Target-​6-phosphogluconate+dehydrogenase+(T.+brucei)

***Target (protein/gene name):** 6-phosphogluconate dehydrogenase
Virtual ToDo: get control ligands

IN GOLD: Dock control ligands against the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site) - Vicky Screen the libraries using this

Dock NADPH back into the NADPH site (use the NADPH from 2IYP aligned in PyMol to define the binding site) - Kavya -- use a LigPrepped version of NADPH from the PubChem site to be what you dock (should end up being 3 versions of NADPH after LigPrep) Determine which NADPH conformation looks best. Use the NAPDH + Tb6PGD as your receptor for a docking job of control ligands into the 5RP site (use 5RP from2IYP aligned in PyMol to define the binding site) Screen the libraries using this

// TDR Targets: // @http://tdrtargets.org/targets/view?gene_id=12199
 * NCBI Gene # or RefSeq#:** 3660922
 * Protein ID (NP or XP #) or Wolbachia#:** XP_827463
 * Organism (including strain):** // Trypanosoma brucei //

// Trypanosoma brucei // is a protozoan that is known to cause African Sleeping sickness in humans. It originally is a endoparasite within the midgut of the tsetse fly, until it migrates to the salivary gland of the fly to later be injected into a mammalian host upon biting. The protist is then an endoparasite in the mammalian bloodstream. African Sleeping sickness is endemic to the subsaharan regions of Africa. The infection is curable with proper medication, but left untreated, it is commonly fatal. The course of the disease have two phases, differentiated by wether the parasite has crossed the blood-brain barrier and has thus infected the central nervous system. In the first phase, the parasite multiples in the tissues, blood, and lymph, and the infected individual suffers from fever, headaches, joint pain, and itching. In the second phase, the neurological phase, changes in behavior, sensory disturbances, poor coordination, confusion, and disruptions of the sleep cycle are common. Treatment of the disease is dependent upon how early the infection is detected and diagnosed. Continued research on the protozoan and the disease is necessary to improve the treatment options of those infected, improve the detection methods to limit the spread, and to develop a vaccination for those living in or traveling to endemic areas.
 * Etiologic Risk Group (see link below):** Appendix B-II-C Risk Group 2 (RG2)- Parasitic Agents
 * Background/Disease Information (sort of like the Intro to your Mini Research Write up):**


 * Essentiality of this protein:** Essential. []
 * Complex of proteins?:** No, but can be a dimer
 * Druggable Target:** Yes []

[] []
 * *EC#: ** 1.1.1.44
 * Link to BRENDA EC# page:** []
 * Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):** reagents
 * -- link to Sigma (or other company) page for assay or assay reagents (substrates)**
 * -- link (or citation) to paper that contains assay information**
 * -- List cost and quantity of substrate reagents and supplier**
 * phosphogluconic acid SIGMA P7877 - $75 for 100mg**

β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate (N-0505) $79.70 for 100mg
Glycylglycine Buffer= $77 per 25g 6-Phosphogluconate= $108 per 25un ß-Nicotinamide Adenine Dinucleotide Phosphate Solution= $473 per kit (30 assays) Magnesium Chloride= $43.60 per 100g

b-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE Sodium Salt Product Number N0505 []®ion=US Preparation Instructionsβ-NADP+ is soluble in water at 50mg/ml.It is also soluble in methanol, much less soluble in ethanol andpractically insoluble in ether and ethyl acetate.Aqueous solutions stored as frozen aliquots are stable for at least one year.Repeated freeze thaw cycles are not recommended.Storage/StabilityStore at–20ºC Enzo Life Sciecnefor NADP+ [] soluble in water to 50mg/ml sesnsitive to alkaline - so store in water that is a little __acidic__.
 * SUBSTRATE INFO**
 * NADP+**

