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Both the NMR and Crystalline structure of Spo0f have both been solved.  In 1997 Dr. John Cavanagh and lab successfully determined the 3 dimensional structure of  spo0f through NRM spectroscopy (1).  Later in 1998 spo0f was successfully crystalized by Zapf, Hoch, Whiteley, Xuong, and Varughese; through the use of X-ray crystal diffraction they were able to characterize the 3-dimensional crystalline structure (4,5).

The sporulation process begins by B. subtilis respoinding to harsh environmantal conditions or increased cellular density (1,2).  Once the specific condtions are met the cell begins to express specific gene clusters to begin translation of proteins for cellular conversion into the spore state (1,2,3,6).  The overall process of spore formation is controlled by a phosphorelay chain; initiated by autophosphorylation of histadine kinase A and B which then phosphorylate protein spo0F (2).

The signal transduction pathway begins by the celularl ATP-dependant autophosphorylation ofhistadine kinase A and B in response to specific envirionmental conditions (2); this mechenism is still uknown.  Spo0F is then phosphorylated by KinA at spo0F’s active site.  This phosphorylation creates a confromtaions change to spo0F (1,2,6) which then activetes its kinase properties.  Once activated, spo0F is able to phospohrylate spo0A through a phosphoramidate intermediate spo0B (1,2).  Spo0B acts as a phosphotransferase between spo0F and A, which reacts reverable between the two kinases (2).  Once phosphorylated, spo0A is in its active transcriptional reulator form and initiates the transcriptions of spoulation proteins and spore formation may begin.

The phosphorylation and hydrolysis of Spo0F is dependant upon a magnesium ion cofactor that binds to the active site, and a histidine autokinase (1,2,7).  It is hypothesized that the Mg ion is used to stablalize the transition state for the phosphoryl-transfer and hydrolysis reactions (1).  A unique characteristic of spo0F in comparison to other response regulatory proteins is the low binding affinity for magnesium, thus leading to a decreased stablility of the transition state.  The decreased stablility of the transition state leads to spo0F having longer phosphrylated life time than other similar enzymes leading to the selectivety of the enzyme.  It is shown the affinity difference between response regulator enzymes, such as spo0F, is due to the non-conserved sequence within the protein (1).  

Sporulation may be mediate in multiple ways through regulation of spo0F. One method is the hydrolysis of phosphorylated spo0F (spo0f-P) by Rap Phosphotases (7).  The Rap phophotases are highly specific within this system and will only dephosphorylate spo0F in this phosphorelay pathway (7), despite the similarities in structure of spo0A and spo0F (2,7).  Even though the Rap does not directly infulence spo0A or transctription it still inhibits sporulation by reducing the conentration of spo0F-p thus reducing production of spoA-p and trancritption, due to the reversible phophotransferase between A and F (2).  Thus, regulation is due to competing phosphorylation of the secondary messangers in the pathway.

The low affinity of Mg binding to the active site of spo0F assists in enzyme regulation as well (1).  The reduced affintiry leads to regulation by only allowing spo0F to phosphorylate phosphoramidates (1) and may only due so when the active cellular concentration of Mg is high enough.  Spo0F is only able to phosphorylate phosphoramidates, such as spo0B and spo0A, due to the ease of transfer and thermodynamic favorability.  Because the intermediate state is not very stable, spo0F is only able to transfer its phosphate group with highly energetically favorable reactions.  This specific transfer leads to the selectivity and regulation of sporlulation and spo0F.  This is also depenant upon the active concetration of magnesium; without high enough concentrations the reaction cannot occur and spo0F is not able to attract Mg under  low concentrations.

