Sandbox Reserved 657

This Sandbox is Reserved from 30/08/2012, through 01/02/2013 for use in the course "Proteins and Molecular Mechanisms" taught by Robert B. Rose at the North Carolina State University, Raleigh, NC USA. This reservation includes Sandbox Reserved 636 through Sandbox Reserved 685.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. Click the 3D button (when editing, above the wikitext box) to insert Jmol. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing For more help, look at this link: http://proteopedia.org/w/Help:Getting_Started_in_Proteopedia spinqualitylabelsall models Protein Spo0F Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image Spo0FSpo0F The protein spo0F is a 124 residue polypeptide found within Bacillus subtilis (1)(4), and is classified as a sporulation response regulatory kinase. Spo0f, is thus, an integral protein for multiple phosphorelay systems (3). It functions as a secondary messenger within sporulation phosphorelay systems and regulates the transition of B. subtilis from the vegetative to sporulated state (3). The protein helps prevent induced dormancy and use of resources when the conditions are not proper for sporulation. Expression of this protein is highly regulated and stimulated by environmental taxis becoming unsuitable for growth. The organism responds by expressing gene clusters, containing spo0f, to induce sporulation. Spo0F functions as a kinase in a phosphorelay pathway. In B. subtilis when environmental conditions become harsh, histadine kinases are autophosphorylated. These phosphorylated kinases are then able to activate spo0f by phosphorylation. Activated spo0F then phosphorylates the transcription factor spo0A by using the intermediate phosphor-transferase spo0B, which then begins transcription for sporulation. Spo0F’s structure is composed of 45% helical secondary structures and 21% beta sheets , with D10, D11, D54, and K104 functioning as the active site (2). It is a single strand phospho-kinase that is functional as a single stranded oligomer. One unique characteristic of spo0F is two hydrophobic portions that are on the external face of the molecule (1,2). These hydrophobic regions on the external faces are what allow for protein protein interactions. 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 crystallized 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). Figure 1: Image of the HSQC results of isolated spo0f; shows the hydrogen-nitrogen correlation spectrum with in the labeled Spo0f. Varian Inova 600 mHz NMR spectrometer ran the prepared 15N labeled spo0f. Sporulation OverviewSporulation Overview Bacteria adapt to changing environmental conditions in two designed states; functioning as a vegetative organism or in a sporulated state. Sporulation is used to send the cells into a dormant, dehydrated state to protect from harsh environmental conditions (2,3). As conditions become unsuitable for growth, gene clusters are expressed activating the sporulation process. The process is mediated by signal transduction pathways and specifically through a phosphorelay system (1,2). It is shown sporulation is specifically controlled and regulated though an expanded two component phosphorelay system (3). This process consumes large amounts of energy to place the cells into the dormant state and is a highly regulated process. Mechanism of SporulationMechanism of Sporulation The sporulation process begins by B. subtilis respoinding to harsh environmental conditions or increased cellular density (1,2). Once the specific conditions 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 histidine kinase A and B which then phosphorylate protein spo0F (2). The signal transduction pathway begins by the cellular ATP-dependant autophosphorylation ofhistadine kinase A and B in response to specific environmental conditions (2); this mechanism is still unknown. Spo0F is then phosphorylated by KinA at spo0F’s active site. This phosphorylation creates a confrontational change to spo0F (1,2,6) which then activates 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 reversible between the two kinases (2). Once phosphorylated, spo0A is in its active transcriptional regulator form and initiates the transcriptions of spoulation proteins and spore formation may begin. Figure 2: The pathways for vegetative or spore development in bacterial cells. The figure presents the phosphorelay involving Histidine Kinase (HK) and the phosphorylation relay between Spo0f/b/a, and the method for cell development to stop sporulation . The phosphorylation and hydrolysis of Spo0F is dependent 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 stability of the transition state. The decreased stability 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). Regulation of mechanismRegulation of mechanism 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 influence spo0A or transctription it still inhibits sporulation by reducing the concentration 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 messengers in the pathway. The low affinity of Mg binding to the active site of spo0F assists in enzyme regulation as well (1). The