P7877 []®ion=US ?? - no info on storage
 * 6-phosphogluconate**

@http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/2/n6505pis.Par.0001.File.tmp/n6505pis.pdf b -NADPH is soluble in 0.01 M sodium hydroxide (50 mg/ml), yielding a clear, light yellow solution. It is recommended to store Products N1630, N7505, and N6505 desiccated at –20 ° C protected from light. Product N9910 can be stored at room temperature. The normal impurities and/or decomposition products are b -NADP and monophosphoadenosine 5 ¢ - diphosphoribose. It is recommended to prepare solutions fresh and use promptly, unless you are sure this is an unnecessary precaution for your work. However, __it has been__ __reported that a 0.5 mM solution in 0.02 M NaOH __ __(pH 12.3) showed no loss of purity in a week at 4 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C or __ __ - <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">85 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C, but a 13% loss at –20 ° <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">C. <span style="font-family: Arial,sans-serif; font-size: 9.33333px;">3 __
 * Substrate Info (Dr. B) - 100113**
 * May also dilute in Tris - talk to Kaarthik.**
 * NADPH** - sigma N6505 (NOT THE RIGHT SUBSTRATE FOR THIS ENZYME UNLESS DOING THE BACK REACTION)
 * <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">Preparation Instructions **
 * <span style="font-family: Arial,sans-serif; font-size: 13.3333px;">Storage/Stability **

For NADH and NADPH - the concern is that these are reduced and don't want them to be oxidized. So, want to reduce their exposure to oxygen - hence the NaOH.

-- PDB #: 1PGJ
 * Structure Available (PDB or Homology model)**


 * Current Inhibitors:** 4-phospho-D-erythronate and 2-deoxy-6-phosphono-D-gluconate are two of the current inhibitors with in vitro anti-parasitic activity.

[]
 * Expression Information (has it been expressed in bacterial cells):**Can be expressed in E.coli bacteria.
 * Purification Method:** 2-step purification mothod: DE-52 cellulose batch preparation followed by 2′ AMP-agarose affinity chromatography


 * Image of protein (PyMol with features delineated and shown separately):**

Figure 2: X-ray structure of 6-phosphogluconate dehydrogenase from the protozoan parasite //T. brucei//. SMDVGVVGLGVMGANLALNIAEKGFKVAVFNRTYSKSEEFMKANASAPFAGNLKAFETMEAFAASLKKPRKALILVQAGA ATDSTIEQLKKVFEKGDILVDTGNAHFKDQGRRAQQLEAAGLRFLGMGISGGEEGARKGPAFFPGGTLSVWEEIRPIVEA AAAKADDGRPCVTMNGSGGAGSCVKMYHNSGEYAILQIWGEVFDILRAMGLNNDEVAAVLEDWKSKNFLKSYMLDISIAA ARAKDKDGSYLTEHVMDRIGSKGTGLWSAQEALEIGVPAPSLNMAVVSRQFTMYKTERQANASNAPGITQSPGYTLKNKS PSGPEIKQLYDSVCIAIISCYAQMFQCLREMDKVHNFGLNLPATIATFRAGCILQGYLLKPMTEAFEKNPNISNLMCAFQ TEIRAGLQNYRDMVALITSKLEVSIPVLSASLNYVTAMFTPTLKYGQLVSLQRDVFGRHGYERVDKDGRESFQWPELQ Figure 3: TMpred graph image of 6-phosphogluconate dehydrogenase shows a few points above zero, but only 1 peak. This predicts 1 possible trans-membrane protein.
 * Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):**
 * length of your protein in Amino Acids:** 478
 * Molecular Weight of your protein in kiloDaltons using the [|Expasy ProtParam]website:** 52.03 kDa
 * Molar Extinction coefficient of your protein at 280 nm wavelength:** 48360
 * TMpred graph Image ** (@http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

However, TDR targets predicted no trans-membrane proteins.

