Two-dimensional crystallization of annexin A5 on phospholipid bilayers andmonolayers: A solid-solid phase transition between crystal forms

Annexin A5 is a soluble protein which binds to negatively charged phospholi pids, such as dioleoylphosphatidylserine (DOPS), in the presence of Ca2+. A solid-solid phase transition between two two-dimensional crystal forms of annexin A5, p6 and p3, previously identified by electron microscopy (EM), w as studied in situ on supported phospholipid bilayers (SPBs) by atomic forc e microscopy (AFM) and ex situ on lipid monolayers by EM. The ability to di rectly vary the protein surface density on SPBs allowed a delicate and syst ematic way to investigate the reversibility of the transition with the AFM. The p6 crystal form was found to form first and to cover the entire lipid surface available, at all DOPS concentrations studied (5-95%). An increase in protein surface density, achievable only on SPBs (or monolayers) contain ing sufficient amounts of DOPS, destabilized the p6 phase. The transition t o the more compact (p3) form occurred via local melting of the p6 phase at defects, such as grain boundaries, initially present in p6 crystals. On mon o- or bilayers with lower DOPS contents, the density-dependent p6-p3 transi tion was not observed, but the p3 crystal form could still be reached by di sturbing the system mechanically.