Engineering of Proteins for Crystallography

Summary

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

Top Publications

  1. pmc Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcription
    Mary 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
  2. pmc Fast docking on graphics processing units via Ray-Casting
    Karen R Khar
    Center for Bioinformatics, University of Kansas, Lawrence, Kansas, United States of America
    PLoS ONE 8:e70661. 2013
  3. ncbi Salvage or recovery of failed targets by mutagenesis to reduce surface entropy
    Lukasz 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

Detail Information

Publications3

  1. pmc Core circadian protein CLOCK is a positive regulator of NF-κB-mediated transcription
    Mary 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...
  2. pmc Fast docking on graphics processing units via Ray-Casting
    Karen 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. ..
  3. ncbi Salvage or recovery of failed targets by mutagenesis to reduce surface entropy
    Lukasz 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. ..

Research Grants30

  1. Interdisciplinary center of excellence for the study of pain and sensory function
    Ian D Meng; Fiscal Year: 2013
    ..Completion of these Aims will develop the research careers of a multidisciplinary group of junior investigators, and establish the core facilities and equipment necessary to constitute a competitive research center. ..
  2. Structural Genomics on Membrane Proteins
    Wayne 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. ..
  3. The Virtual Physiological Rat Project
    Daniel 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. ..
  4. 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. ..
  5. TransportPDB: Center for the X-ray Structure Determination of Human Transporters
    Michael H B Stowell; Fiscal Year: 2013
    ..abstract_text> ..
  6. Multi-Level Optimization of Membrane Proteins for Crystallography
    Michael Wiener; Fiscal Year: 2013
    ..abstract_text> ..
  7. Center for Structure of Membrane Proteins
    Robert 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. ..
  8. Fundamental Studies of RNA Folding
    Daniel Herschlag; Fiscal Year: 2013
    ..abstract_text> ..
  9. New Ion Mobility and Crosslinking Technologies for Analysis of Protein Complexes
    Brandon T Ruotolo; Fiscal Year: 2013
    ..Importantly, it will also provide a basis for developing methods for following interaction dynamics in protein complexes. ..
  10. Spatial and Temporal Regulation of Angiogenesis
    HAROLD FISHER DVORAK; Fiscal Year: 2013
    ..abstract_text> ..
  11. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013
    ..The Equipment Core (Core A) supports the specialized comprehensive suite of instrumentation for the Program. The Administrative Core (Core B) administers the Program Project. ..
  12. Primary Immuno-Deficiencies Affecting Specific Stages of the Immune Response
    CORNELIS P TERHORST; Fiscal Year: 2013
    ..abstract_text> ..
  13. Midwest Center for Structural Genomics
    ANDRZEJ JOACHIMIAK JOACHIMIAK; Fiscal Year: 2013
    ..The platform provides for rapid model validation and deposition in PDB. In PSI:Biology, these steps will be further advanced and integrated using LIMs and databases into a system capable of determining 200+ structures per year. ..
  14. GPCR Network
    Raymond 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. ..
  15. Structure, Dynamics and Activation Mechanisms of Chemokine Receptors
    Tracy M Handel; Fiscal Year: 2013
    ..All of these studies will be augmented with computational modeling methods in order to rationally guide the experimental construct design and to interpret the biophysical data in a 3D context. ..
  16. Structural Genomics of Eukaryotic Domain Families
    Gaetano T Montelione; Fiscal Year: 2013
    ..abstract_text> ..
  17. Membrane protein structures by solution NMR
    James J Chou; Fiscal Year: 2013
    ..abstract_text> ..
  18. The Transmembrane Protein Center
    Brian G Fox; Fiscal Year: 2013
    ..Expanded options for crystallization screening, access to synchrotrons, and improvements in software used to solve structures will also contribute to the increased throughput necessary to achieve PSI:Biology goals. ..
  19. The NYSGRC: A Large Scale Center for PSI:Biology
    Steven C Almo; Fiscal Year: 2013
    ..abstract_text> ..
  20. Toward a Universal Influenza Virus Vaccine
    Peter Palese; Fiscal Year: 2013
    ..These epitopes will then be used to guide the design of vaccine constructs which induce cross-protective immune responses against many different influenza virus variants. ..