Brittany deRonde/Sandbox 1

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One of the CBI Molecules being studied in the University of Massachusetts Amherst Chemistry-Biology Interface Program at UMass Amherst and on display at the Molecular Playground.

1 Introduction
2 Cell Penetrating Peptides
3 Tat-mediated Transduction
4 Transduction
5 Endocytosis
6 My Research Interests
7 Acknowledgments
8 References

Introduction

HIV Tat, or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.^[1] Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.^[2]^[3]

HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain

Drag the structure with the mouse to rotate

It is released by HIV infected cells in order to enhance replication of the virus.^[4]^[5] Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.^[6] When the protein enters non-infected cells, the transcription efficiency increases. It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.^[7] Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.^[8]^[9] These were the first known reports of a cell penetrating peptide (CPP).

Cell Penetrating Peptides

Cell penetrating peptides (CPPs) are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.^[10] Such cargo can be associated via covalent or non-covalent interactions. Tat is considered a CPP because it contains a protein transduction domain (PTD). PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.^[11]

These sequences improve interactions between CPPs and cellular membranes and can help them enter cells. The (amino acid ) of HIV-1 Tat comprise the protein transduction region of this protein. This arginine rich, 11 amino-acid sequence, is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.

Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. ^[12]^[13] The PTD sequences for these proteins can be found in the table below.

Tat-mediated Transduction

The PTD in HIV Tat is highly cationic. When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases. It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.^[14]^[15] Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.^[16] Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.^[17] As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.^[18]^[19] The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.

Transduction

Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes. However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis. In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. ^[20] Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.^[21] Some groups have also studied transduction of Tat using model vesicles. ^[22]

Endocytosis

Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis. This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules. ^[23] During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.

My Research Interests

As part of the Tew Research Group in the Department of Polymer Science and Engineering at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery. Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics. Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.

Acknowledgments

The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.

References

↑ Sodroski et al. Science. 1985. 227, 171-173. [1] http://www.jstor.org/stable/1695050
↑ Arya et al. Science 1985. 229, 69-73.
↑ Sodroski et al. Science. 1985. 229, 74-77.
↑ Dayton et al. Cell. 1986. 44, 941-947.
↑ Fisher et al. Nature. 1986. 320, 367-371.
↑ Xiao et al. Proc. Natl. Acad. Sci. USA. 2000. 97, 11466-11471.
↑ Ensoli et al. Nature. 1990. 345, 84-86.
↑ Green et al. Cell. 1988. 55, 1179-1188.
↑ Frankel et al. Cell. 1988. 55, 1189-1193.
↑ Futaki et al. J. Biol. Chem. 2001. 276, 5836-5840.
↑ Wender et al. Adv. Drug Deliv. Rev. 2008. 60, 452-472.
↑ Futaki et al. J. Biol. Chem. 2001. 276, 5836-5840.
↑ Prochiantz et al. Adv. Drug Deliv. Rev. 2008. 60, 448-451.
↑ Ziegler et al. Biochemistry 2003. 42, 9185-9194.
↑ Goncalves et al. Biochemistry. 2005. 44, 2692-2702.
↑ Ziegler et al. Adv. Drug Deliv. Rev. 2008. 60, 580-597.
↑ Wender et al. Proc. Natl. Acad. Sci. 2000. 97, 13003-13008.
↑ Mitchell et al. J. Pept. Res. 2000. 56, 318-325.
↑ Futaki et al. J. Biol. Chem. 2001. 276, 5836-5840.
↑ Richard et al. J. Biol. Chem. 2003. 278, 585-590.
↑ Drin et al. J. Biol. Chem. 2003. 276, 31192-31201.
↑ Ziegler et al. Biochemistry. 2003. 278, 9185-9194.
↑ Gump et al. Trends in Molecular Medicine. 2007. 13, 443-448.

Proteopedia Page Contributors and Editors (what is this?)

Brittany deRonde, Coralie Backlund

Retrieved from "http://52.214.119.220/wiki/index.php/Brittany_deRonde/Sandbox_1"

