Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS.

Crystallographic efforts often fail to produce suitably diffracting protein crystals. Unstructured regions of proteins play an important role in this problem and considerable advantage can be gained in removing them. We have developed a number of enhancements to amide hydrogen/high-throughput and high-resolution deuterium exchange MS (DXMS) technology that allow rapid identification of unstructured regions in proteins. To demonstrate the utility of this approach for improving crystallization success, DXMS analysis was attempted on 24 Thermotoga maritima proteins with varying crystallization and diffraction characteristics. Data acquisition and analysis for 21 of these proteins was completed in 2 weeks and resulted in the localization and prediction of several unstructured regions within the proteins. When compared with those targets of known structure, the DXMS method correctly localized even small regions of disorder. DXMS analysis was then correlated with the propensity of such targets to crystallize and was further used to define truncations that improved crystallization. Truncations that were defined solely on DXMS analysis demonstrated greatly improved crystallization and have been used for structure determination. This approach represents a rapid and generalized method that can be applied to structural genomics or other targets in a high-throughput manner.

[1]  Virgil L. Woods,et al.  Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS) , 2003, Protein science : a publication of the Protein Society.

[2]  A. Coker,et al.  Mycobacterium tuberculosis Chaperonin 10 Heptamers Self-Associate through Their Biologically Active Loops , 2003, Journal of bacteriology.

[3]  Ashley M Deacon,et al.  Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein. , 2003, Structure.

[4]  Virgil L. Woods,et al.  Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Virgil L. Woods,et al.  Dynamics of cAPK type IIbeta activation revealed by enhanced amide H/2H exchange mass spectrometry (DXMS). , 2003, Journal of molecular biology.

[6]  Virgil L. Woods,et al.  Phosphorylation driven motions in the COOH-terminal Src kinase, CSK, revealed through enhanced hydrogen-deuterium exchange and mass spectrometry (DXMS). , 2002, Journal of molecular biology.

[7]  Glen Spraggon,et al.  Computational analysis of crystallization trials. , 2002, Acta crystallographica. Section D, Biological crystallography.

[8]  M. Deinzer,et al.  Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor‐beta (rhM‐CSFβ) , 2002 .

[9]  Adam Godzik,et al.  Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  D. J. Reed,et al.  Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry , 2002, Protein science : a publication of the Protein Society.

[11]  M. Deinzer,et al.  Mass-spectrometric analysis of agonist-induced retinoic acid receptor γ conformational change , 2002 .

[12]  B. Gowen,et al.  ATP-Bound States of GroEL Captured by Cryo-Electron Microscopy , 2001, Cell.

[13]  D. J. Reed,et al.  Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by hydrogen/deuterium exchange and electrospray ionization mass spectrometry. , 2001, Biochemistry.

[14]  M. Deinzer,et al.  Structural comparison of recombinant human macrophage colony stimulating factor β and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry , 2001, Protein science : a publication of the Protein Society.

[15]  R. Stevens,et al.  Global Efforts in Structural Genomics , 2001, Science.

[16]  D. J. Reed,et al.  Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry. , 2001, Journal of the American Chemical Society.

[17]  Raymond C. Stevens,et al.  Industrializing Structural Biology , 2001, Science.

[18]  J. R. Engen,et al.  Investigating protein structure and dynamics by hydrogen exchange MS. , 2001, Analytical chemistry.

[19]  J. Mandell,et al.  Solvent accessibility of the thrombin-thrombomodulin interface. , 2001, Journal of molecular biology.

[20]  E. Goldsmith,et al.  Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[21]  H. Dyson,et al.  Intrinsically unstructured proteins: re-assessing the protein structure-function paradigm. , 1999, Journal of molecular biology.

[22]  K. Resing,et al.  Modeling deuterium exchange behavior of ERK2 using pepsin mapping to probe secondary structure , 1999, Journal of the American Society for Mass Spectrometry.

[23]  J. Sodroski,et al.  Probability Analysis of Variational Crystallization and Its Application to gp120, The Exterior Envelope Glycoprotein of Type 1 Human Immunodeficiency Virus (HIV-1)* , 1999, The Journal of Biological Chemistry.

[24]  J. Mandell,et al.  Identification of protein-protein interfaces by decreased amide proton solvent accessibility. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  E. Gouaux,et al.  Probing the ligand binding domain of the GluR2 receptor by proteolysis and deletion mutagenesis defines domain boundaries and yields a crystallizable construct , 1998, Protein science : a publication of the Protein Society.

[26]  J. Mandell,et al.  Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry. , 1998, Analytical chemistry.

[27]  T. Sosnick,et al.  Hydrogen exchange: The modern legacy of Linderstrøm‐Lang , 1997, Protein science : a publication of the Protein Society.

[28]  Zhongqi Zhang,et al.  Probing the non-covalent structure of proteins by amide hydrogen exchange and mass spectrometry. , 1997, Journal of mass spectrometry : JMS.

[29]  Steven L. Cohen,et al.  Probing the solution structure of the DNA‐binding protein Max by a combination of proteolysis and mass spectrometry , 1995, Protein science : a publication of the Protein Society.

[30]  Yawen Bai,et al.  [15] Thermodynamic parameters from hydrogen exchange measurements , 1995 .

[31]  John C. Wootton,et al.  Non-globular Domains in Protein Sequences: Automated Segmentation Using Complexity Measures , 1994, Comput. Chem..

[32]  S. Englander,et al.  Structure and energy change in hemoglobin by hydrogen exchange labeling. , 1994, Methods in enzymology.

[33]  L Mayne,et al.  Primary structure effects on peptide group hydrogen exchange. , 1972, Proteins.

[34]  J. R. Rogero,et al.  Protein hydrogen exchange studied by the fragment separation method. , 1985, Analytical biochemistry.

[35]  Yawen Bai,et al.  Primary structure effects on peptide group hydrogen exchange , 1993, Biochemistry.

[36]  S. Englander,et al.  Hydrogen-tritium exchange. , 1972, Methods in enzymology.