<span class="ff_line">ATGTCAATGGATGTCGGTGTTGTCGGCCTCGGGGTGATGGGCGCGAACCTGGCCTTGAACATTGCGGAGA AAGGGTTTAAAGTTGCTGTGTTCAACCGTACGTACTCTAAGAGCGAGGAATTCATGAAAGCGAATGCCTC TGCTCCATTTGCGGGTAATTTGAAGGCGTTTGAAACTATGGAGGCATTTGCAGCGTCACTCAAGAAACCT CGAAAGGCCCTCATCCTGGTGCAGGCGGGCGCGGCTACGGACTCAACAATTGAACAACTTAAGAAAGTGT TTGAGAAGGGAGATATCCTCGTTGATACCGGTAACGCGCATTTTAAGGATCAGGGACGCCGCGCCCAGCA GCTGGAGGCGGCAGGTCTCCGGTTTCTTGGGATGGGCATATCCGGGGGCGAGGAGGGTGCACGCAAAGGG CCAGCCTTTTTCCCTGGAGGGACGCTTAGTGTGTGGGAGGAAATACGACCAATTGTTGAGGCCGCCGCAG CTAAGGCAGATGATGGCCGGCCCTGTGTGACGATGAACGGCAGCGGCGGCGCGGGATCATGCGTGAAGAT GTACCACAATTCGGGTGAATACGCCATTTTGCAAATTTGGGGTGAGGTTTTTGACATCCTTCGGGCAATG GGACTGAACAACGATGAAGTTGCTGCCGTTCTTGAAGATTGGAAATCAAAGAACTTCTTGAAGTCTTATA TGCTCGATATCTCAATTGCAGCCGCGCGGGCAAAGGATAAGGATGGAAGTTATCTTACGGAGCACGTGAT GGATCGTATTGGATCGAAGGGCACCGGCTTATGGTCCGCCCAAGAGGCTCTCGAGATTGGAGTCCCTGCG CCCAGTTTGAACATGGCTGTCGTATCGCGGCAGTTCACAATGTATAAAACTGAGCGTCAAGCGAATGCCA GCAATGCACCCGGTATTACTCAATCCCCTGGATACACTCTCAAAAACAAAAGCCCCAGTGGGCCCGAAAT TAAGCAGCTCTACGACTCTGTGTGCATTGCCATTATCTCATGCTACGCTCAAATGTTTCAGTGCCTGCGT GAGATGGACAAGGTGCATAACTTCGGACTCAATCTTCCAGCTACCATTGCAACTTTCCGCGCCGGTTGCA TTTTGCAGGGCTACCTTTTAAAACCCATGACTGAGGCATTCGAAAAGAATCCCAACATTAGCAATCTCAT GTGTGCATTCCAAACCGAGATCAGGGCAGGACTACAGAATTACCGCGATATGGTGGCACTTATCACATCA AAGTTGGAAGTGTCCATTCCTGTGCTGTCGGCCTCCCTCAATTACGTTACTGCGATGTTTACGCCAACAC TCAAGTATGGGCAACTTGTGTCGTTGCAGCGGGATGTGTTCGGTCGGCACGGCTACGAAAGGGTGGATAA AGACGGCCGCGAATCATTCCAATGGCCTGAGTTGCAATAA
 * CDS Gene Sequence (paste as text only):**