Spo0F is a 14KDa (2), 36 by 34 by 31 aongstrom single domain globular protein (6) containing 124 residue (4).  The protein contists of 45% helical secondary structures and 21% beta sheets structure (4).  There are 5 helixies and 5 beta sheets present within the polypeptide collectively made using 57 residues and 27 residues respectively (4).  The structure is further classified as an alpha and beta protein in the CheY superfamily, with folds classified as Flavodoxin-like folding (4).  The terriary structure is a “double wound (beta/alpha) motif formed [by the] five parallel beta strands flanked by alpha helicies” (2).  The helicies and strands are parallel in comparison to themselves however in contrast to eachother, i.e, beta to helix, they are opposite (6).The hydrophobic core of spo0F is made up of  beta strand one, three, four, and five; each of these helicies being amphiphathic (6, 1). The mojority of the protein surface is covered with hydrophilic residues, however there are two hyrdophobic regions present on the face of the peptide.  These hydrophobic surface regions are found by the N-terminus of helix five; it is speculated that this interaction is important in the response regulation of spo0F and protein protein interaction (6, 1, 7).  AS well, the side chains are highly ordered is the classical staggereing conformation.  From the most energetically enssamble of potential structures the side chains are withing 30 degrees of the staggered ordering (1).

The active site of spo0F is present within buried and exposed portions of the secondary structure found at the C-terminus of the central beta sheet (6).  The active site is made up of four residues, three alanines (D10,D11,D54) and a lysine (K104), with all the active residues on a single face (1,2, 6).  D54 is shown to be the specific site of phosphorylation in spo0F (6)  The active site itself accomidates a magnesium ion, to be used as a cofactor in the phosphate transfer, and Cl- for stability (6).  The calcium bound at the aspartate pocket stabalizes a octahedral structure of the binding pocket by interacting with the carboxylate residues of D11 and D54, and carbonyl group of K56.  This calcium ion is also shown to stimulate the enzymatic process.  The other binding postitions are bound to water molecules for increased stability(6).  However, the phosphorylation reaction is dependant upon Thr82 and its hydroxyl group, this residue may sometimes may be functinally mutated to a Ser residue.  The Thr82 is esential in the phosphotransfer reaction but does not assist in recognition and binding of other proteins in the phospohrelay chain (2).   

The active site itself is regulated by phospohrylation.  If spo0F is not phosphorylated the active site is inaccesable ; when phospohorylated a conformational change occurs opening the active site (6).  This pohsphyrlation is regulated by a histadine kinase (3).  Otherwise the protein itself lacks an effector domain (6).

Spo0F is able to interact with other proteins due to the hydrophobic residues found on the external faces of the molecule.  These regions are found on the N-teminus of alpha hleix 1 and alpha helix 5 along with the loop between beta sheet 4 and alpha helix 4 (6,1).  Spo0F must be able to interact with kinases, phosphatases, and phosphotransferases for functionality in the phosphorelay system.  There is however some flexibilty in the protein protein interaction of spo0F and other enzymes.  Both Kinase A and spo0B bind to the same site of spo0F; however these enzymes are sequentially unrelated (1).  This shows that the protein intereaction is specific, yet the binding site has a degenerate nature.

The overall process of analasis of spo0F includes proliferation, isolation, purification, and analysis.  One specific method of analysis will be described.  The proliferation process can accomplished by transfoming competent  E. coli cells through heat shock treatment.  E. coli is able to relatively easily uptake plamids and may be induced to over express the protein.  The plasmid used in the transformation requires a his-tagged spo0F gene, an antibiotic resistance gene, and a transcription initiation sequence.  A common initiation sequence is one that is activated by isopropyl β-D-1-thiogalactopyranoside (IPTG) activation sequence.  Once the cells are transformed cellular proliferation is the next step.  To isolate the transformed cell, the cells are cultured on a selective medium using the antibiotic that the plasmid provides resistance to.

Once large quantities of transformed cells have been obtained, overexpression of the protein is induced.  This process is dependent upon the initiation sequence within the plasmid.  For example, with the use of the ITPG initiation sequence addition of ITPG will induce expression of the gene with the specific tag.  Once the protein is proliferated to the desired quantity the cells are harvested, lysed, and centrifuged to remove large organic waste.  Finally the protein needs to be isolated, commonly through the use of chromatography.  If the His tag is used, spo0F may be isolated through the use of a nickel resin gravity column and an imidazole salt gradient.  Once isolated and purified the protein may then be analyzed through NMR, or crystalized for X-ray diffraction analysis.