* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download 122486 - IDEALS @ Illinois
Structural alignment wikipedia , lookup
Circular dichroism wikipedia , lookup
Protein design wikipedia , lookup
Rosetta@home wikipedia , lookup
List of types of proteins wikipedia , lookup
Homology modeling wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Protein folding wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein structure prediction wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Protein purification wikipedia , lookup
Protein moonlighting wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Western blot wikipedia , lookup
Protein domain wikipedia , lookup
IspH – RPS1 “Rosetta Stone” Protein Presented By Armaan Haleem, Oldfield Group Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Urbana-Champaign Introduction . Cytoscape Visualization and Comparison of IspH Protein Sizes IspH-RPS1 is a fusion protein that contains the IspH domain which functions as the last essential reducing enzyme in the methylerithritol phosphate pathway. This pathway is part of the isoprenoid biosynthesis pathway and is an alternative to the mevalonate pathway found in humans. The IspH-RPS1 protein is found in organsims that cause severe diseases such as Clostridium tetani and Clostridium botulinism MALDI-TOF Spectrum, UV-Vis Spectrum, Michaelis Mentin Kinetics, Activity Comparison, EPR Spectrum Proposed Function of IspH-RPS1 Possible IspH-RPS1 Bacterial Ribosome Interaction Comparison of the Mevalonate and Methylerithritol Pathways Fig. 1 Sequence similarity network of the IspH family proteins with 15660 members constructed at an expectation-value (e-value) of 10−120. A single node represents sequences with at least 90% similarity. Major clusters of interest include: (a) gammaproteobacteria (dark cyan, upper left) and betaproteobacteria (blue, lower right); (b) alphaproteobacteria (cyan); (c) firmicutes (purple); (d) firmicutes (with very long sequences, red); (e) bacteroidetes (yellow); (f) actinobacteria (orange); (g) cyanobacteria and plants (green); (h) apicomplexa (pink, genus Plasmodium, the malaria parasites) and (i) desulfobacterales (brown, an order of deltaproteobacteria) (j) the architectures of IspH–RPS1 with 4 (top) and 6 (bottom) S1 repeats. Immethun, C. M., Hoynes-O’Connor, A. G., Balassy, A., & Moon, T. S. (2013). Microbial Production of Isoprenoids Enabled by Synthetic Biology. Front. Microbiol. Frontiers in Microbiology, 4. doi:10.3389/fmicb.2013.00075 The methylerithritol pathway and mevalonate pathway are parallel pathways in isoprenoid biosynhesis. The methylerithritol pathway does not exist in humans and thus serves as a potential drug target. Analysis Proposed Structure of IspH-RPS1 The IspH-RPS1 fusion protein is decidedly a “Rosetta” stone protein because of the unaffected function of the IspH domain. This is further shown through the retained Kcat of the IspH domain on the fusion protein from Clostridium thermocellum [56min-1] which is very similar to the Kcat of IspH expressed from aquifex aeolicus. Additionally, no other genes were found on the genomes to code for these proteins indicating that the fusion is likely due to complementary functions of the domain. The IspH Catalzyed Reaction of HMBPP DMAPP and IPP Scheme 1 IspH catalysis. The reaction involves the 2H+/2e− reductive dehydroxylation of (E)-1-hydroxy-2methylbut-2-enyl-4-diphosphate (3) to dimethylallyl diphosphate (1) and isopentenyl diphosphate (2). Fig.3 Characterization of CthIspH–RPS1 and its truncation mutants. (a) MALDI-TOF spectrum of purified CthIspH. M1: CthIspH–RPS1; M2: EcRho, see text for more details. (b) UV-Vis spectrum of CthIspH–RPS1 after iron–sulfur cluster reconstitution (black) and after dithionite reduction (red). (c) Michaelis–Menten kinetics of CthIspH–RPS1. (d) Cartoon of the truncation proteins. D0: no S1 repeat, D1: one S1 repeat, D2: two S1 repeats, D3: three S1 repeats. WT has the four S1 repeats. (e) Relative activities of truncation mutants compared to wildtype protein. Error bars are from n = 3 replicates. (f) EPR of reduced