Biophysical Society Thematic Meeting - October 13-15, 2015

Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling

Poster Abstracts

17-POS Board 17 Structural, Morphological and Kinetic Studies of Human Carbonic Anhydrase II Aggregation Preeti Gupta .Shashank Deep. Indian Institute of Technology, Delhi, New Delhi, India. Investigation of physico-chemical factors that modulate protein aggregation is important not only to understand and mitigate amyloid-related pathologies, but also for the manufacture, storage, and administration of protein based therapeutics. In the present work, we investigated the effect of two different environmental determinates i.e. an organic solvent, trifluoroethanol (TFE) and salt (NaCl) ions on the aggregation propensity of human carbonic anhydrase II (HCA II). Along with aggregation kinetics, we also examined the morphological properties of aggregates induced at above-mentioned solution conditions. Our studies with TFE indicate that HCA II undergoes a transition from β-sheet to α-helix on addition of alcohol. TFE exhibited a bell-shaped dependence of aggregation on the cosolvent concentration. At intermediate [TFE], protein adopts partially structured, extended non-native β- sheet conformation with maximum aggregation propensity. Also, protein aggregates induced by TFE possess amyloid-like features as revealed by ThT binding and TEM studies. Our results suggest that TFE concentration and polypeptide backbone conformation are critical for protein aggregation. The second condition explored was the effect of salt ions on the heat-induced aggregation kinetics of HCA II. The aggregation kinetic trace of protein displayed a typical single-transition (sigmoid) profile suggesting a nucleation dependent polymerization. Strikingly, on addition of salt, the biphasic aggregation kinetic behaviour with two distinct transitions was observed. The extent as well as rate of aggregation of both the transitions was extremely receptive to NaCl concentration. The biphasic aggregation pattern was also observed with other salts (NH 4 Cl, MgCl 2 ), but they affect two transitions differently. Protein solubility studies and SEM analysis suggest the assemblage of monomeric protein into small, coalesced spherical particles during first transition, whereas there is only fusion of existing spheres into less packed aggregate clusters during second transition without any recruitment of protein from the solution.

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