European Biophysics Journal

, Volume 35, Issue 7, pp 577–583

Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase–inhibitor complex

  • M. P. Blakeley
  • A. Mitschler
  • I. Hazemann
  • F. Meilleur
  • D. A. A. Myles
  • A. Podjarny


Protonation states determination by neutron (2.2 Å at room temperature) and X-ray (0.66 Å at 100 K) crystallographic studies were compared for a medium size enzyme, human aldose reductase (MW = 36 kDa), complexed with its NADP+ coenzyme and a selected inhibitor of therapeutic interest. The neutron resolution could be achieved only with the ab initio fully deuterated protein and the subsequent crystallization in D2O of the complex. We used the largest good-quality crystal (1.00 × 0.67 × 0.23 mm, i.e. volume of 0.15 mm3) that we were able to grow so far. Both studies enable the determination of protonation states, with a clear advantage for neutrons in the case of less-ordered atoms (B > 5 Å2). Hydrogen atoms are best determined by a complementary analysis of the Fourier maps obtained from both methods.


  1. Ames J, Mathies R (1990) The role of back-reactions and proton uptake during the N–O transition in bacteriorhodopsin’s photocycle: a kinetic resonance Raman study. Biochemistry 29:7181–7190CrossRefGoogle Scholar
  2. Arzt S, Campbell JW, Harding MM, Hao Q, Helliwell JR (1999) LSCALE—the new normalization, scaling and absorption correction program in the Daresbury Laue software suite. J Appl Cryst 32:554–562CrossRefGoogle Scholar
  3. Blakeley MP, Kalb (Gilboa) AJ, Helliwell JR, Myles DAA (2004) The 15-K neutron structure of saccharide-free concanavalin A. PNAS 101(47):16405–16410CrossRefADSGoogle Scholar
  4. Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang J-S, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Cryst D54:905–921Google Scholar
  5. Budayova-Spano M, Fisher S, Dauvergne M, Agbandje-McKenna M, Silverman D, Myles D, McKenna R (2006) Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II. Acta Crystallogr F Biol Crystallogr 62:6–9CrossRefGoogle Scholar
  6. Campbell JW (1995) LAUEGEN, an X-windows-based program for the processing of Laue X-ray diffraction data. J Appl Cryst 28:228–236CrossRefGoogle Scholar
  7. Campbell J, Hao Q, Harding M, Nguti ND, Wilkinson C (1998) LAUEGEN version 6.0 and INTLDM. J Appl Cryst 31:496–502CrossRefGoogle Scholar
  8. Cipriani F, Castagna J, Wilkinson C, Oleinek P, Lehmann M (1996) Cold neutron protein crystallography using a large position-sensitive detector based on image-plate technology. J Neutron Res 4:79–85CrossRefGoogle Scholar
  9. Coates L, Erskine P, Wood S, Myles D, Cooper J (2001) A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism. Biochemistry 40:13149–13157CrossRefGoogle Scholar
  10. Dauter Z, Lamzin V, Wilson K (1997) The benefits of atomic resolution. Curr Opin Struct Biol 7:681–688CrossRefGoogle Scholar
  11. Deacon A, Gleichmann T, Kalb (Gilboa) AJ, Price H, Raftery J, Bradbrook G, Yariv J, Helliwell JR (1997) The structure of concanavalin A and its bound solvent determined with small-molecule accuracy at 0.94 Å resolution. J Chem Soc Faraday Trans 93(24):4305–4312CrossRefGoogle Scholar
  12. Dioumaev A (2001) Infrared methods for monitoring the protonation state of carboxylic amino acids in the photocycle of bacteriorhodopsin. Biochemistry (Mosc) 66:1269–1276CrossRefGoogle Scholar
