Abstract
Hydrogen bonds are key constituents of biomolecular structures, and their response to external perturbations may reveal important insights about the most stable components of a structure. NMR spectroscopy can probe hydrogen bond deformations at very high resolution through hydrogen bond scalar couplings (HBCs). However, the small size of HBCs has so far prevented a comprehensive quantitative characterization of protein hydrogen bonds as a function of the basic thermodynamic parameters of pressure and temperature. Using a newly developed pressure cell, we have now mapped pressure- and temperature-dependent changes of 31 hydrogen bonds in ubiquitin by measuring HBCs with very high precision. Short-range hydrogen bonds are only moderately perturbed, but many hydrogen bonds with large sequence separations (high contact order) show greater changes. In contrast, other high-contact-order hydrogen bonds remain virtually unaffected. The specific stabilization of such topologically important connections may present a general principle with which to achieve protein stability and to preserve structural integrity during protein function.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Raman, S. et al. NMR structure determination for larger proteins using backbone-only data. Science 327, 1014–1018 (2010).
Kang, Y. Which functional form is appropriate for hydrogen bond of amides? J. Phys. Chem. B 104, 8321–8326 (2000).
Desiraju, G. R. A bond by any other name. Angew. Chem. Int. Ed. 50, 52–59 (2011).
Dingley, A. & Grzesiek, S. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J. Am. Chem. Soc. 120, 8293–8297 (1998).
Shenderovich, I. et al. Nuclear magnetic resonance of hydrogen bonded clusters between F− and (HF)n: experiment and theory. Ber. Bunsen Phys. Chem. 102, 422–428 (1998).
Cordier, F. & Grzesiek, S. Direct observation of hydrogen bonds in proteins by interresidue 3hJNC′ scalar couplings. J. Am. Chem. Soc. 121, 1601–1602 (1999).
Cornilescu, G., Hu, J. & Bax, A. Identification of the hydrogen bonding network in a protein by scalar couplings. J. Am. Chem. Soc. 121, 2949–2950 (1999).
Grzesiek, S., Cordier, F., Jaravine, V. & Barfield, M. Insights into biomolecular hydrogen bonds from hydrogen bond scalar couplings. Prog. Nucl. Magn. Res. Spectrosc. 45, 275–300 (2004).
Cornilescu, G. et al. Correlation between 3hJNC′ and hydrogen bond length in proteins. J. Am. Chem. Soc. 121, 6275–6279 (1999).
Scheurer, C. & Bruschweiler, R. Quantum-chemical characterization of nuclear spin–spin couplings across hydrogen bonds. J. Am. Chem. Soc. 121, 8661–8662 (1999).
Barfield, M. Structural dependencies of interresidue scalar coupling h3JNC′ and donor 1H chemical shifts in the hydrogen bonding regions of proteins. J. Am. Chem. Soc. 124, 4158–4168 (2002).
Sass, H.-J., Schmid, F. F.-F. & Grzesiek, S. Correlation of protein structure and dynamics to scalar couplings across hydrogen bonds. J. Am. Chem. Soc. 129, 5898–5903 (2007).
Wilkens, S. J., Westler, W. M., Weinhold, F. & Markley, J. L. Trans-hydrogen-bond h2JNN and h1JNH couplings in the DNA A–T base pair: natural bond orbital analysis. J. Am. Chem. Soc. 124, 1190–1191 (2002).
Kawahara, S., Kojima, C., Taira, K. & Uchimaru, T. A theoretical study of correlation between hydrogen-bond stability and J-coupling through a hydrogen bond. Helv. Chim. Acta 86, 3265–3273 (2003).
Cordier, F. & Grzesiek, S. Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR. J. Mol. Biol. 317, 739–752 (2002).
Hawley, S. A. Reversible pressure–temperature denaturation of chymotrypsinogen. Biochemistry 10, 2436–2442 (1971).
Privalov, P. L., Griko, Y. V., Venyaminov, S. Y. & Kutyshenko, V. P. Cold denaturation of myoglobin. J. Mol. Biol. 190, 487–498 (1986).
Smeller, L. Pressure-temperature phase diagrams of biomolecules. Biochim. Biophys. Acta 1595, 11–29 (2002).
Kalbitzer, H. R. et al. 15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure. Protein Sci 9, 693–703 (2000).
Kitahara, R., Yamada, H., Akasaka, K. & Wright, P. E. High pressure NMR reveals that apomyoglobin is an equilibrium mixture from the native to the unfolded. J. Mol. Biol. 320, 311–319 (2002).
Kitahara, R. & Akasaka, K. Close identity of a pressure-stabilized intermediate with a kinetic intermediate in protein folding. Proc. Natl Acad. Sci. USA 100, 3167–3172 (2003).
Kitahara, R., Yokoyama, S. & Akasaka, K. NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar–3 kbar. J. Mol. Biol. 347, 277–285 (2005).
Wilton, D. J., Tunnicliffe, R. B., Kamatari, Y. O., Akasaka, K. & Williamson, M. P. Pressure-induced changes in the solution structure of the GB1 domain of protein G. Proteins 71, 1432–1440 (2008).
Inoue, K. et al. Pressure-induced local unfolding of the Ras binding domain of RalGDS. Nature Struct. Biol. 7, 547–550 (2000).
Li, H., Yamada, H., Akasaka, K. & Gronenborn, A. M. Pressure alters electronic orbital overlap in hydrogen bonds. J. Biomol. NMR 18, 207–216 (2000).
