Figure 4. Role of EGF-like domain in TC-CD320 complex formation.

(a) SEC profile of TC-CD320(Gly₄Ser)₄ construct, revealing higher mass peaks indicating multimeric species. Arrow indicates position of 1:1 TC-CD320 peak. Peak fractions indicated above SEC profile correspond to lanes in SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Note that TC appears as a double band depending on polyacrylamide percentage and SDS–PAGE running conditions. (b) Native electrospray ionization mass spectrometry (ESI-MS) analysis of TC-CD320(Gly₄Ser)₄ complex. Each peak cluster comprises different charged species of a single macromolecular assembly formed due to the presence of residual salt from protein purification and correspond to approximate molecular weights (MWs) of 65 and 120 for the lower and higher molecular weight species, respectively (left panel). Analysis of Individually purified SEC peaks shows isolated high MW (middle) and low MW (right) species. (c) Model of properly oriented LDLR-A1 and LDLR-A2 domains (strong yellow) acting in tandem to specifically bind a single TC molecule. Dashed line indicates a flexible linker with proposed second TC molecule (faded yellow) binding.