@@ Line 1: / Line 1: @@
-<Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat' scene='HIV Tat' />
+One of the [[CBI Molecules]] being studied in the  [http://www.umass.edu/cbi/ University of Massachusetts Amherst Chemistry-Biology Interface Program] at UMass Amherst and on display at the [http://www.molecularplayground.org/ Molecular Playground].
-<applet load='1ema' size='450' frame='true' align='right' scene='Green_Fluorescent_Protein/1ema_gfp_default/2' caption='Green fluorescent protein complex with peptide-derived chromophore ([[1ema]])' />
-'''HIV Tat''' is a [[bioluminescent]] polypeptide consisting of 238 residues isolated from the body of [[Aequorea victoria]] jellyfish.<ref name="PDBsum">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=main.html], Protein Database (PDBsum): 1ema.  European Bioinformatics (EBI); 2009.</ref> GFP converts the blue chemiluminescent of [[aequorin]] in the jellyfish into green fluorescent light.<ref name="Yang">[http://www-bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html], Yang F, Moss LG, Phillips GN Jr.  1996.  The molecular structure of green fluorescent protein.  Biotechnology.  14: 1246-1251.  DOI 10.1038/nbt1096-1246.</ref> It remains unclear why these jellyfish use fluorescence, why green is better than blue, or why they produce a separate protein for green fluorescence as opposed to simply mutating the present aequorin to shift its wavelength,<ref name="Tsien" /> but in the laboratory, GFP can be incorporated into a variety of biological systems in order to function as a marker protein. Since its discovery in 1962, GFP has become a significant contributor to the research of monitoring gene expression, localization, mobility, traffic, interactions between various membrane and cytoplasmic proteins, as well as many others.<ref name="Haldar">[http://www.springerlink.com/content/wvg513864266g77n/fulltext.pdf], Haldar S, Chattopadhyay A.  2009.  The green journey.  J Fluoresc.  19:1-2.  DOI 10.1007/s10895-008-0455-6; biographical background on [http://en.wikipedia.org/wiki/Douglas_Prasher Douglas Prasher], [http://en.wikipedia.org/wiki/Martin_Chalfie Martin Chalfie] and [http://en.wikipedia.org/wiki/Roger_Tsien Roger Tsien].</ref>
-==History==
+== '''Introduction''' ==
-''Aequorea victoria'' was first discovered and investigated for its bioluminescence by Frank Johnson, who invited Osamu Shimomura to work with him in on a small island not far from British Columbia, where the jellyfish is abundant.<ref name="Shimomura">[http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura_lecture.pdf], Shimomura O.  The discovery of green fluorescent protein.  Nobel Prize Lecture; 2009;; biographical background at [http://en.wikipedia.org/wiki/Osamu_Shimomura Wikipedia].</ref> Found off the west coast of the United States between British Columbia and central California,<ref name="Cowles">[http://www.wallawalla.edu/academics/departments/biology/rosario/inverts/Cnidaria/Class-Hydrozoa/Hydromedusae/Aequorea_victoria.html],Cowles D, Cowles J.  ''Aequorea victoria''.  2007.  Walla Wall University.</ref> the jellyfish was considered a local phenomenon as it would drift in and out of the harbors.<ref name="Shimomura" />
+'''HIV Tat''', or simply Tat, is a human immunodeficiency virus (HIV) gene that regulates transcription of HIV dsRNA.<ref>Sodroski ''et al. Science.'' '''1985'''. 227, 171-173. [http://www.jstor.org/stable/1695050] http://www.jstor.org/stable/1695050</ref>  Tat, which stands for trans-activator of transcription, contains 86 amino acid residues in its sequence.<ref>Arya ''et al. Science'' '''1985'''. 229, 69-73.</ref><ref>Sodroski ''et al. Science.'' '''1985'''. 229, 74-77.</ref> <Structure load='1TIV' size='500' frame='true' align='right' caption='HIV Tat. Grey residues = polar groups, Pink residues= hydrophobic residues, and Blue residues= protein transduction domain' scene='Brittany_deRonde/Sandbox_1/Hiv_tat/2' /> It is released by HIV infected cells in order to enhance replication of the virus.<ref>Dayton ''et al. Cell.'' '''1986'''. 44, 941-947.</ref><ref>Fisher ''et al. Nature.'' '''1986'''. 320, 367-371.</ref>  Nanomolar concentrations of Tat have been reported in the blood of HIV-1 infected people.<ref>Xiao ''et al. Proc. Natl. Acad. Sci. USA.'' '''2000'''. 97, 11466-11471.</ref> When the protein enters non-infected cells, the transcription efficiency increases.  It is estimated that transcription levels are 10 to 100 fold higher during the transcription elongation stage when compared to normal basal transcription.<ref>Ensoli ''et al. Nature.'' '''1990'''. 345, 84-86.</ref>  Green and Lowenstein, and Frankel and Pabo independently published studies in 1988 that demonstrated that Tat had the ability to cross cellular membranes and initiate transcription of HIV dsRNA.<ref>Green ''et al. Cell.'' '''1988'''. 55, 1179-1188.</ref><ref>Frankel ''et al. Cell.'' '''1988'''. 55, 1189-1193.</ref>  These were the first known reports of a '''cell penetrating peptide (CPP)'''.