1 ATGTCTATGGACGTTGGTGTAGTTGGCCTGGGTGTTATGGGTGCGAACCTGGCGCTGAAT 61 ATTGCGGAAAAAGGTTTCAAAGTTGCGGTTTTCAACCGTACCTACTCTAAATCTGAAGAA 121 TTTATGAAAGCGAACGCTTCTGCACCGTTTGCGGGCAACCTGAAGGCGTTTGAGACCATG 181 GAAGCATTTGCGGCGTCTCTCAAGAAACCGCGTAAAGCGCTCATTCTGGTACAAGCGGGT 241 GCGGCCACCGACTCTACCATCGAACAGCTCAAAAAAGTATTTGAAAAGGGTGACATCCTG 301 GTCGACACCGGTAACGCGCATTTCAAGGACCAGGGTCGTCGTGCGCAGCAGCTGGAGGCT 361 GCGGGTCTCCGTTTCCTCGGCATGGGTATTTCTGGTGGTGAAGAAGGTGCGCGTAAGGGT 421 CCGGCGTTCTTCCCGGGTGGTACCCTGTCTGTATGGGAAGAGATTCGTCCGATCGTTGAA 481 GCTGCCGCTGCGAAAGCGGACGATGGTCGCCCGTGCGTAACGATGAATGGTTCCGGTGGT 541 GCTGGTTCTTGCGTTAAAATGTACCACAACAGCGGTGAATACGCGATCCTGCAAATCTGG 601 GGTGAAGTATTCGATATCCTGCGCGCGATGGGCCTGAATAATGACGAAGTAGCCGCGGTT 661 CTGGAAGACTGGAAAAGCAAAAACTTTCTCAAATCCTATATGCTGGACATCTCCATCGCT 721 GCGGCACGCGCTAAAGACAAGGACGGCTCCTACCTGACCGAGCACGTGATGGACCGTATC 781 GGTAGCAAAGGTACCGGTCTGTGGTCTGCCCAGGAAGCGCTGGAAATCGGTGTACCTGCG 841 CCATCCCTGAACATGGCGGTAGTTTCCCGCCAATTCACCATGTACAAGACCGAACGTCAG 901 GCGAATGCGTCTAACGCTCCGGGTATCACCCAGTCTCCTGGTTACACCCTGAAAAACAAA 961 TCTCCGTCTGGTCCGGAAATCAAACAACTGTACGACTCTGTTTGCATTGCGATCATCTCT 1021 TGCTACGCCCAAATGTTCCAATGCCTGCGTGAAATGGACAAAGTTCACAATTTCGGCCTC 1081 AACCTCCCAGCGACTATCGCGACCTTTCGTGCGGGCTGTATCCTCCAGGGTTACCTGCTG 1141 AAACCGATGACGGAGGCATTCGAGAAAAATCCGAACATCTCTAACCTCATGTGCGCATTC 1201 CAAACTGAGATCCGTGCCGGCCTCCAAAACTATCGTGATATGGTTGCTCTGATTACCTCT 1261 AAACTGGAAGTTTCTATCCCAGTTCTGAGCGCGAGCCTGAACTACGTAACCGCTATGTTC 1321 ACTCCGACGCTGAAATATGGCCAGCTCGTTTCTCTGCAGCGTGACGTTTTCGGTCGTCAT 1381 GGTTACGAACGTGTTGACAAAGATGGCCGCGAATCTTTTCAGTGGCCGGAGCTGCAATAA 1441
 * GC% Content for gene:** 51.39%
 * CDS Gene Sequence (codon optimized) - copy from output of Primer Design**
 * Protocol (paste as text only):**


 * GC% Content for gene (codon optimized): 49%**

Do Not Need this info for Spring (but still copy these lines to your Target page for now) https://drive.google.com/?tab=mo&authuser=0#folders/0B4O2KqKh2q_-UnZrYzVIWlhRZVU -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
 * Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):**
 * ( link to DNA Works output text file - **that should be saved in your Google Docs folder after you did the primer design protocol)

<span style="font-family: 'Times New Roman','serif'; font-size: 13.3333px;">Forward Primer: <span style="font-family: 'Times New Roman','serif'; font-size: 13.3333px;">5’ TAC TTC CAA TCC ATG TCT ATG GAC GTT GGT GT 3’
 * Primer design results for 'tail' primers (this is just 2 sequences):**

<span style="font-family: 'Times New Roman','serif'; font-size: 13.3333px;">Reverse Primer (complement): <span style="font-family: 'Times New Roman','serif'; font-size: 13.3333px;">5’ TAT CCA CCT TTA CTG TTA TTG CAG CTC CGG CCA CT 3’

Resources:
Expression and Purification:[] Druggability: [] Essentiality: [] Brenda EC# [] XP #: [] http://en.wikipedia.org/wiki/Trypanosoma_brucei http://en.wikipedia.org/wiki/African_trypanosomiasis