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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Mutants of an Amyloidogenic Human Cystatin C inPressure-Induced Denaturation Studies UsingHydrogen Exchange Mass SpectrometryElzbieta Jankowska 1 , Marta Orlikowska 1 , Marta Sosnowska 1 ,Aneta Szymanska 1 , Piotr Stefanowicz 2 , Karolina Kowalewska 2 ,and Zbigniew Szewczuk 21 Faculty of Chemistry, University of Gdansk, Sobieskiego 18, 80-952, Gdansk, Poland2 Faculty of Chemistry, University of Wroclaw, Joliot-Curie 14, 50-383, Wroclaw, PolandIntroductionHuman cystatin C (hCC) is a 120 amino acids containing protein, ubiquitous in all bodyfluids. Its main physiological function is regulation of activity of cysteine proteases, eitherreleased from damaged or dying lysosomes or originated from microbial invasion. Besidesits inhibitory function, hCC plays a role in development of a neurodegenerative diseasecalled amyloid angiopathy, caused by pathological aggregation of the wild-type protein orits naturally occurring L68Q variant [1]. The mechanism of cystatin C amyloid formationhas not been elucidated till now. It is postulated, however, that aggregation and fibrilizationproceed through propagated 3D domain-swapping process, which was evidenced as amechanism of hCC dimerization [2]. The dimer reconstructs in duplicate the general fold ofthe monomeric protein with the exception of the hinge region encompassing residuesQ 55 IVAG 59 , which links the swapped subdomain to the rest of the molecule. With the aimto check implications of greater or decreased stability of this region for dimerization andaggregation propensity of human cystatin C, we designed and constructed hCC mutantswith Val57 residue replaced by Asp, Asn or Pro, respectively. The obtained proteins weresubsequently the subject of hydrogen exchange mass spectrometry analysis combined withhigh pressure-induced denaturation to monitor stability and an unfolding/refolding processof the mutants in comparison to the wild-type human cystatin C.Results and DiscussionHCC variants, obtained by protein engineering methods [3], were dissolved in a deuteratedbuffer and incubated under elevated pressure. After 30 min of incubation during which theprotein underwent unfolding and deuteration, each sample was decompressed and dilutedwith a non-deuterated buffer, what initialized the deuterium-hydrogen (D/H) backexchange. Following this process by mass spectrometry we were able to gain someknowledge about stability of different variants of hCC and their refolding process.Fig. 1. Number of unexchangeable deuterons in hCC variants depicted as a function ofthe applied pressure.284