-[[Image:GFP mice.png|thumb|left|450x200px|Mice with GFP inserted into their genomes for neurology studies.]]
-Shimomura was originally looking only to isolate the blue luminescent protein of ''Aequorea victoria'', traditionally thought to be [[luciferase]], but it would soon become apparent that the glow was in fact due to aequorin, a substance related, but slightly varying from luciferase.<ref name="Haldar" /><ref name="Shimomura" /> However, the light emitted from aequorin still differed from the light emitted from the wild jellyfish. This quandary led to the discovery of the green fluorescent protein responsible for this disparity, but sufficient amounts of the protein could not be collected for study until 1979. The journey to discover the nature of GFP had begun.<ref name="Shimomura" />
+== '''Cell Penetrating Peptides''' ==
-In the 1990’s, Douglas Prasher, Frank Predergast, and co-workers successfully cloned the gene that encoded for GFP.  Martin Chalfie further pursued this line of work and was eventually able to express GFP in heterologous systems such as E. coli and C. elegans.  Chalfie’s research provided the first evidence that GFP was unique as it did not require the presence of any exogenous substance or cofactor for fluorescence.<ref name="Haldar" /> The lack for the need for a cofactor proved that the cloned GFP gene contained all the information necessary for posttranslational synthesis of the chromophore. <ref name="Tsien" />
+'''Cell penetrating peptides (CPPs)''' are proteins with the ability to cross cellular membranes and facilitate the uptake of various cargo, such as small molecules, siRNA, and small DNA fragments.<ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref>  Such cargo can be associated via covalent or non-covalent interactions.  Tat is considered a CPP because it contains a protein transduction domain (PTD).  PTDs are cation-rich sequences of 10-30 residues, usually containing several Lysine and/or Arginine residues.<ref>Wender ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 452-472.</ref>
-Roger Tsien and co-workers were intrigued by the absence of a necessary cofactor and began to research the structure of GFP and how it relates to its fluorescence.  They discovered that a helix within the beta barrel structure of GFP actually contained a fluorophore responsible for fluorescence.  In researching its structure, they were able to develop GFP derivatives with improved fluorescence and photo-stability.  Shimomura, Chalfie, and Tsien were each recognized for their work involving GFP with the Nobel Prize in 2008.<ref name="Haldar" />  In the time since the work of these three researchers, GFP has been successfully expressed and utilized in bacteria, yeast, slime mold, plants, drosophila fruit flies, zebra-fish, and mammalian cells.<ref name="Yang" />  Below, mice have had GFP inserted into their genomes for studies in neurology.
+[[Image:Arginine1.jpg]] [[Image:Lysine1.jpg]]
-==Structure==
+These sequences improve interactions between CPPs and cellular membranes and can help them enter cells.  The  (amino acid <scene name='Brittany_deRonde/Sandbox_1/Hiv_tat/3'>residues 47-57</scene>) of HIV-1 Tat comprise the protein transduction region of this protein. This arginine rich, 11 amino-acid sequence, <scene name='47/475184/Tat_arg/3'>YGRKKRRQRRR</scene>  is now referred to as the TAT peptide, and has be shown to have improved cellular uptake compared to Tat.
-===Primary & Secondary Structure===
-{{STRUCTURE_1ema |  SIZE=400 |PDB=1ema  |  SCENE=Green_Fluorescent_Protein/1ema_gfp_default/2 |CAPTION = 1ema, resolution 1.90&Aring; (<scene name='Green_Fluorescent_Protein/1ema_gfp_default/2'>default scene</scene>).}}
-Green fluorescent protein (<scene name='Green_Fluorescent_Protein/1ema_gfp_default/2'>default scene</scene>) is a 21 kDa protein consisting of 238 residues strung together<ref>Primary structure at [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=protein.html&r=wiring&l=1&chain=A www.ebi.aci.uk].</ref> to form a
-<scene name='Green_Fluorescent_Protein/1ema_gfp_barrel/2'>secondary structure</scene> of five α-helices and one eleven-stranded β-pleated sheet,<ref name="PDBsum" /> where each strand contains nine to thirteen residues each.<ref name="Ormo" />  (To view the primary and secondary structure of GFP, go to .)  These β-strands display an almost “seamless symmetry” in which only two of the strands vary in structural content.<ref name="Phillips">PMID: 9434902</ref>  This β-sheet conforms itself through regular hydrogen bonding into a β-barrel.<ref name="Yang" />  In GFP, the structure is so regular that <scene name='Green_Fluorescent_Protein/Water_stripes/1'>"stripes"</scene> of water molecules (red) can be seen following the structure of the barrel.<ref name="Phillips" />  Together with the α-helices at either end of the molecule, a nearly perfect cylinder is produced, 42Å long and 24Å in diameter,<ref name="Ormo" /> creating what is referred to as a “β-can” formation.<ref name="Phillips" />  The short helical segments at either end of the cylinder form “caps” to further protect the interior of the β-barrel.<ref name="Phillips" />  Overall stability is maintained by this β-can structure, helping to resist unfolding from heat and other denaturants.<ref name="Yang" />
