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SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES

SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES Anna Grzechnik 1 , Dennis Carlton 1 , Heath Klock 2 Mark W. Knuth 2 and Scott A. Lesley 1,2* 1 The Joint Center for Structural Genomics (JCSG), The Scripps Research Institute 2 JCSG, The Genomics Institute of the Novartis Research Institute

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SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES

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  1. SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES Anna Grzechnik1, Dennis Carlton1, Heath Klock2 Mark W. Knuth2 and Scott A. Lesley1,2* 1 The Joint Center for Structural Genomics (JCSG), The Scripps Research Institute 2 JCSG, The Genomics Institute of the Novartis Research Institute *slesley@gnf.org NIH Bottlenecks Meeting 4/15/08

  2. Protein families come in all shapes and sizes • Common traits which allow us to recognize them but individual characteristics can vary greatly

  3. Protein families are collections of related sequences. • Draft 1 and 2 Pfam families are large with many potential targets.

  4. We want these targets. Not these targets. With 100 or more potential family members, we have the pick of the litter

  5. http://ffas.burnham.org/XtalPred-cgi/xtal.pl Target Selection Within Pfam Assignments Practical Filters: genomic DNA, #met, #cys 87 targets pursued from PF07726 8 targets with crystals to beamline PG1132I - 2.00 A resolution

  6. Just doing more targets is not enough. Select 82 families from 400 Draft 1 and 2 targets which failed using multiple targets. Find the best possible targets from all genomes and process through a full panel of salvage strategies.

  7. Low Temperature Expression 188 Targets from failed 82 families Microscreen expression and purification with total yield solubility cutoff 37C 25C

  8. Low Temperature Expression Micro-ANSEC analysis. Highly parallel, quick (12 min) run times, minimal resolution for aggregation testing

  9. Low Temperature Expression

  10. Making Truncations • PIPE cloning facilitates making truncations and point mutations • What truncations to make? • nested N- and C-terminal • bioinformatic predictions • experimentally determined Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 7: 982-94.

  11. Partial Proteolysis PT03787B-mth-267-18-182 1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG 150 151 ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267 Uncleaved Cleaved Fragment was reconstructed, re-expressed and is currently in crystal trials. 31790.801 PT03787B-mth-267-46-127 18035.201 1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG150 151ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267 Overlapping fragment not pursued further. 9017.601

  12. Deuterium Exchange Mass Spectrometry (DXMS) DXMS identifies regions of disorder and flexibility by mapping the location of rapid hydrogen/deuterium exchange to peptides derived from targets of interest. The information can be used to design expression constructs with improved crystallization properties (Spraggon et al., 2004). PFAM 6249: Ethanolamine utilization protein eutQ from Salmonella typhimurium LT2 FL-protein: poor crystallization Truncation: 1.9Å structure

  13. I II III IV V

  14. Surface Mutagenesis

  15. Nucleotide-Like Ligand Library: ADP GDP ADP Ribose GTP AMP NAD ATP NADP CDP NADPH CMP Tryptophan CTP cAMP Ligand Screening Target: PG1132F • Methods of ligand screening used: • Thermofluor: fluorescent detection of melting temperature • DSC: detection of differences in protein heat capacity • Stargazer: light scattering detection of aggregation 185 Targets Target PG1132F binds Blue resin suggesting nucleotide binding. Target is screened against nucleotide ligand library by thermofluor. Binding to ADP and GDP is observed. 111 Targets bind Affi-Gel Blue Method courtesy of Chang Yub-Kim 74 Non binding targets Targets that do not bind to the resin are screened against a 300 ligand library

  16. Reductive Methylation Target PS03963 RM No mountable crystals were available from native protein. After reductive methylation, two conditions produced harvestable crystals which are currently in finescreening. TargetPS04248 RM Structure of PS04248 was solved with Reductive Methylation

  17. UCSD & Burnham Bioinformatics Core John Wooley Adam Godzik Lukasz Jaroszewski Sri Krishna Subramanian Andrew Morse Tamara Astakhova Lian Duan Piotr Kozbial Dana Weekes Natasha Sefcovic Prasad Burra Josie Alaoen Cindy Cook GNF & TSRI Crystallomics Core Scott Lesley Mark Knuth Heath Klock Dennis Carlton Thomas Clayton Christina Trout Marc Deller Daniel McMullan Polat Abdubek Julie Feuerhelm Joanna Hale Jessica Paulsen Thamara Janaratne Hope Johnson Edward Nigoghossian Linda Okach Sebastian Sudek Glen Spraggon Sanjay Agarwalla Anna Grzechnik Regina Gorski Connie Chen Dustin Ernst Stanford /SSRL Structure Determination Core Keith Hodgson Ashley Deacon Mitchell Miller Herbert Axelrod Hsiu-Ju (Jessica) Chiu Kevin Jin Christopher Rife Qingping Xu Silvya Oommachen Henry van den Bedem Scott Talafuse Ronald Reyes Abhinav Kumar Christine Trame The JCSG is supported by the NIH Protein Structure Initiative (PSI) Grant U54 GM074898 from NIGMS (www.nigms.nih.gov). TSRI NMR Core Kurt Wüthrich Reto Horst Maggie Johnson Amaranth Chatterjee Michael Geralt Wojtek Augustyniak Pedro Serrano Bill Pedrini William Placzek TSRI Administrative Core Ian Wilson Marc Elsliger Gye Won Han David Marciano Henry Tien Lisa van Veen

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