  13. Esposito L, Vitagliano L, Mazzarella L (2002) Recent advances in atomic resolution protein crystallography. Protein Pept Lett 9:95–106CrossRefGoogle Scholar
  14. Habash J, Raftery J, Nuttall R, Price H, Wilkinson C, Kalb AJ, Helliwell JR (2000) Direct determination of the positions of the deuterium atoms of the bound water in -concanavalin A by neutron Laue crystallography. Acta Crystallogr D Biol Crystallogr 56:541–50CrossRefGoogle Scholar
  15. Harris TK, Turner GJ (2002) Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53:85–98CrossRefGoogle Scholar
  16. Hazemann I, Dauvergne M, Blakeley M, Meilleur F, Haertlein M, Van Dorsselaer A, Mitschler A, Myles D, Podjarny A (2005) High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase. Acta Crystallogr D Biol Crystallogr 61:1413–1417CrossRefGoogle Scholar
  17. Helliwell JR, Habash J, Cruickshank DW, Harding MM, Greenhough TJ, Campbell JW, Clifton IJ, Elser M, Machin PA, Papiz MZ, Zurch S (1989) The recording and analysis of synchrotron X-radiation Laue diffraction photographs. J Appl Cryst 22:483–497CrossRefGoogle Scholar
  18. Howard EI, Sanishvili R, Cachau RE, Mitschler A, Chevrier B, Barth P, Lamour V, Van Zandt M, Sibley E, Bon C, Moras D, Schneider TR, Joachimiak A, Podjarny A (2004) Ultra-high resolution drug design I: details of interactions in human aldose reductase-inhibitor complex at 0.66 Å. Proteins 55:792–804CrossRefGoogle Scholar
  19. Meilleur F, Contzen J, Myles D, Jung C (2004) Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101). Biochemistry 43:8744–8753CrossRefGoogle Scholar
  20. Meilleur F, Dauvergne MT, Schlichting I, Myles DAA (2005) Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam. Acta Crystallogr D Biol Crystallogr 61:539–544CrossRefGoogle Scholar
  21. Minasov F, Wang X, Shoichet BK (2002) An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation. J Am Chem Soc 124:5333–5340CrossRefGoogle Scholar
  22. Myles D, Bon C, Langan P, Cipriani F, Castagna JC, Lehmann MS, Wilkinson C (1998) Neutron Laue diffraction in macromolecular crystallography. Physica B 241–243:1122–1130Google Scholar
  23. Petrova T, Podjarny A (2004) Protein crystallography at subatomic resolution. Rep Prog Phys 67:1565–1605CrossRefADSGoogle Scholar
  24. Rosenbaum G, Alkire R, Evans G, Rotella F, Lazarski K, Zhang R, Ginell S, Duke N, Naday I, Lazarz J, Molitsky M, Keefe L, Gonczy J, Rock L, Sanishvili R, Walsh M, Westbrook E, Joachimiak A (2006) The structural center 19ID undulator beamline: facility specifications and protein crystallographic results. J Sync Rad 13:30–45CrossRefGoogle Scholar
  25. Schmidt A, Lamzin V (2002) Veni, vidi, vici—atomic resolution unravelling the mysteries of protein function. Curr Opin Struct Biol 12:698–703CrossRefGoogle Scholar
  26. Shu F, Ramakrishnan V, Schoenborn B (2000) Enhanced visibility of hydrogen atoms by neutron crystallography on fully deuterated myoglobin. Proc Natl Acad Sci USA 97:3872–3877CrossRefADSGoogle Scholar

Copyright information

© EBSA 2006

Authors and Affiliations

  • M. P. Blakeley
    • 1
  • A. Mitschler
    • 2
  • I. Hazemann
    • 2
  • F. Meilleur
    • 3
  • D. A. A. Myles
    • 4
  • A. Podjarny
    • 2
  1. 1.EMBLGrenobleFrance
  2. 2.IGBMC, CNRS/INSERM/ULPStrasbourg-IllkirchFrance
  3. 3.ILLGrenobleFrance
  4. 4.Oak Ridge National LaboratoryOak RidgeUSA

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