-One <scene name='Green_Fluorescent_Protein/Central_helix/1'>α-helix</scene> can be found running through the central axis of the β-barrel,<ref name="Haldar" /> roughly <scene name='Green_Fluorescent_Protein/Perpendicular/1'>perpendicular</scene> to the symmetry axis of the barrel.<ref name="Ormo">Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ.  1996.  Crystal structure of the ''Aequorea victoria'' green fluorescent protein.  Science.  273(5280):1392-1395.  DOI 10.1126/science.273.5280.1392.</ref>  This helix is extremely important as it contains the fluorophore responsible for fluorescence.<ref name="Yang" /><ref name="Haldar" />  This α-helix in particular is highly stabilized by the many <scene name='Green_Fluorescent_Protein/Spacefill/1'>contacts</scene> that are made with each strand of the barrel.<ref name="Andrews">PMID:18713871</ref>
+Since the discovery of HIV Tat, other Arginine-rich, natural CPPs have been discovered including, penetratin from Drosophila antennapedia protein, sweet arrow peptide (SAP), HIV-1 Rev, flock house virus (FHV) coat, brome mosaic virus (BMV) Gag, human T-cell lymphotrophic virus (HTLV)-II Rex, and the nuclear localization signal (NLS) from nucleoplasmin. <ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref><ref>Prochiantz ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 448-451.</ref> The PTD sequences for these proteins can be found in the table below.
-{{Link Toggle FancyCartoonHighQualityView}}.
-===The Chromophore===
+[[Image:Arginine_Rich_Peptides.png]]
-The <scene name='Green_Fluorescent_Protein/Chromophore/1'>chromophore</scene> (<scene name='Green_Fluorescent_Protein/1ema_gfp_chromophorezoom/6'>top view</scene>) of GFP is located at the center of the β-barrel with a wild-type excitation peak of 395 nm, and a minor peak at 475 nm (about three times less intense<ref name="Tsien" />) <ref name="Yang" /><ref name="Cubitt" /><ref name="Ormo" /><ref name="Phillips" /> with extinction coefficients of approximately 30,000 and 7,000 M<sup>-1</sup> cm<sup>-1</sup>, respectively.<ref name="Yang" /><ref name="Phillips" />  Interestingly, the ''Aequorea victoria'' jellyfish utilizes the smaller of the two excitation peaks as pure aequorin emits a light of 470 nm.<ref name="Tsien">Tsien, Roger Y.  1998.  The Green Fluorescent Protein.  Annual Review in Biochemistry.  67:509-544.</ref>  The relative amplitudes of these two excitation peaks can vary depending on environmental factors and previous illumination.<ref name="Ormo" />  For example, continued excitation leads to a diminution of the 395 nm excitation peak with a reciprocal amplification of the 475 nm peak.<ref name="Phillips" />  Regardless of absorption, the chromophore of GFP emits light of 508 nm.<ref name="Yang" /><ref name="Cubitt" /><ref name="Ormo" /><ref name="Phillips" />
+== '''Tat-mediated Transduction''' ==
-Three amino residues in the central α-helix constitute the fluorophore of GFP: Ser<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> (see below).  Tsien et al. discovered that this tri-peptide sequence is post-translationally modified by internal cyclization and oxidation<ref name="Haldar" /> to produce a <scene name='Green_Fluorescent_Protein/Chromophore_structure/1'>4-(p-hydroxybenzylidene)-imidazolidin-5-one</scene> structure.<ref name="Yang" />  Studies with E. coli proposed a sequential mechanism for the formation of the fluorophore that was initiated by a rapid cyclization between Ser<sup>65</sup> and Gly<sup>67</sup> to form an imidazolin-5-one intermediate.<ref name="Yang" />  This rapid cyclization is carried out via nucleophilic attack of the amino group from Gly<sup>67</sup> on the carbonyl group of Ser<sup>65</sup> to form a five-membered ring.  The loss of water then forms the imidazolin-5-one intermediate.<ref name="Cubitt" />  Cyclization is succeeded by a much slower rate-limiting oxygenation of the Tyr<sup>66</sup> hydroxybenzyl side chain by atmospheric oxygen (No fluorescence was seen in anaerobically grown E. coli.), resulting in the 4-(p-hydroxybenzylidene)-imidazolidin-5-one stucture.<ref name="Yang" /><ref name="Cubitt" /><ref name="Phillips" />  The double bond that results from this series of reactions results in the linkage of the two π-systems of the rings, forming a larger conjugated system essential for fluorophore stability. <ref name="Bublitz"> Bublitz G, King BA, Boxer SG.  1998.  Electronic structure of the chromophore in green fluorescent protein (GFP).  Journal of the American Chemical Society.  120(36): 9370-9371.  DOI 10.1021/ja98160e.</ref>
