Fig. 1
From: Epsin and Sla2 form assemblies through phospholipid interfaces
Native MS of ENTH/PIP2 complexes suggests an allosteric binding mechanism. a For analyzing lipid binding to ENTH domains, proteins were measured in presence of PIP2 and cytochrome c as reference. Raw spectra show free cytochrome c and unspecific attachment of 1 PIP2 (gray), while ENTH domains (here C. thermophilum) bind 0–3 PIP2. b Signal intensities from MS were summed over all charge states (back) and corrected for unspecific PIP2 clustering based on the ratio of bound/unbound reference protein (front). Data of at least three independent measurements were normalized to the corrected signal of unbound ENTH and the averages of the relative signal intensities and their standard deviations were plotted. The signal for ENTH with three PIP2 observed in raw spectra disappears after correction. c Schematic illustration of microscopic and macroscopic dissociation constants of two PIP2 molecules (orange) binding independently to ENTH (blue). For the first binding event of PIP2, two pathways with the microscopic dissociation constants kd,1 and kd,2 are available, leading to one apparent species of ENTH+PIP2. Combined, they account for the macroscopic dissociation constant KD,1. The second macroscopic dissociation constant KD,2 describes PIP2 binding to the thus far unoccupied binding site that yields the product ENTH+2PIP2. Again, this binding event can be partitioned into two pathways with the microscopic dissociation constants kd,1 and kd,2. If kd,1 and kd,2 are unaltered in the first and second binding event, binding sites are independent. Binding sites are represented by rounded rectangles
