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From CellBiology

Green Fluorescent Protein (GFP)

Introduction

GFP is the Green Fluorescent Protein found in jellyfish "Aquorea victoria". This is a protein of 238 amino acids that emits green flurescence at 509nm in the presence of blue or UV light. It is extremely useful in fluorescence microscopy as in the presence of oxygen the protein spontaneously forms a fluorescent chromophore. No substrates are necessary, and fusion of GFP to a protein rarely affects the proteins activity or mobility. GFP is also resistant to heat, alkali pH, detergents, photobleaching, salts and many proteases [1]

There are a few negative aspects of using GFP in mitochondria research - slow chromophore formation, requirement of oxygen, and difficulty distinguishing GFP fluorescence from background fluorescence when GFP is not highly expressed. [2]

By attaching it to a specific gene, "protein expression, localisation, movement, interaction and activity in a cell, tissue or organism can be visualised under UV light." [3] Fluorescent mice , fishand insects have been created through the use of GFP as a protein tag.

GFP has a unique 11 beta-barrel like structure. The beta-sheets form a cylindrical structure, with an alpha-helix running diagonally through the cylinder. It is thought that the barrel structure protects the chromophore which is located in the middle of the beta-barrel. A 2D image of the structure can be found here.

There are other types of GFPs found in numerous organisms, however Aequorea victoria GFP was the first GFP to be cloned and expressed.[4]

Figure 1. 3D structure of GFP. PBD ID 1w7s

History

  • 1955 - Aequorea victoria jellyfish reported as fluorescing under UV light
  • 1979 - Shimomura deduced the structure of GFP
  • 1992 - Prasher cloned and sequenced GFP
  • 1994 - Chalfie used GFP as a fluorescent tag
  • 1995-6 - Tsien created mutants of GFP - different colours and faster fluorescing [5]
  • 2008 - Nobel Prize in Chemistry awarded to Shimomura, Chalfie and Tsien "for the discovery and development of the green fluorescent protein, GFP" [6] Also, first mutant of Aequorea victoria GFP that forms a red chromophore was studied [7] [8]
Figure 2. Molecular Structure of GFP chromophore

Mechanism

The central peptide of GFP contains a tyrosine subunit, which is "spontaneously modified to yield a chromogenic entity that is responsible for the colourful appearance of the proteins." [9] It is this subunit which is able to be modified to produce different colour fluorescence.

Two main catalytic units aid in creating the fluorescence. The Gly67 subunit is essential for chromophore formation, with Arg96 and Glu222 being catalytic molecules. Research has shown that Arg96 stabilised Gly67, with the role of Glu222 being a base catalyst. [10]

For the GFP fluorescence to be expressed in mitochondria, a gene encoding a specific protein would preferably have an importing signal to the mitochondria on the upstream side of the GFP gene. Examples of these are cytochrome c oxidase subunit VIII, and cDNA of cytochrome C, as these are associated with the mitochondrial membrane. The GFP gene and the importing signal can be next to each other, or separated by several nucleotides - but must be in the same reading frame.[11]

GFP tagging has been used to visualise mitochondria under microscopic conditions. It has been used to track mitochondria location, movement and interaction.

Figure 3. Applications of fluorescent proteins. Left: promoter activation, Middle: protein labelling, Right: protein-protein interaction using FRET

Use in Mitochondria research

There are five main applications of GFP:

"1. visualisation of target-gene promoter up and down regulation

2. protein labelling

3. detection of protein-protein interactions

4. tracking protein movement and

5. monitoring cellular parameters."[12]

There is a specific method used for tagging GFP to certain proteins, with two main steps:

1. Construction of expression vectors

In this initial stage, the signal sequences of the target gene and the GFP gene are separately amplified using PCR. They are then ligated together to form a gene sequence tagged with GFP, along with a promoter and a poly-A tail. [13]

2. Transfection

With the use of a transfection reagent such as FuGENE 6or Lipofectamine, the newly tagged target gene can be integrated in to a normal cell. This means it will translate the DNA to RNA with a GFP tag. However, if creating transgenic animals (a living animal with a GFP tagged protein), the recombinant DNA is inserted into pronucleus stage eggs of the female, and the offspring is tested to see whether it has the GFP tag.