+The PTD in HIV Tat is highly cationic.  When charged residues (Arginine or Lysine) are replaced with neutral residues (Alanine), cellular uptake decreases.  It is likely that the cationic charge is necessary for electrostatic interactions with components of the cellular membrane, such as lipid head groups and proteoglycans.<ref>Ziegler ''et al. Biochemistry'' '''2003'''. 42, 9185-9194.</ref><ref>Goncalves ''et al. Biochemistry.'' '''2005'''. 44, 2692-2702.</ref>  Other interactions may also play a role in interactions with the cellular membrane, such as hydrophobic and non-covalent interactions.<ref>Ziegler ''et al. Adv. Drug Deliv. Rev.'' '''2008'''. 60, 580-597.</ref>  Although cellular uptake is not directly dependent upon architecture (branched vs. linear), it is dependent upon the number of Arginine residues. Polymers with less than five arginine residues are unable to enter cells.<ref>Wender ''et al. Proc. Natl. Acad. Sci.'' '''2000'''. 97, 13003-13008.</ref>  As the number of residues increases, cellular uptake improves up until 15 residues where polymers can enter cells but with hindered efficiencies.<ref>Mitchell ''et al. J. Pept. Res.'' '''2000'''. 56, 318-325.</ref><ref>Futaki ''et al. J. Biol. Chem.'' '''2001'''. 276, 5836-5840.</ref>  The exact mechanism of cellular uptake is still highly debated in the literature, but a couple of the proposed methods are discussed below.
-[[Image:GFP Chromophore.png|center|489x360px]]
-The process is completely auto-catalytic such that there are no known co-factors or enzymatic components required.<ref name="Yang" />  Despite the stability of the final product, while the chromophore is forming, the environmental temperature cannot drop below 30°C or the yield of viable GFP will decrease substantially.<ref name="Yang" /><ref name="Phillips" />  This, of course, is not an issue for the protein in nature as the jellyfish is unlikely to encounter waters of this degree in the Pacific Northwest.<ref name="Tsien" />  Such a temperature sensitivity is only relevant during formation as the stability of the final product is maintained through a network of close contacts surrounding the fluorophore.<ref name="Yang" />  This, however, can and has been used in [[pulse-chase experiments]] in which the GFP-expressing cells are exposed to varying temperatures in place of labeled vs. unlabeled trials.<ref name="Tsien" />
+==Transduction==
+Early studies indicated that HIV Tat primarily entered cells through transduction, meaning that the protein directly crossed the cell membranes.  However, for most of these studies, cells were fixed with formaldehyde prior to sample analysis.  In 2003, it was discovered that by fixing cells prior to analysis, it permeablized the cell membranes and resulted in artificially high cellular uptake values. Fixed cells showed higher concentrations of Tat in nuclei compared to non-fixed cells where it was localized in the cytoplasm. <ref>Richard ''et al. J. Biol. Chem.'' '''2003'''. 278, 585-590.</ref>  Studies also showed that FACS could not tell the difference between internalized cells and those proteins that were only bound to the cellular membranes. Cellular uptake studies performed at 4°C and under conditions of ATP depletion show that a small percentage of CPPs can enter cells and also suggests that a majority of the protein enters cells through energy dependent pathways.<ref>Drin ''et al. J. Biol. Chem.'' '''2003'''. 276, 31192-31201.</ref>  Some groups have also studied transduction of Tat using model vesicles. <ref>Ziegler ''et al. Biochemistry.'' '''2003'''. 278, 9185-9194.</ref>
-<applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/2' name='2'/>
+==Endocytosis==
+Another proposed mechanism for cellular uptake is endocytosis, specifically macropinocytosis.  This form of fluid phase endocytosis is less known that other types (phagocytosis, clathrin, and coated pit), it is believed to occur in all cell types and enable the uptake of large molecules.  <ref>Gump ''et al. Trends in Molecular Medicine.'' '''2007'''. 13, 443-448.</ref>  During this process, actin protrusions fold around molecules and the surrounding medium and enable cellular uptake.
-As the central α-helix is not located directly in the center of the β-barrel, cavities of differing area exist on either side of the chromophore.  The larger cavity, consisting of about 135 Å,<ref name="Ormo" /> does not open out to the bulk solvent, but rather houses <scene name='Green_Fluorescent_Protein/Water_molecules/1'>four water molecules</scene>.<ref name="Ormo" /><ref name="Van">van Thor JJ, Sage, JT.  2006.  Charge transfer in green fluorescent protein.  Photochemical & Photobiological Sciences.  5:597-602.  DOI 10.1039/b516525c.</ref>  Had this space not been occupied, it would be expected to considerably destabilize the protein as a whole.  The hydrogen bonding created by the presence of the water molecules, however, helps to link the buried <scene name='Green_Fluorescent_Protein/Gln69_glu222/1' target='2'>side chains</scene> of Glu<sup>222</sup> and Gln<sup>69</sup> that would otherwise be actively polar.<ref name="Ormo" />  Therefore, the water molecules are extremely important in establishing a hydrogen bonding network about the chromophor.<ref name="Lammich">PMID: 17040991</ref>
