investigation ofthe Ub-UBD interactions.
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