Figure 2. Analysis of LAP subdomain interaction with Hsp60.

(A; left panel) SDS-PAGE (7.5% acrylamide) gel showing affinity-purified LAP subdomains. All purified proteins showed expected band size of approximately 45–54 kDa, which includes LAP subdomains; N1 (24 kDa), N2 (21 kDa), C1 (28 kDa) and C2 (26 kDa) and endogenous His, S and Trx tags derived from pET-32a cloning vector (Novagen). (A, right panel) represents ligand overlay assay showing purified Hsp60 binding to the N2 subdomain (arrow). As a positive control, full length LAP (F-LAP) also reacted with Hsp60 but showing two bands, second 53 kDa band is thought to be a breakdown product. (B) Immunofluorescence assay of LAP subdomains (N1, N2, C1, and C2) and full-length LAP (F-LAP) interaction with Hsp60. Of the 4 varying concentrations tested, the N2 subdomain showed significantly higher Hsp60 interaction (P<0.05) relative to other LAP subdomains. Values labeled with different letters (a, b, c) for a given protein concentration are significantly different at P<0.05.(C) Interaction of N2 and F-LAP with Hsp60 using values from 2B above. A logarithmic fitting curve was observed when values for each subdomain were plotted against their respective concentrations. An R² value of greater than 0.9 was used for curve fitting. Binding affinity (K _D) values for N2 and F-LAP interactions with Hsp60 were calculated using the 1-site binding equation in Graphpad. K _D values are the average of 2 independent curve-fitting experiments and were estimated to be 9.51±2.59 nM and 7.17±0.53 nM for N2 and F-LAP, respectively (average ± standard error of the mean [SEM]).