+== '''My Research Interests''' ==
+As part of the [http://www.pse.umass.edu/gtew/index.html Tew Research Group] in the [http://www.pse.umass.edu Department of Polymer Science and Engineering] at the University of Massachusetts, Amherst, I am interested in the design of Tat-inspired CPPs for drug and gene delivery.  Using functionalized oxanorbornene derivatives as monomers (as shown below), ring opening metathesis polymerization (ROMP) is performed using Grubbs' 3rd generation catalyst to synthesize cationic CPP mimics.  Hydrophobicity, charge, aromaticity, and pi-electronics of the monomer structures can be optimized to achieve better cellular uptake and cell viability by changing R1 and R2.
-The opposite side of the chromophore, however, is within close proximity of several aromatic and polar side chains.  Several <scene name='Green_Fluorescent_Protein/Polar_interactions/2'>polar interactions</scene> between the surrounding residues and the chromophore are present including: hydrogen bonds of His<sup>148</sup>, Thr<sup>203</sup>, and Ser<sup>205</sup> with the phenolic hydroxyl of Tyr<sup>66</sup>; Arg<sup>96</sup> and Gln<sup>94</sup> with the carbonyl of the imidazolidinone ring; and hydrogen bonds of Glu<sup>222</sup> with the side chain of Thr<sup>65</sup>.  Additional hydrogen bonding in the area around the chromophore helps to stabilize Arg<sup>96</sup> in the protonated form, which suggests the presence of a partial negative charge on the carbonyl oxygen of the imidazolidinone ring in the deprotonated fluorophore.<ref name="Ormo" />  Arg<sup>96</sup> and Gln<sup>94</sup> in turn help to steady the imidazolidone.<ref name="Yang" />  Therefore, it is thought that Arg<sup>96</sup> is essential for the formation of the fluorophore by catalyzing the initial ring closure.<ref name="Ormo" />  Tyr<sup>145</sup> provides a stabilizing
+[[Image:norbornenederivatives.png]]
-<scene name='Green_Fluorescent_Protein/Edge_face_interaction/1'>edge-face interaction</scene><ref> [http://www.tim.hi-ho.ne.jp/dionisio/ Information about edge-face (CH/π) interactions].</ref> with the benzyl ring of the chromophore.<ref name="Ormo" />  The stability provided by the internal polar interactions are further augmented by the surrounding β-barrel.
-The β-barrel provides a highly constrained environment that protects the chromophore from the bulk solvent,<ref name="Haldar" /> nearly creating the atmosphere of a vacuum.<ref name="Lammich" />  This is most likely responsible for the small [[Stoke’s shift]], or the small wavelength difference between excitation and emission.<ref name="Ormo" />
+== '''Acknowledgments''' ==
+The Tew research group is gratefully acknowledged for their contributions to the design and content of this page.
-{{Link Toggle FancyCartoonHighQualityView}}.
-Findings show that fluorescence will not occur from a naked chromophore, but rather requires the protection of the β-can structure.<ref name="Cubitt" />  However, ''in crystallum'' GFP will exhibit a nearly identical fluorescence spectrum and lifetime when compared with aqueous GFP.  These two elements point to a fluorescence that is not inherent to the isolated fluorophore,<ref name="Yang" /><ref name="Phillips" /> but rather from the auto-catalytic cyclization of the polypeptide sequence Ser<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> and subsequent oxidation of Tyr<sup>66</sup>.<ref name="Phillips" />  However, this sequence is found in many proteins - why does GFP fluoresce?  According to Phillips (1997), fluorophore formation is due to the close proximity of the backbone atoms between Ser<sup>65</sup>. and Gly<sup>67</sup> gained through a lack of sterical hindrance by the hydrogen atom side chain of glycine.  In fact, no functional fluorescent proteins have been found in which any other amino acid other than glycine was found at position 67.  Even so, there are still proteins that have this specific sequence, therefore, there must be another inherent property to GFP that is still left misunderstood.<ref name="Phillips" />
-This quandary led Phillips to study the acid/base chemistry catalyzing the initial cyclization of the chromophore.  He found that Arg<sup>96</sup> actually acts as a
-<scene name='Green_Fluorescent_Protein/Arg96/1' target='2' >base</scene> by withdrawing electrons through hydrogen bonding with the carbonyl oxygen of Ser<sup>65</sup> to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen of Gly<sup>67</sup>.  This mechanism was further supported by ''ab initio'' calculations, as well as database searches of similar compounds and protein sequences.  Through acid/base chemistry, the chromophore is stabilized by resonance.<ref name="Phillips" />  Femtosecond Raman spectroscopy has been used to map the alteration of the structure close the chromophore during excited-state protein transfer and shown that chromophore wagging is orchestrated by the protein environment.<ref name="Fang">PMID: 19907490</ref>
-===Mutant Studies===
-<applet load='1ema' size='400' frame='true' align='right' scene='Green_Fluorescent_Protein/1ema_gfp_barrel/2' name='A'/>