CthIspH–RPS1, its truncation mutants, and E. coli IspH. (g) EPR of CthIspH–RPS1_1-171, reduced protein, with substrate HMBPP (1) and several ligands. The small peak at g = 2.0 in the oxidized protein sample (blue) is due to the small amount of [Fe3S4]+. Fig. 2 IspH structures and IspH–fusion protein structure predictions. (a) X-ray structure of E. coli IspH (PDB ID code: 3KE8) (b) X-ray structure of P. falciparum IspH (PDB code: 4N7B) (c) Phyre2 structure prediction of CthIspH–RPS1. The IspH domains are in orange while the 4 S1 repeats are colored green, purple, blue, or cyan. The identification of the IspH domain function as unaltered in the IspH-RPS1 fusion is essential to the classification of this protein as a “Rosetta Stone” protein. Fig. 4 Possible IspH–RPS1/RelA/Rho ribosome interactions. Upon binding to the ribosome through the RPS1 domain, IspH could interact with RelA and affect its activity. The ppGpp released modulates a series of cell activities, for example inhibiting RNA polymerase (RNAP). The transcription termination factor Rho could also be affected upon interacting with IspH–RPS1 (as suggested from the MALDI-TOF experiments). Conclusions The IspH-RPS1 was first given attention due to the identification of an IspH domain in a protein 300-550 amino acid residues longer than the IspH domain. When analyzed using various methods, the domain was identified to have the same function in the fusion protein as an independent IspH. This leads to the conclusion that IspH-RPS1 is a “Rosetta Stone” protein because it is likely that these two proteins have complementary functions to each other. Acknowledgments Guodong Rao Bing O’ Dowd Dr. Eric Oldfield Jikun Li Ke Wang IspH Poster Presentation Introduction IspH is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (essential reducing enzyme) that contains a 4Fe-4S cluster. In this cluster, the fourth iron will carry out the reducing activity with the substrate. This enzyme is the last reducing enzyme in the methylerithritol phosphate pathway, which is an alternative to the mevalonate isoprenoid biosynthesis pathway that exists in humans. The pathway that exists in humans is called the mevalonate pathway. The substrate for IspH is HMBPP which will produce both DMAPP (dimethylallyl pyrophosphate) + IPP (Isopentenyl diphosphate). The subject of this presentation is the analysis of the fusion of IspH to another protein known as Ribosomal Protein S1. Many anaerobic bacteria found in the human stomach utilize IspH-RPS1 and some of these bacteria are pathogenic. IspH domains were found on proteins that were twice the size of usual IspH proteins. IspH has been studied before and found to typically be around 300 residues and the IspH’s of concern were found in proteins with a size of 600-850 residues. Analysis was done using the Enzyme Similarity Tool to find sequence similarity. Additionally, the location of the clusters was used to visualize the (similarities? – in reference to Enzyme Similarity Tool) through the software “Cytoscape”. Growing/Purifying/Re-folding IspH proteins CthispH-RPS1 and mutants were amplified from genomic DNA of Clostridium thermocellum. This was done because thermophilic bacteria were intended to produce enzymes that are more stable due to the nature of their environments. The DNA was then digested with SacIHF and SalIHF and cloned into the pET28a(+) vector. The vector was transformed into E. coli – RIPL (used for “rare” proteins, and is different due to large amounts of tRNA allow for the rare codons to be translated). Then the bacteria was grown to the necessary OD and induced with IPTG (which inhibits the lac operon to allow transcription). Afterwards, the pellets were collected through centrifugation and lysed by sonication. Then the lysate was run through a Ni 2+ column to bind with the histidine tag. These Ni-NTA binding sites are washed with imidazole in varying concentrations to wash the column and elute the protein. The iron sulfur clusters need to be reconstituted after this process. Azobacter vinelandii IscS protein is utilized. This enzymes hsCBA (domain?) acts as a chaperone to mature Fe-S clusters. IspH-RPS1 was concentrated down to 2 mL and is degassed by bubbling nitrogen and transferred to a glovebox. The buffer used to reconstitute is .5mM Fe(NH4)2(SO4)2, 2.5 mM L-cysteine, 5mM DTT and .1mM IscS (this is not dialysed out because it is not considered a significant portion of the total protein). Analysis of IspH-RPS1 As stated before, the first noticeable difference between the fusion IspH’s was that the protein contained many more residues but still contained an IspH domain. These additional residues were found to comprise 4-6 RPS1 domains. These were found in anaerobic bacteria. [majority of these IspH-RPS1 containing bacteria are firmicutes – Gram +, low G/C, and also members of Clostridium] 37% of IspH-RPS1s are present in human gut bacteria. Clostridium is the 7th most prevalent gut bacteria and Eubacterium is the 9th most prevalent. The function of RPS1 in the RPS1-IspH fusion is not proven but the high level of hybrids indicates a related functioning. Of the RPS1-IspH containing bacteria, it was found that the Gram + bacteria had four repeats of RPS1 and the Gram – bacteria had six repeats. The first two repeats bound to the ribosome and the last four were for mRNA binding. The fusion protein was simulated to have a trefoil or cloverleaf structure (E. coli and P. falciparum). Alpha and beta domains composed the trefoil/cloverleaf with the catalytic 4Fe-4S cluster located in the center. Linked to this protein were three Cys thiols that are essential for function. The other coordinating sites were bound by substrate/inhibitor/water molecules. The bacteria that contained the IspH-RPS1 fusion proteins did not show any other RPS1 or IspH genes. This would indicate that IspH does perform it’s previously assumed role as these bacteria do not utilize the mevalonate pathway (they lack the genes to code for necessary proteins). The Kcat [56min-1] of the fusion protein from Clostridium thermocellum proved to be very similar to the Kcat of IspH (not fused) that was expressed from aquifex aeolicus, and the Km [15 uM] was 2 fold larger. These measurements were taken using the methylviolagen assay, where the substrate HMBPP is used by the enzyme to create the products at a 1:5 ratio. (determine mechanism of violagen assay –to create the violagen radical 606 nm). The results were further confirmed by liquid chromatography-mass spectroscopy and NMR. When an EPR spectrum was taken of the IspH-RPS1 fusion protein, the longer sequence was again identified to contain IspH as they demonstrated a similar signal as “normal” IspHs. To further prove that RPS1 does not affect the function of the IspH domain, four truncations were performed of the RPS1 domains and none of these affected the IspH activity. This collective data suggests that the proteins are true Rosetta-Stone proteins in that their complement the other’s activity. Role of IspH-RPS1 These complementary roles are only hypothesized and not proven. It has been shown that IspH-RPS1 binds close to the RelA site on bacterial ribosomes. RelA is responsible for the biosynthesis of alarmone, which is a regulator for the bacterial stringent response. The stringent response is activated when stress is detected by the bacteria and will cause the bacteria to stop spreading and instead focus on the survival of it’s current cells. It is possible that IspH-RPS1 would detect stressors such as oxygen or iron and contribute to the response. This would be logical due to the location of binding on the bacterial ribosome. Conclusion IspH is an essential protein in the methylerythritol isoprenoid biosynthesis pathway. This experiment establishes IspH-RPS1 as a RosettaStone protein in which the functions of the two proteins are complementary. In analyzing the hybrid IspH-RPS1 further studies and experiments can be done to determine the exact function of this fusion protein. References 1. Oldfield, E., Dr., O'Dowd, B., Rao, G., Li, J., & Wang, K. (2015). IspH–RPS1 and IspH–UbiA: “Rosetta stone” proteins. Chemical Science, 6813-6822. doi:10.1039/c5sc02600h