Difference between revisions of "C2AB: A Molecular Glue for Lipid Vesicles with a Negatively Charged Surface"

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(New page: Original entry: William Bonificio, AP 225, Fall 2009 ==Information== Organic Field Effect Transistor Using Pentacene Single Crystals Grown by a Liquid-Phase Crystallization Process. Yasuo...)
 
(Soft matter keywords)
 
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==Information==
 
==Information==
Organic Field Effect Transistor Using Pentacene Single Crystals Grown by a Liquid-Phase Crystallization Process. Yasuo Kimura, Michio Niwano, Naohiko Ikuma, Kenichi Goushi, Kingo Itaya. Langmuir 2009 25 (9), 4861-4863
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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==
 
==Soft matter keywords==
Liquid-Phase Crystallization, Pentazene, [[Organic Field Effect Transistor]], Pentacene, Tricholorbenzene.
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[[Liposome]], [[C2AB]], [[Vesicle]], [[Protein]], [[Epoxy]].
  
 
==Summary==
 
==Summary==
The purpose of this study was to investigate a different fabrication technique for creating single crystals of pentacene to be used for organic field effect transistors.  The liquid-phase crystallization process creates single crystals by dissolving pentacene in solution, then precipitating it as a single crystalThe pentacene crystals formed by this method then had their semiconducting properties measured and these properties were compared to pentacene crystals grown using different methods.
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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 liposomesWhen liposomes, C2AB and <math>Ca^{2+}</math> are combined the liposomes fuse.
  
 
==Soft matter discussion==
 
==Soft matter discussion==
  
[[Image:Pentacene2.jpg|500px|thumb|right|The structure of pentacene.]]
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[[Image:C2AB.jpg|500px|thumb|left|Schematic illustration of experiment.  Immobilized vesicles 'glued' to mobile vesicles via C2AB.]]
  
  
[[Image:Pentacene1.jpg|500px|thumb|left|The top image is an atomic force microscopy image of pentacene single crsytal. The bottom image is the height along the white arrow from the top image.]]
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[[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>]]
  
Organic field effect transistors (OFETs) have many favorable properties over current field effect transistors (FETs). The most popular OFET, due to its optimal electronic properties derived from its delocalized pi bond, is pentacene.  These properties however, are degraded by the existence of grain boundaries in polycrystalline pentacene as a result of the reduced mobility in charge carriers. Therefore, single crystals of pentacene are primarily used. The most popular way of synthesizing the single crystalline pentacene is currently by physical vapor deposition.
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[[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]]
  
An alternative method of creating pentacene single crystals is by liquid-phase crystallization.  In this process pentacene dissolved in trichlorobenzene is purified via various methodsThen the pentacene solution is heated and very slowly cooled so that precipitation occurs as a single crystal.  In fact, in this experiment the pentacene was heated to 200C, then cooled at 0.1C per hourThe phase change from solute to solid single crystal is clearly applicable to the soft matter discussion.  The image on the left shows the single crystal as seen from AFM.  Each step shown is a single molecule step.  The entire image has an area of 4umx4um.
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The academic group conducting this research is experimenting with liposomes as a drug delivery vehicleThey 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 efficacyThey 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.
  
The crystalline pentacene was then fabricated onto a thin film of heavily doped silicon oxide to create the OFET.  The OFET electrical properties were measured by standard methods.  The most important of these properties was the field effect mobility which was measured to be <math>0.4 \frac{cm^2}{Vs}</math>. This number is similar to PVD single crystal pentacene which shows that the fabrication technique was a successLiquid-phase crystallization is a practical method in producing pentacene single crystals for OFETs.
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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 liposomesThe charge and conformation taken together cause the C2AB to pierce through the liposome and the C2AB secures itself in within the membrane.
  
There are some differences between this method and PVDFor example, there seems to be a large anisotropy in the method used hereThat means that the orientation of the crystal, and the direction by which you measure your voltage across makes a differenceThis created mobilities that were both higher and lower than expected.  Perhaps this can be tuned to create an even better OFET.
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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 wavelengthWhen 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 bindingThe <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.
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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 paperFinally, it was noted that the more negatively charged the liposome surface was, the more binding that occurred with C2AB.

Latest revision as of 13:16, 29 November 2011

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

Schematic illustration of experiment. Immobilized vesicles 'glued' to mobile vesicles via C2AB.


(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>
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.