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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.