Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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 29:7181–7190
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 32:554–562
Blakeley MP, Kalb (Gilboa) AJ, Helliwell JR, Myles DAA (2004) The 15-K neutron structure of saccharide-free concanavalin A. PNAS 101(47):16405–16410
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 D54:905–921
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 62:6–9
Campbell JW (1995) LAUEGEN, an X-windows-based program for the processing of Laue X-ray diffraction data. J Appl Cryst 28:228–236
Campbell J, Hao Q, Harding M, Nguti ND, Wilkinson C (1998) LAUEGEN version 6.0 and INTLDM. J Appl Cryst 31:496–502
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 4:79–85
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 40:13149–13157
Dauter Z, Lamzin V, Wilson K (1997) The benefits of atomic resolution. Curr Opin Struct Biol 7:681–688
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):4305–4312
Dioumaev A (2001) Infrared methods for monitoring the protonation state of carboxylic amino acids in the photocycle of bacteriorhodopsin. Biochemistry (Mosc) 66:1269–1276
Esposito L, Vitagliano L, Mazzarella L (2002) Recent advances in atomic resolution protein crystallography. Protein Pept Lett 9:95–106
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 56:541–50
Harris TK, Turner GJ (2002) Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53:85–98
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 61:1413–1417
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 22:483–497
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 55:792–804
Meilleur F, Contzen J, Myles D, Jung C (2004) Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101). Biochemistry 43:8744–8753
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 61:539–544
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 124:5333–5340
Myles D, Bon C, Langan P, Cipriani F, Castagna JC, Lehmann MS, Wilkinson C (1998) Neutron Laue diffraction in macromolecular crystallography. Physica B 241–243:1122–1130
Petrova T, Podjarny A (2004) Protein crystallography at subatomic resolution. Rep Prog Phys 67:1565–1605
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 13:30–45
Schmidt A, Lamzin V (2002) Veni, vidi, vici—atomic resolution unravelling the mysteries of protein function. Curr Opin Struct Biol 12:698–703
Shu F, Ramakrishnan V, Schoenborn B (2000) Enhanced visibility of hydrogen atoms by neutron crystallography on fully deuterated myoglobin. Proc Natl Acad Sci USA 97:3872–3877
Acknowledgements
We thank the staff of the EMBL and ILL, Grenoble, France and of the SBC, APS, Argonne, USA, for their help in data collection, and of the IGBMC for their support. This work was supported by the Centre National de la Recherché Scientifique (CNRS), by the CNRS-DFG collaboration (CERC3), by the Institut National de la Santé et de la Recherché Médicale and the Hôpital Universitaire de Strasbourg (H.U.S).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Blakeley, M.P., Mitschler, A., Hazemann, I. et al. Comparison of hydrogen determination with X-ray and neutron crystallography in a human aldose reductase–inhibitor complex. Eur Biophys J 35, 577–583 (2006). https://doi.org/10.1007/s00249-006-0064-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-006-0064-8