View source for C2AB: A Molecular Glue for Lipid Vesicles with a Negatively Charged Surface

Original entry: William Bonificio, AP 225, Fall 2009

==Information==
C2AB: A Molecular Glue for Lipid Vesicles with a Negatively Charged Surface. Jiajie Diao, Tae-Young Yoon, Zengliu Su, Yeon-Kyun Shin, Taekjip Ha. Langmuir 2009 25 (13), 7177-7180

==Soft matter keywords==
[[Liposome]], [[C2AB]], [[Vesicle]], [[Protein]], [[Epoxy]].

==Summary==
The purpose of this study was to test the efficacy of a calcium binding protein, currently found in cells, to fuse liposomes to one another.  The binding protein, C2AB, binds calcium at both ends of it.  This binding gives it a positive charge on both sides, which in turn gives the C2AB an affinity to negatively charged liposomes.  When liposomes, C2AB and <math>Ca^{2+}</math> are combined the liposomes fuse.

==Soft matter discussion==

[[Image:C2AB.jpg|500px|thumb|left|Schematic illustration of experiment.  Immobilized vesicles 'glued' to mobile vesicles via C2AB.]]


[[Image:C2AB3.jpg|500px|thumb|left|(a) shows TIR images before and after the introduction of <math>Ca^{2+}</math>. (b) and (c) show TEM images of the liposomes before and after the addition of <math>Ca^{2+}</math>]]

[[Image:C2AB4.jpg|500px|thumb|right|Open and closed conformations of C2AB, from the paper: Structure of Human Synaptotagmin 1 C2AB in the Absence of Ca2+ Reveals a Novel Domain Association. Kerry L. Fuson,, Miguel Montes,, J. Justin Robert, and, R. Bryan Sutton. Biochemistry 2007 46 (45), 13041-13048]]

The academic group conducting this research is experimenting with liposomes as a drug delivery vehicle.  They believe they can solve the problem of targeting the liposome to the correct cells, however there is a perceived problem that the concentration of the drug in each liposome will not have the necessary efficacy.  They hope to be able to fuse the liposomes together at the site of the target to ensure that the drug concentration is high enough to provide a sufficient dosage.

They are collaborating this work with a group that has been studying the protein C2AB which is used in cells as a calcium sensor in synaptic vesicle exocytosis.  The protein has two active sites, one of which binds 2 <math>Ca^{2+}</math>, the other binding 3 <math>Ca^{2+}</math>. When this protein binds the <math>Ca^{2+}</math> it undergoes a conformational change that creates two positively charged ends that are very strongly attracted to negatively charged liposomes.  The charge and conformation taken together cause the C2AB to pierce through the liposome and the C2AB secures itself in within the membrane.

The group created negatively surface charged liposomes and anchored them to a quartz substrate.  They then fluorescently labeled these liposomes.  After this they fluorescently labeled other liposomes with a molecule emitting a different wavelength.  When they put these two liposomes together, they did not bind to eachother as determined by fluorimetry.  After adding C2AB to the mixture, in the absence of <math>Ca^{2+}</math> still no binding occurred.  However, when <math>Ca^{2+}</math> was added, binding took place immediately, within two seconds.  They then used <math>Mg^{2+}</math> in place of <math>Ca^{2+}</math> to determine if it was the existence of any divalent cation that caused the binding.  The <math>Mg^{2+}</math> however did not cause binding.  This shows that the C2AB is only activated as a 'glue' in the presence of <math>Ca^{2+}</math>, and it is the positive charge of the <math>Ca^{2+}</math> along with the conformational change of the C2AB that is causing this activation.

They also studied how strong the binding was by doing repeated washings, and it was shown to be a strong bond, although this was only mentioned qualitatively, not qauntitatively in the paper.  Finally, it was noted that the more negatively charged the liposome surface was, the more binding that occurred with C2AB.