Abstract
Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed “hot spots” in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.
Highlights
More than 50 years after its discovery in human body fluids (Clausen, 1961) and 30 years after determination of its amino acid sequence (Grubb and Löfberg, 1982), cystatin C still attracts the attention of many groups of scientists
IMMUTABILITY OF THE HYDROPHOBIC INTERIOR ESSENTIAL TO CYSTATIN C STABILITY We have studied the impact of the second “hot spot” in the Human cystatin C (hCC) sequence on its conformational stability
DIRECTIONS The results obtained for two groups of cystatin C variants: the hinge loop and the hydrophobic core mutants, show that the main stabilization force of the hCC molecule comes from the www.frontiersin.org hydrophobic interactions responsible for holding together the αhelix and the C-terminal part of the molecule
Summary
More than 50 years after its discovery in human body fluids (Clausen, 1961) and 30 years after determination of its amino acid sequence (Grubb and Löfberg, 1982), cystatin C (hCC) still attracts the attention of many groups of scientists. As it was mentioned above, the naturally occurring L68Q cystatin C variant shows increased dimerization propensity (Nilsson et al, 2004) but is highly amyloidogenic (Abrahamson and Grubb, 1994) As it was proposed by Janowski, polar and sterically bigger glutamine residue, introduced into the hydrophobic core of the molecule, can disrupt the network of hydrophobic interactions stabilizing the α/β interface, triggering unfolding and subsequent domain swapping (Janowski et al, 2001). Another factor, accounting for increased dimerization propensity of the hCC L68Q variant may be lowering of the unfolding energy barrier due to reduction of the unfavorable solvent contacts in the unfolded (or partially unfolded), more polar cystatin C variant comparing to the wild-type protein. Minute amounts obtained from the inclusions bodies mimicked rather the properties of the hydrophobic core, than the hinge loop variants displaying low thermal stability and high propensity to first dimerization and precipitation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
