Engineering of Proteins for Crystallography
Principal Investigator: Adam Godzik
Abstract: DESCRIPTION (provided by applicant): In spite of its enormous successes, structural biology is severely limited by the low success rates of macromolecular crystallization, and by the poor performance of many samples in NMR. Many valuable, biomedically important targets elude crystallization attempts, and overall high costs of structure determination are due primarily to the labor and time-intensive screening of targets at the stages of protein production, crystallization and initial HSQC spectra collection. Our research will address this bottleneck, through development of protein engineering strategies that allow for rational design of target variants with enhanced solubility, stability, and crystallizability or performance in NMR, and implementation of web-based, publicly available servers that facilitate application of these strategies. During the previous phase, we demonstrated that protein crystallization can be rationally induced by surface engineering based on the premise of surface entropy reduction (SER), i.e. mutagenesis of large, polar and solvent exposed amino acids, such as Lys, Glu and Gln, with small residues, e.g. Ala. Further, we designed and implemented the first generation XtalPred and SERp servers, which offer automated evaluation of protein's propensity to crystallize and design of variants with enhanced crystallizability based on the SER strategy. These tools have been used successfully by thousands of investigators world-wide, and helped solve nearly 170 crystal structures, including those of novel globular and membrane proteins, complexes and drug- targets in drug design pipelines. We now propose to pursue further experimental and computational studies of the relationships between physical chemistry of the protein surface and its solution properties. Specifically, we will investigate how surface entropy reduction affects protein solubility and stability, and how protein surface properties impact on the quality of heteronuclear NMR spectra. We will design and implement second generation XtalPred and SERp algorithms, with numerous new features, to achieve higher success rates for prediction of protein crystallizability and for design of variants with higher crystallizability, solubility, and with enhanced performance in NMR spectroscopy. The two second generation servers will be integrated into an interoperable network with the fold-prediction server FFAS03, and we will design a Protein Construct Design Metaserver (PCDM) making it possible to access the entire toolbox from a single GUI, proceeding from prediction to interactive design of primers and/or synthetic genes for expression of targets. Finally, to validate the methods, we will test them using a selection of biologically relevant protein targets.
Funding Period: 2011-09-30 - 2015-07-31
more information: NIH RePORT
- Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcriptionMary L Spengler
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA
Proc Natl Acad Sci U S A 109:E2457-65. 2012..These findings establish a molecular link between two essential determinants of the circadian and immune mechanisms, the transcription factors CLOCK and NF-κB, respectively...
- Fast docking on graphics processing units via Ray-CastingKaren R Khar
Center for Bioinformatics, University of Kansas, Lawrence, Kansas, United States of America
PLoS ONE 8:e70661. 2013..We anticipate that all three of these enhanced approaches, which now become tractable, will lead to improved screening results. ..
- Salvage or recovery of failed targets by mutagenesis to reduce surface entropyLukasz Goldschmidt
UCLA DOE Institute for Genomics and Proteomics, Howard Hughes Medical Institute, University of California, Los Angeles, CA, USA
Methods Mol Biol 1140:201-9. 2014..Three representative cases of the application of the SER strategy, assisted by the automated prediction of the mutation sites using the SER prediction (SERp) server are described. ..
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- Structural Genomics on Membrane ProteinsWayne A Hendrickson; Fiscal Year: 2013..The project will be managed to optimize output and to integrate effectively with the PSI-Biology network and with other membrane protein structure efforts. ..
- The Virtual Physiological Rat ProjectDaniel A Beard; Fiscal Year: 2013..This proposal targets the grand challenge of understanding complex multi-faceted disease phenotypes through experiments and simulations that capture the complex genotype-environment-phenotype relationship. ..
- Center for the Spatiotemporal Modeling of Cell Signaling (STMC)Bridget S Wilson; Fiscal Year: 2013..The Center will strongly support translation of new technical and computational tools to other signaling systems linked to human disease, especially other immune diseases and cancer. ..
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- Multi-Level Optimization of Membrane Proteins for CrystallographyMichael Wiener; Fiscal Year: 2013..abstract_text> ..
- Center for Structure of Membrane ProteinsRobert M Stroud; Fiscal Year: 2013..3.1, one of the world's most productive protein crystallography facilities. Overall, the combined expertise of principal investigators provides a unique environment to achieve the proposed aims. ..
- Fundamental Studies of RNA FoldingDaniel Herschlag; Fiscal Year: 2013..abstract_text> ..
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- Spatial and Temporal Regulation of AngiogenesisHAROLD FISHER DVORAK; Fiscal Year: 2013..abstract_text> ..
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- GPCR NetworkRaymond C Stevens; Fiscal Year: 2013..Based on the experience of the CMPD investigators, preference will be for human or eukaryotic membrane proteins to maximally leverage the CMPD capabilities. ..
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- Membrane protein structures by solution NMRJames J Chou; Fiscal Year: 2013..abstract_text> ..
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