-Many mutant green fluorescent proteins have been developed in order to further understand the structure and mechanism of the fluorophore.  The first mutagenesis studies simply
-<scene name='Green_Fluorescent_Protein/Truncated_ends/2' target='A'>truncated the ends</scene> of the amino acid sequence (<scene name='Green_Fluorescent_Protein/1ema_gfp_barrel/2' target='A'>see without truncated ends</scene>.  NOTE: The structure represented here is already truncated at the carbonyl terminus).  Shortening the polypeptide by more than seven amino acids from either terminus lead to a total loss of fluorescence, as well as a complete failure to absorb light at the traditional wavelengths.   This is most likely due to the structure of the protein.  The last seven amino acid residues of the carboxyl terminus are roughly disordered, and thus do not interfere with the overall structure.  After seven residues, however, the capping α-helix structure is disrupted, leading to an unstable or unformed chromophore.  The <scene name='Green_Fluorescent_Protein/Amino_terminus/2' target='A'>amino terminus</scene> is less understood, but the same principle still applies even though the β-barrel does not begin until residue ten or eleven.<ref name="Yang" />
-Point mutations have also been extensively studied in order to examine their effects on the chromophore.  In general, most point mutations lead to a diminished excitation, especially in regions of the sequence adjacent to the fluorophore or those that interact with the fluorophore.  An exception to this trend is the Ser<sup>65</sup>Thr<sup>66</sup> mutant (normal Ser<sup>65</sup>Tyr<sup>66</sup>), which actually increases fluorescence intensity, although the reason is unclear.<ref name="Yang" />
-An interesting mutation discovered by Ormo et al. (1996) was the Thr<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> mutant, which produces an α-helical conformation in the chromophore opposed to the normal conformation, which is nearly perpendicular to the helical axis, due to its interaction with Arg<sup>96</sup>.  This further supports the idea that Arg<sup>96</sup> is an important factor in the structural arrangement required for cyclization, perhaps by promoting the attack of Gly<sup>67</sup> on the carbonyl carbon of Thr<sup>65</sup>.<ref name="Ormo" />
-In high protein concentrations, GFP has been found to dimerize under the influence of high ionic strength between the two monomers.  In ''Aequorea victoria'', the aequorin is able to bind to the <scene name='Green_Fluorescent_Protein/1gfl/1' target='A'>dimer</scene> ([[1gfl]]), but not the monomer.  Therefore, dimerization is a very important structural feature in terms of its function, as it also assists the GFP to absorb energy at the excitation wavelength of aequorin even though GFP has only a “modest” extinction coefficient.  As a result, dimers, and often even higher <scene name='Green_Fluorescent_Protein/1w7s/1' target='A'>multimers</scene> ([[1w7s]]), are predominant protein populations within the jellyfish.<ref name="Cubitt">[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCV-40W0TN7-50&_user=4187488&_coverDate=11%2F30%2F1995&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000062504&_version=1&_urlVersion=0&_userid=4187488&md5=e92730038bb92b1dfbd4af45a0283cce],Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien R.  1995.  Understanding, improving, and using green fluorescent protein.  Trends in Biochemical Sciences.  20(11): 448-455.  DOI 0.1016/S0968-0004(00)89099-4.</ref>
-{{Link Toggle FancyCartoonHighQualityView}}.
-== Using GFP as a Research Tool ==
-A description of some of the ways GFP is being used as a tool in research is at [[Green_Fluorscent_Protein:_Research_Tool]].
-==3D Structures of Green Fluorescent Protein==
-''Update November 2011''
-[[2qu1]], [[2h9w]] – jGFP - jellyfish<br />
-[[3la1]], [[3i19]], [[2wur]], [[3gex]], [[2qrf]], [[2qt2]], [[2qz0]], [[2gj1]], [[2gj2]], [[3cb9]], [[3cbe]], [[3cd1]], [[3cd9]], [[2hjo]], [[2hqz]], [[2hrs]], [[2okw]], [[2oky]], [[2q57]], [[2due]], [[2duf]], [[2dug]], [[2duh]], [[2dui]], [[2q6p]], [[2hcg]], [[2hfc]], [[2hgd]], [[2hgy]], [[2awj]], [[2awk]], [[2awl]], [[2awm]], [[2g16]], [[2g2s]], [[2g3d]], [[2g5z]], [[2g6e]], [[2ah8]], [[2aha]], [[2fwq]], [[2fzu]], [[2b3p]], [[2b3q]], [[1z1p]], [[1z1q]], [[1yhg]], [[1yhh]], [[1yhi]], [[1yj2]], [[1yjf]], [[1s6z]], [[1q4a]], [[1q4b]], [[1q4c]], [[1q4d]], [[1q4e]], [[1q73]], [[1qyf]], [[1qyo]], [[1qyq]], [[1qst]], [[1qy3]], [[1cv7]], [[1jc0]], [[1jc1]],  [[1jby]], [[1jbz]], [[1kp5]], [[1kyp]], [[1hcj]], [[1h6r]], [[1b9c]], [[1c4f]], [[1emc]], [[1eme]], [[1emf]], [[1emk]], [[1eml]], [[1emm]], [[2emd]], [[2emn]], [[2emo]], [[1emb]], [[1gfl]], [[1ema]], [[2y0g]], [[3gj1]], [[3gj2]], [[3p28]], [[1qxt]] – jGFP (mutant)<br />
-[[3evp]] – jGFP circular permutation<br />
-[[2h6v]] – jGFP+imidazole derivative<br />
-[[1rm9]], [[1rmm]], [[1rmo]], [[1rmp]], [[1rrz]] – jGFP containing fluorotryptophan<br />
