pH effect on protein stability and dynamics of frataxin from Psychromonas ingrahamii

ERNESTO A. ROMAN; RODOLFO M. GONZALEZ LEBRERO; PATRICIO O. CRAIG; JAVIER SANTOS

San Pablo

Simposio; Symposium on Current Topics in Molecular Biophysics; 2014

p { margin-bottom: 0.08in; direction: ltr; color: rgb(0, 0, 255); widows: 2; orphans: 2; }p.western { font-family: "Arial",sans-serif; font-size: 12pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 12pt; }p.ctl { font-family: "Arial",sans-serif; font-size: 10pt; } ntroduction. Frataxin is a protein that participates in iron binding and delivery to other proteins. Its absence in humans yields Friedreich's Ataxia. In our laboratory we study iron binding and folding mechanism of human and Psychromonas ingrahamii frataxin (pFXN). Goal: Here, we will discuss our results on the stability and dynamics of pFXN using equillibrium and rapid kinetics experiments and computational simulations. Materials and methods: We performed stopped-flow experiments to study its folding kinetics, protein crystallography, and computer simulations. In this in silico studies, residues were reduced to 3 atoms over which the bonded and non bonded forces were projected. In this model only the residues that make contact in the native state interact favorably whereas all the others interact only repulsively through excluded volume effect. The model was supplemented with a coulombic electrostatic potential to account for the effect of pH on the stability of the protein. Discussion and results: We previously reported that pFXN stability is highly modulated by pH in the range 6-8 (Roman EA at al.BBA, 2013). Then, we performed stopped-flow experiments to study its folding kinetics. Our results indicate that in the range between 6-8, both transition and native state ensambles are affected by a shift in pH, increasing protein stability at the lowest pH. Protein crystals of pFXN revealed that the position histidine residues in the structure could be important for pFXN stability. We did simulation experiments using structure based models. Our results show that protonation of histidine residues highly modulate protein stability. Conclusion: Our results indicate that protonable residues in the pH range between 6-8 are crucial to protein stability. Computational experiments suggest that histidine might be playing an important role but other protonable residues with abnormal pKa could be participating in the stabilization phenomena.