Cordier, F., Nisius, L., Dingley, A. J. & Grzesiek, S. Direct detection of N–H[…]O=C hydrogen bonds in biomolecules by NMR spectroscopy. Nature Protoc. 3, 235–241 (2008).
Jaravine, V., Alexandrescu, A. & Grzesiek, S. Observation of the closing of individual hydrogen bonds during TFE-induced helix formation in a peptide. Protein Sci. 10, 943–950 (2001).
Baldwin, R. L. Temperature dependence of the hydrophobic interaction in protein folding. Proc. Natl Acad. Sci. USA 83, 8069–8072 (1986).
Kauzmann, W. Some factors in the interpretation of protein denaturation. Adv. Protein Chem. 14, 1–63 (1959).
Tanford, C. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J. Am. Chem. Soc. 84, 4240–4247 (1962).
Kauzmann, W. Protein stabilization—thermodynamics of unfolding. Nature 325, 763–764 (1987).
Hummer, G., Garde, S., García, A. E., Paulaitis, M. E. & Pratt, L. R. The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. Proc. Natl Acad. Sci. USA 95, 1552–1555 (1998).
Grigera, J. R. & McCarthy, A. N. The behavior of the hydrophobic effect under pressure and protein denaturation. Biophys. J. 98, 1626–1631 (2010).
Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531–544 (1987).
Cornilescu, G., Marquardt, J., Ottiger, M. & Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J. Am. Chem. Soc. 120, 6836–6837 (1998).
Magalhaes, A., Maigret, B., Hoflack, J., Gomes, J. N. & Scheraga, H. A. Contribution of unusual arginine–arginine short-range interactions to stabilization and recognition in proteins. J. Protein Chem. 13, 195–215 (1994).
Sheppard, D., Li, D.-W., Godoy-Ruiz, R., Brüschweiler, R. & Tugarinov, V. Variation in quadrupole couplings of alpha deuterons in ubiquitin suggests the presence of C(α)–H(α)…O=C hydrogen bonds. J. Am. Chem. Soc. 132, 7709–7719 (2010).
Kiel, C. & Serrano, L. The ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes. J. Mol. Biol. 355, 821–844 (2006).
Makhatadze, G. I., Lopez, M. M., Richardson, J. M. & Thomas, S. T. Anion binding to the ubiquitin molecule. Protein Sci. 7, 689–697 (1998).
Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nature Rev. Mol. Cell. Biol. 6, 610–621 (2005).
Lange, O. F. et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 1471–1475 (2008).
Herberhold, H. & Winter, R. Temperature- and pressure-induced unfolding and refolding of ubiquitin: a static and kinetic Fourier transform infrared spectroscopy study. Biochemistry 41, 2396–2401 (2002).
Day, R. & García, A. E. Water penetration in the low and high pressure native states of ubiquitin. Proteins 70, 1175–1184 (2007).
Bai, Y., Sosnick, T. R., Mayne, L. & Englander, S. W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).
Khorasanizadeh, S., Peters, I. D. & Roder, H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nature Struct. Biol. 3, 193–205 (1996).
Babu, C. R., Hilser, V. J. & Wand, A. J. Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation. Nature Struct. Mol. Biol. 11, 352–357 (2004).
Fitzkee, N. C. et al. Are proteins made from a limited parts list? Trends Biochem. Sci. 30, 73–80 (2005).
Quinlan, R. J. & Reinhart, G. D. Baroresistant buffer mixtures for biochemical analyses. Anal. Biochem. 341, 69–76 (2005).
Delaglio, F. et al. NMRPipe—a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Garrett, D., Powers, R., Gronenborn, A. & Clore, G. A Common-sense approach to peak picking in 2-dimensional, 3-dimensional, and 4-dimensional spectra using automatic computer-analysis of contour diagrams. J. Magn. Reson. 95, 214–220 (1991).
Acknowledgements
The authors acknowledge H.-R. Kalbitzer for introducing them to high-pressure NMR technology and R. Peterson (Daedalus Innovations) for many helpful discussions. This work was supported by SNF grant 31-132857 (to S.G.) and a stipend from the Boehringer Ingelheim Fonds (to L.N.). This article is dedicated to Anna and Joachim Seelig, celebrating their 40 years of science and life at the Biozentrum Basel.
Author information
Authors and Affiliations
Contributions
L.N. and S.G. performed the experiments, analysed the data and wrote the Article.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary information
Supplementary information (PDF 484 kb)
Rights and permissions
About this article
Cite this article
Nisius, L., Grzesiek, S. Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network. Nature Chem 4, 711–717 (2012). https://doi.org/10.1038/nchem.1396
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchem.1396
This article is cited by
-
Screening cyclooxygenase-2 inhibitors from Allium sativum L. compounds: in silico approach
Journal of Molecular Modeling (2022)
-
Validating the CHARMM36m protein force field with LJ-PME reveals altered hydrogen bonding dynamics under elevated pressures
Communications Chemistry (2021)
-
Polyol and sugar osmolytes can shorten protein hydrogen bonds to modulate function
Communications Biology (2020)
-
Molecular simulations by generalized-ensemble algorithms in isothermal–isobaric ensemble
Biophysical Reviews (2019)
-
Orthotropic Piezoelectricity in 2D Nanocellulose
Scientific Reports (2016)