-[[2o24]], [[2o29]], [[2o2b]], [[1w7u]], [[1w7t]], [[1w7s]], [[1emg]] – jGFP (mutant)+imidazole derivative<br />
-[[1kyr]] – jGFP (mutant)+imidazole derivative+Cu<br />
-[[1kys]] – jGFP (mutant)+imidazole derivative+Zn<br />
-[[3ogo]] – jGFP+cGFP nanobody – camel<br />
-[[3g9a]], [[3k1k]] – jGFP+minimize nanobody – ''Lama pacos''<br />
-[[2qle]] – GFP (mutant) – ''Azotobacter vinelandii''<br />
-[[2rh7]] – GFP – ''Renilla reniformis''<br />
-[[3adf]] – monomeric azami green – ''Galaxea fascicularis''<br />
-[[2vzx]] – GFP DENDRA2 – Dendronephthya<br />
-[[2gw3]] – GFP KAEDE – ''Trachiphyllia geoffroyi''<br />
-[[2pox]], [[2gx0]], [[2gx2]], [[2iov]], [[2ie2]] – FP DRONPA – Echinophyllia<br />
-[[2dd7]] – CpGFP - ''Chiridius poppei''<br />
-[[2dd9]] – CpGFP (mutant)<br />
-[[2c9i]] – saGFP – sea anemone<br />
-[[1xmz]] – saGFP (mutant)<br />
-[[2c9j]] – GFP – ''Cerianthus membranaceus''<br />
-[[2hpw]] – GFP – ''Clytia gregaria''<br />
-[[2g3o]] – PpGFP – ''Pontellina plumata''<br />
-[[2g6x]], [[2g6y]] – PpGFP (mutant)<br />
-[[3lva]], [[3lvc]], [[3lvd]] – GFP (mutant) – ''Aequoarea coerulescens''<br />
-===Yellow fluorescent protein===
-[[3dpw]], [[3dpx]], [[3dpz]], [[3dq1]], [[3dq2]], [[3dq3]], [[3dq4]], [[3dq5]], [[3dq6]], [[3dq7]], [[3dq8]], [[3dq9]], [[3dqa]], [[3dqc]], [[3dqd]], [[3dqe]], [[3dqf]], [[3dqh]], [[3dqi]], [[3dqj]], [[3dqk]], [[3dql]], [[3dqm]], [[3dqn]], [[3dqo]], [[3dqu]], [[1myw]], [[1huy]], [[2yfp]], [[1yfp]] – jGFP (mutant)<br />
-[[1f09]], [[1f0b]] – jGFP (mutant)+imidazole derivative+I<br />
-[[2ogr]] – Z-FP - Zoanthus<br />
-[[2pxs]], [[2pxw]], [[1xa9]], [[1xae]] – Z-FP (mutant)<br />
-[[2jad]] – jGFP/glutaredoxin<br />
-===Red fluorescent protein===
-[[2icr]], [[2ojk]] –  Z-RFP <br />
-[[2fl1]] – Z-RFP (mutant)<br />
-[[3bx9]], [[3bxa]], [[3bxb]], [[3bxc]], [[3e5t]], [[3e5w]], [[1uis]], [[3ip2]], [[3pj5]], [[3pj7]], [[3pjb]], [[3pib]] -  EnRFP  – ''Entacmaea quadricolor''<br />
-[[3e5v]], [[3rwt]] – EnRFP (mutant)<br />
-[[1zgo]], [[2vad]], [[2vae]], [[1ggx]] – DiRFP – ''Discosoma''<br />
-[[1zgp]], [[1zgq]], [[2h8q]], [[2v4e]], [[1g7k]] – DiRFP (mutant)<br />
-[[3cfa]] – AsRFP – ''Anemonia sulcata''<br />
-[[3nt3]], [[3nt9]] – RFP – artificial gene<br />
-[[1yzw]] – RFP – ''Heteractis''
-===Cyan fluorescent protein===
-[[2wsn]], [[2wso]] - jGFP<br />
-[[2otb]] – cyan C-FP – Clavularia<br />
-[[2ote]] - cyan C-FP (mutant)<br />
-[[2zo6]], [[2zo7]] – cyan FP – ''Fungia concinna''<br />
-[[1oxd]], [[1oxe]], [[1oxf]] – cyan FP (mutant) – marker plasmid<br />
-===Blue fluorescent protein===
-[[1bfp]] – jGFP (mutant)
-===Photoconvertible fluorescent protein===
-[[2vvh]], [[2vvi]], [[2vvj]], [[3p8u]] –  LhGFP (mutant) – ''Lobophyllia hemprichii''<br />
-[[1zux]] –  LhGFP<br />
-[[2btj]] -  LhGFP+imidazole derivative<br />
-[[2ddc]], [[1xss]] –  FfFP – ''Favia favus''<br />
-[[2ddd]] -  FfFP (mutant)<br />
-[[3cff]], [[3cfh]] – AsGFP (mutant)
-===Green fluorescent protein chimera===
-[[3ai4]] – jGFP/mPolymerase iota ubiquitin binding motif - mouse<br />
-[[3ai5]] - jGFP/m ubiquitin<br />
-[[3o77]], [[3o78]], [[3ek4]], [[3ek7]] - jGFP/myosin light chain kinase/calmodulin<br />
-[[3evr]], [[3evu]], [[3evv]] - jGFP/myosin light chain kinase/calmodulin+Ca<br />
-[[3ek8]], [[3ekh]], [[3ekj]] - jGFP/myosin light chain kinase/calmodulin (mutant)<br />
-[[3osq]], [[3osr]] – jGFP/maltose-binding protein
-==Reference for this Structure==
-Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ.  1996.  Crystal structure of the ''Aequorea victoria'' green fluorescent protein.  Science.  273(5280):1392-1395.  [http://www.sciencemag.org/cgi/content/abstract/273/5280/1392 DOI 10.1126/science.273.5280.1392].
-==References==
+== '''References''' ==
 {{Reflist}}
-==Additional Resources==
-*For additional information, see: [[Colored & Bioluminescent Proteins]]
-*[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1ema First Glance]
-*PDBsum: [http://www.ebi.ac.uk/pdbsum/1ema 1ema]
-*RCSB PDB [http://www.rcsb.org/pdb/explore.do?structureId=1ema 1ema]
-*[http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ema OCA]
-*UniProt: [http://www.uniprot.org/uniprot/P42212 P42212]
-*Scop: [http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.e.gc.b.b.b.html P42212]
-*CATH: [http://www.cathdb.info/domain/1emaA00 1emaA00]
-*Pfam: [http://pfam.sanger.ac.uk/family?acc=PF01353 PF01353]
-*InterPro: [http://www.ebi.ac.uk/interpro/ISearch?query=IPR000786 IPR000786]
-*[http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb42_1.html GFP featured] at the '''[http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/index.html Molecule of the Month]''' series of tutorials by [[User:David_S._Goodsell|David Goodsell]].
-* [http://www.nature.com/nature/journal/v462/n7270/edsumm/e091112-05.html Inside green fluorescent protein] - editor's summary that accompanied [http://www.nature.com/nature/journal/v462/n7270/covers/ structural detail of GFP chromophore on the cover] of Nature.
-[[he:GFP_(Hebrew)]]
-[[Category:Topic Page]]

Brittany deRonde/Sandbox 1

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