Prolines Affect the Nucleation Phase of Amyloid Fibrillation Reaction; Mutational Analysis of Human Stefin B.

Samra Hasanbašić,Selma Berbić,Vida Puizdar,Ajda Taler-Verčič,Andrej Vilfan,Magda Tušek Žnidarič,Eva Žerovnik,Veronika Stoka

doi:10.1021/acschemneuro.8b00621

Abstract

Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.

Highlights

The primary aim of studying the mechanism of ordered aggregation of proteins into amyloid fibrils is to gain insight into the basic molecular processes underlying proteinopathies, called conformational diseases.[1]
We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B
This study confirms that cis to trans isomerization of critical Pro residues contributes to amyloid fibril formation, starting with domain swapping.[8]

Summary

■ INTRODUCTION

The primary aim of studying the mechanism of ordered aggregation of proteins into amyloid fibrils is to gain insight into the basic molecular processes underlying proteinopathies, called conformational diseases.[1]. Pro-limited folding reactions can be investigated using site-directed mutagenesis.[16] In this case changes in folding kinetics can be related to Pro residues only if a cis prolyl bond is substituted with a trans nonprolyl bond, which leads to an increase of the fast refolding amplitude and the disappearance of slow, Prolimited reaction.[14] The stefin B fibrillation reaction is proline isomerization-dependent, which was shown by the addition of the peptidyl prolyl cis/trans isomerase, CypA. Similar cases showing the importance of Pro cis/trans isomerism in fibrillation have been reported in other proteins, for example, in β2-microglobulin (β2m), a protein related to dialysis-related amyloidosis (DRA) The His 31−Pro 32 bond in β2m has an unfavorable cis conformation, with cis to trans isomerism of Pro 32 leading directly to amyloid fibril formation.[36] To our knowledge, no systematic analysis of proline mutagenesis and its influence on fibrillation has been reported. We systematically mutated the Pro residues of human stefin B to understand their influence in both oligomer and amyloid fibril formation (Table 1)

RESULTS AND DISCUSSION

CONCLUSIONS

METHODS

■ ACKNOWLEDGMENTS

■ REFERENCES