Protein Fibrillation Lag Times During Kinetic Inhibition

PAGANO, R.S (CO-FIRST AUTHOR); LÓPEZ MEDUS, M. (CO-FIRST AUTHOR); GÓMEZ, G.E (SECOND AUTHOR); COUTO, PM; LABANDA, MS; LANDOLFO, L; D ALESSIO, C; CARAMELO, JJ

BIOPHYSICAL JOURNAL

CELL PRESS

Año: 2014 vol. 107 p. 711 - 720

0006-3495

Protein aggregation is linked to more than 30 human pathologies, including Alzheimer?s and Parkinson?sdiseases. Since small oligomers that form at the beginning of the fibrillation process probably are the most toxic elements,therapeutic strategies involving fibril fragmentation could be detrimental. An alternative approach, named kinetic inhibition,aims to prevent fibril formation by using small ligands that stabilize the parent protein. The factors that govern fibrillation lag timesduring kinetic inhibition are largely unknown, notwithstanding their importance for designing effective long-term therapies. Inhibitor-bound species are not likely to be incorporated into the core of mature fibrils, although their presence could alter the kineticsof the fibrillation process. For instance, inhibitor-bound species may act as capping elements that impair the nucleation processand/or fibril growth. Here, we address this issue by studying the effect of two natural inhibitors on the fibrillation behavior oflysozyme at neutral pH. We analyzed a set of 79 fibrillation curves obtained in lysozyme alone and a set of 37 obtained inthe presence of inhibitors. We calculated the concentrations of the relevant species at the beginning of the curves using theinhibitor-binding constants measured under the same experimental conditions. We found that inhibitor-bound protein speciesdo not affect fibrillation onset times, which are mainly determined by the concentration of unbound protein species present inequilibrium. In this system, knowledge of the fibrillation kinetics and inhibitor affinities suffices to predict the effect of kineticinhibitors on fibrillation lag times. In addition, we developed a new methodology to better estimate fibrillation lag times fromexperimental curves.