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Human cystatin C as a protein containing eight Pro residues possesses 111 amide protonspotentially available for the hydrogen-deuterium exchange (in V57P mutant this number isreduced to 110, due to additional Pro residue in the sequence). Besides the backbone amideprotons, also protons from the amino acid side chain functionalities containing O-H andN-H bonds can be substituted by deuterons, overall 107 protons in the wild-type hCC, 109in V57N and 108 in V57D mutant. To the group of labile protons belong also the protonsfrom the terminal amine and carboxylic functions in each protein. Summarizing, the totalnumber of the exchangeable protons amounts to 218 in case of the wild-type cystatin C,217 in V57P mutant, and 219 and 220, respectively, for V57D and V57N hCC variants.Application of increasing pressure forced the unfolding of hCC molecules, graduallyexposing more and more buried protons to the solvent and allowing their exchange intodeuterons. Because pressure denaturation is a reversible process, after decompression theproteins started to refold. Subsequent dilution of the samples in a non-deuterated solventcaused substitution of the available deuterons by protons, however, the deuterons whichwere captured inside the refolded protein were protected against the back-exchange andstayed at their positions. Figure 1 depicts dependence of a number of unexchangeabledeuterons upon the applied pressure for the equilibrium point (60 min after decompressionand dilution) and reflects differences between the wild-type and mutated cystatin C inregard to their structural stability. The pressure-induced changes in the wild-type proteinand V57N mutant began above 200 MPa, at 400 MPa both proteins adopted stable unfoldedconformation and further pressure increase did not cause any additional perturbations intheir structure. In the case of V57D mutant, the unfolding process started earlier – at about150 MPa, but complete unfolding took place at the same moment as for the wild-type hCC– at a pressure of 400 MPa. Lack of any plateau in the first part of the denaturation curve ofV57P clearly indicates that the unfolding of this protein started at the very beginning of thedenaturation process, and points out the proline mutant as the least stable hCC variant,without any resistance to the applied pressure. Simultaneously, V57P mutant occurred toattain the final unfolded conformation at lower pressure (300MPa) than the other studiedproteins.Only 32 deuterons were trapped in the molecule of V57P mutant after its recoveryfrom the completely unfolded structure whereas V57N, V57D and the wild-type hCC in theidentical experiments captured from 6 to 8 deuterons more (Figure 1). These results suggestthat V57P did not refold with the same rate as the rest of the studied proteins or was evenunable to refold correctly. Its structure was penetrable by solvent molecules for an extendedperiod of time allowing free re-exchange of the incorporated deuterons into protons.Delayed establishing of the equilibrium of the D/H back-exchange process, resulting in alower number of the trapped deuterons, indicated evidently that proline residue at the hingeregion made the protein structure more flexible and dynamic, and prolonged its existence ina kind of an ‘open’ state. Flexibility of the hCC V57P mutant may be interpreted as a resultof the reported broadening of the hinge loop L1 caused by V→P substitution and thepreference of Pro residue to adopt the dihedral angles characteristic for an extendedstructure [4]. Quite easy achievable and apparently fairly stable ‘open’ conformationprobably allows V57P molecule to exist in this form in solution for the time long enough tomeet another ‘open’ molecule and swap domains with it, giving rise to the dimericstructure. Enhanced stability of the ‘open’ conformation explains the observation that V57Pdimerizes the most eagerly among all hCC mutants [3].AcknowledgmentsThe work was supported by grants BW/8000-5-0253-9 and 1264/B/H03/2009/37.References1. Olafsson, I., Grubb, A. Amyloid 7, 70-79 (2000).2. Janowski, R., Kozak, M., Jankowska, E., Grzonka Z., Grubb, A., Abrahamson, M., Jaskólski M.Nature Struct. Biol. 8, 316-320 (2001).3. Szymanska, A., Radulska, A., Czaplewska, P., Grubb, A., Grzonka, Z., Rodziewicz-Motowidło, S.Acta Biochim. Pol. 56, 455-463 (2009).4. Rodziewicz-Motowidło, S., Iwaszkiewicz, J., Sosnowska, R., Czaplewska, P., Sobolewski, E.,Szymanska, A., Stachowiak, K., Liwo, A. Biopolymers 91, 373-383 (2009).285