reduced affinity 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 concentration of magnesium; without high enough concentrations the reaction cannot occur and spo0F is not able to attract Mg under low concentrations. StructureStructure Spo0F is a 14KDa (2), 36 by 34 by 31 angstrom single domain globular protein (6) containing 124 residue (4). The protein consists of 45% helical secondary structures, , and 21% beta sheets structure, (4). There are 5 helices 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 tertiary structure is a “double wound (beta/alpha) motif formed [by the] five parallel beta strands flanked by alpha helicies” (2). The helices and strands are parallel in comparison to themselves however in contrast to each other, 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 helices being amphiphathic (6, 1). The majority of the protein surface is covered with hydrophilic residues, however there are two hydrophobic 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 staggering 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 accommidates 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 stabilizes 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 positions are bound to water molecules for increased stability(6). However, the phosphorylation reaction is dependent upon Thr82 and its hydroxyl group, this residue may sometimes may be functionally mutated to a Ser residue. The Thr82 is essential 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 inaccessible ; when phospohorylated a conformational change occurs opening the active site (6). This phosphorylation 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-terminus of alpha helix 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 flexibility 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 interaction is specific, yet the binding site has a degenerate nature. Methods of AnalysisMethods of Analysis The overall process of analysis of spo0F includes proliferation, isolation, purification, and analysis. One specific method of analysis will be described. The proliferation process can accomplished by transforming competent E. coli cells through heat shock treatment. E. coli is able to relatively easily uptake plasmids 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 crystallized for X-ray diffraction analysis. ImplicationsImplications Spo0F may be as a site of drug design. The adaptive benefit of sporulation in bacteria is an important cellular response to harsh environments, and makes the organism much more difficult to kill. Drug design can be used to inhibit the sporulation process, thus making spo0F a potential target for drug design. If sporulation can be prevented then infection can be inhibited. So if spo0F, or enzymes like it, can be inhibited from its kinase properties then the entire sporulation process may be halted. Another implication of spo0F is the ease at which it is isolated and analyzed. The protein is a single stranded oligomer that is easily expressed. The simply process of analysis provides experience with lab techniques and structural analysis. For beginner lab technicians it is a good starting point to learn how equipment and procedures are accomplished. Works Cited:Works Cited: Feher, Victoria A., James W. Zapf, James A. Hoch, John M. Whiteley, Lawrence P. McIntosh, Mark Rance, Nicholas L. Skelton, Frederick W. Dahlquist, and John Cavanagh. "High Resolution NMR Structure and Backbone Dynamics of the Bacillus Subtilis Response Regulator, Spo0f: Implications for Phosphorylation and Molecular Recognition." Biochemistry 36(1997): 10015-10025. 2 Tzeng, Yih-ling, and James A. Hoch. "Molecular Recognition in Signal Transduction: the Interaction Surfaces of the Spo0F Response Regulator with Its Cognate Phosphorelay Proteins Revealed by Alanine Scanning Mutagenesis. “Journal of Molecular Biology 272(1997): 200-212. 3. Varughese, Kottayil I., Haiyan Zhao, Vidya Velfore, and James Zapf. "Sporulation Phosphorelay Proteins and Their Complexes: Crystallographic Characterization." Methods in Enzymology 422(2007): 102-123. 4 Madhusudan, Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.H., and Varughese, K.I.. "CRYSTAL STRUCTURE OF SPOOF FROM BACILLUS SUBTILIS." PDB - Protein Data Bank. NSF, 1997. Web. 23 Oct. 2012. http://www.rcsb.org/pdb/explore/explore.do?structureId=1NAT Madhusudan, M., Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.H., Varughese, K.I. “A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis.” Biochemistry 36(1997): 12739-12745 Madhusudan, James Z., John M. Whiteley, James A. Hoch, Nguyen H. Xuong, and Kottayil I. Varughese. "Crystal Structure of a Phosphatase-resistant Mutant of Sporulation Response Regulator Spo0F from Bacillus Subtilis." Current Biology 4(1996): 679-690. Web. 24 Nov. 2012. <http://www.sciencedirect.com.prox.lib.ncsu.edu/science/article/pii/S0969212696000743>. Perego, M., Hanstein, C., Welsh, K. M., Diavakhishvili, T., Glaser, P. & Hoch, J. A. (1994). Multiple proteinaspartate phosphatases provide a mechanism for the integration of diverse signals in the control of development in B. subtilis. Cell, 79, 1047-1055.