As shown in Figure 4, Kohler et al (1997) [14] transformed the GFP downstream from from the gene enscribing cytochrome oxidase subunit IV (associated with mitochondrial membrane), so it would be 'tagged' with the fluorescent protein when transcribed and translated. In this instance, GFP was used to visualise plant mitochondria.

In another lab, Hou & Hsu (2005) [15] tagged the pro-apopotic protein BAX to trace its movement into the mitochondria of cardiac cells during apoptosis.

Figure 4. Constructs used for transformation of tobacco NT1 suspension cells and Petit Havana plants. Adapted from Figure 1. The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Köhler RH, Zipfel WR, Webb WW, Hanson MR. Plant J. 1997

There are a few different way mitochondria can be viewed after being tagged with GFP.

  • FRAP: by bleaching a certain tagged area of protein, it can be determined protein movement within the cell. This is done by tracking the bleached proteins throughout the cell or along the membrane.
  • FRET: this is used to determine whether certain proteins interact. An example of this is one protein labelled with a blue fluorescent protein (donator), and one with GFP (acceptor). If they interact within 10nm of each other, one protein will donate photons to the other - the donator will tim, and the acceptor will brighten, thus a green fluorescence will be seen.
  • Confocal Microscopy this is a commonly used method when wanting to primarily observe GFP tagged mitochondria. The use of a pinhole aperture makes the confocal much more powerful than upright microscopes, as it fluoresces any photos in focus. It is also able to amplify fluorescent signals, along with separation of fluorochromes down to 2nm [16]

References

  1. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Zimmer M. Chem Rev. 2002 Mar;102(3):759-81. Review. No abstract available. PMID: 11890756
  2. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Zimmer M. Chem Rev. 2002 Mar;102(3):759-81. Review. No abstract available. PMID: 11890756
  3. [Fluorescent proteins as a toolkit for in vivo imaging.Chudakov DM, Lukyanov S, Lukyanov KA. Trends Biotechnol. 2005 Dec;23 12):605-13. Epub 2005 Nov 2. Review.PMID: 16269193]
  4. Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Zimmer M. Chem Rev. 2002 Mar;102(3):759-81. Review. No abstract available. PMID: 11890756
  5. GFP - history, Conneticut College website. Maintained by M. Zimmer (author of 'GFP: applications, structure, and related photophysical behaviour'
  6. http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/
  7. GFP- Timeline, Conneticut College website
  8. The first mutant of the Aequorea victoria green fluorescent protein that forms a red chromophore. Mishin AS, Subach FV, Yampolsky IV, King W, Lukyanov KA, Verkhusha VV. Biochemistry. 2008 Apr 22;47(16):4666-73. Epub 2008 Mar 27. PMID: 18366185
  9. Chromogenic cross-link formation in green fluorescent protein. Wachter RM.Acc Chem Res. 2007 Feb;40(2):120-7.PMID: 17309193
  10. Fluorescent proteins: maturation, photochemistry and photophysics.Remington SJ.Curr Opin Struct Biol. 2006 Dec;16(6):714-21. Epub 2006 Oct 24. Review.PMID: 17064887
  11. http://www.freepatentsonline.com/7227052.html
  12. Fluorescent proteins as a toolkit for in vivo imaging.Chudakov DM, Lukyanov S, Lukyanov KA. Trends Biotechnol. 2005 Dec;23 12):605-13. Epub 2005 Nov 2. Review.PMID: 16269193
  13. Non-invasive visualization of sperm mitochondria behavior in transgenic mice with introduced green fluorescent protein (GFP).Shitara H, Kaneda H, Sato A, Iwasaki K, Hayashi J, Taya C, Yonekawa H.FEBS Lett. 2001 Jun 29;500(1-2):7-11.PMID: 11434917
  14. The green fluorescent protein as a marker to visualize plant mitochondria in vivo. Köhler RH, Zipfel WR, Webb WW, Hanson MR. Plant J. 1997 Mar;11(3):613-21.PMID: 9107047
  15. Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis.Hou Q, Hsu YT.Am J Physiol Heart Circ Physiol. 2005 Jul;289(1):H477-87.PMID: 15961378
  16. Cell Biology Microscopy notes 2009



In the lecture I learnt that there are chromosome territories, and these territories can overlap

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