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[[[[Green Fluorescent Protein]]]]<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]])' />
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].
'''Green fluorescent protein (GFP)''' 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==


''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" />
==Your Heading Here (maybe something like 'Structure')==<StructureSection load='1dq8' size='300' side='right' caption='Structure of HMG-CoA reductase (PDB entry [[1dq8]])' scene=''>Anything in this section will appear adjacent to the 3D structure and will be scrollable.
[[Image:GFP mice.png|thumb|left|450x200px|Mice with GFP inserted into their genomes for neurology studies.]]
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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" />
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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" />
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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.
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==Structure==


===Primary & Secondary Structure===
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{{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>


{{Link Toggle FancyCartoonHighQualityView}}.
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===The Chromophore===
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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" /> 
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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>
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[[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" />
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<applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/2' name='2'/>
7
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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>
8


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
9
<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" /> 


{{Link Toggle FancyCartoonHighQualityView}}.
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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" /> 
11


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
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<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'/>
</StructureSection>
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
<Structure load='1L8Q' size='300' frame='true' align='right' caption='1L8Q 76-399' scene='User:Jing_Liu/Sandbox_1/1/4' />
<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" /> 
This is a <scene name='User:Jing_Liu/Sandbox_1/1/4'>DnaA monomer strucure(residues 76-399)</scene>, which contains an <scene name='User:Jing_Liu/Sandbox_1/1/5'>ADP</scene> in it.


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" />
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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>
----
=== DnaA function in Prokaryotes===
DnaA is an AAA+ protein in prokaryotes for DNA replication initiation. To ensure correct timing of DNA replication, intracellular DnaA is under multi-level control, including changing expression/degradation level, regulator control and functional activation/inactivation by nucleotide-depedent conformational change. DnaA is a conserved protein in prokaryotes. In ''E. coli'', There are four regions in this protein[http://www.ncbi.nlm.nih.gov/pubmed/10572294]:


{{Link Toggle FancyCartoonHighQualityView}}.
I: residues 1-85. DnaB loading domain;


== Using GFP as a Research Tool ==
II: residues 86-133. linker region;


A description of some of the ways GFP is being used as a tool in research is at [[Green_Fluorscent_Protein:_Research_Tool]].
III: residues 134-372. ATPase domain;


 
IV: residues 373-467. DNA binding domain;
==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==
DnaA function is regulated by different nucleotide binding state. In ATP bound state, DnaA can recognize more binding elements on the chromosome(called DnaA box), and initiate replication by unwinding specific region in the DNA and recruit DnaB helicase. However, after the ATP hydrolysis, DnaA-ADP becomes an inactive protein. This activity change was shown to be associated with its oligomeric assembly state. 


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].
=== DnaA Monomer Structure ===
The <scene name='User:Jing_Liu/Sandbox_1/1/4'>structure of DnaA</scene> in ''Aquifex aeolicus'' was solved in 2002[http://www.ncbi.nlm.nih.gov/pubmed/12234917?dopt=Abstract], and it contains the linker region, AAA+ domain and DNA binding domain(DBD). The interaction with DNA is through the recognization of a conserved 9 bp DNA recognition sequence (TTA/TTNCACC), and this was shown by the crystal structure of the <scene name='User:Jing_Liu/Sandbox_1/2/1'>complex</scene> in ''Mycobacterium tuberculosis''[http://www.ncbi.nlm.nih.gov/pubmed/21620858?dopt=Abstract].  


==References==
=== Distinct Assembly State of DnaA ===
<Structure load='1L8Q' size='300' frame='true' align='right' caption='2HCB tetramer' scene='User:Jing_Liu/Sandbox_1/3/3' />
The crystal structure of AMP-PCP-bound DnaA reveals a <scene name='User:Jing_Liu/Sandbox_1/3/3'>right-handed superhelix</scene> structure[http://www.ncbi.nlm.nih.gov/pubmed/16829961?dopt=Abstract]. Each monomer contact with another two monomers to form a filamentous structure. The engagement of ATP involved the arginine figure at position 230.


{{Reflist}}
=== Reference ===
1. Messer, W., et al., Functional domains of DnaA proteins. Biochimie, 1999. 81(8-9): p. 819-25.


==Additional Resources==
2. Erzberger, J.P., et al., The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation. EMBO J. 2002 Sep 16;21(18):4763-73.
*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)]]
3. Tsodikov OV and Biswas T. Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA. J Mol Biol. 2011 Jul 15;410(3):461-76. Epub 2011 May 18.


[[Category:Topic Page]]
4. Erzberger, J.P., et al., Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling. Nat Struct Mol Biol. 2006 Aug;13(8):676-83. Epub 2006 Jul 9.

Latest revision as of 19:01, 10 October 2012

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.


==Your Heading Here (maybe something like 'Structure')==

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Structure of HMG-CoA reductase (PDB entry 1dq8)

Drag the structure with the mouse to rotate

1L8Q 76-399

Drag the structure with the mouse to rotate

This is a , which contains an in it.

DnaA function in ProkaryotesDnaA function in Prokaryotes

DnaA is an AAA+ protein in prokaryotes for DNA replication initiation. To ensure correct timing of DNA replication, intracellular DnaA is under multi-level control, including changing expression/degradation level, regulator control and functional activation/inactivation by nucleotide-depedent conformational change. DnaA is a conserved protein in prokaryotes. In E. coli, There are four regions in this protein[1]:

I: residues 1-85. DnaB loading domain;

II: residues 86-133. linker region;

III: residues 134-372. ATPase domain;

IV: residues 373-467. DNA binding domain;


DnaA function is regulated by different nucleotide binding state. In ATP bound state, DnaA can recognize more binding elements on the chromosome(called DnaA box), and initiate replication by unwinding specific region in the DNA and recruit DnaB helicase. However, after the ATP hydrolysis, DnaA-ADP becomes an inactive protein. This activity change was shown to be associated with its oligomeric assembly state.

DnaA Monomer StructureDnaA Monomer Structure

The in Aquifex aeolicus was solved in 2002[2], and it contains the linker region, AAA+ domain and DNA binding domain(DBD). The interaction with DNA is through the recognization of a conserved 9 bp DNA recognition sequence (TTA/TTNCACC), and this was shown by the crystal structure of the in Mycobacterium tuberculosis[3].

Distinct Assembly State of DnaADistinct Assembly State of DnaA

2HCB tetramer

Drag the structure with the mouse to rotate

The crystal structure of AMP-PCP-bound DnaA reveals a structure[4]. Each monomer contact with another two monomers to form a filamentous structure. The engagement of ATP involved the arginine figure at position 230.

ReferenceReference

1. Messer, W., et al., Functional domains of DnaA proteins. Biochimie, 1999. 81(8-9): p. 819-25.

2. Erzberger, J.P., et al., The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation. EMBO J. 2002 Sep 16;21(18):4763-73.

3. Tsodikov OV and Biswas T. Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA. J Mol Biol. 2011 Jul 15;410(3):461-76. Epub 2011 May 18.

4. Erzberger, J.P., et al., Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling. Nat Struct Mol Biol. 2006 Aug;13(8):676-83. Epub 2006 Jul 9.

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