Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network

Journal name:
Nature Chemistry
Volume:
4,
Pages:
711–717
Year published:
DOI:
doi:10.1038/nchem.1396
Received
Accepted
Published online

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.

At a glance

Figures

  1. Pressure and temperature stability of proteins according to Hawley's theory.
    Figure 1: Pressure and temperature stability of proteins according to Hawley's theory.

    This theory assumes a simple two-state model of protein folding according to equation (2)16, 18. The free-energy difference ΔG between folded and unfolded states corresponds to a paraboloid over the temperature and pressure axes (top). The phase boundary between unfolded and folded states is given by the condition ΔG = 0 and adopts the form of an ellipse in the pT plane (bottom).

  2. High-sensitivity quantitative detection of hydrogen bonds in human ubiquitin by h3JNC′ correlation spectroscopy.
    Figure 2: High-sensitivity quantitative detection of hydrogen bonds in human ubiquitin by h3JNC′ correlation spectroscopy.

    Using the large-volume high-pressure NMR tube, hydrogen bonds can be detected at high pressures with very high sensitivity using a long-range two-dimensional 1H/13C-HNCO-TROSY experiment. a, Long-range two-dimensional 1H/13C-HNCO-TROSY spectrum of uniformly 2H/13C/15N-labelled (amide-protonated) ubiquitin at 35 °C and 2,500 bar (experimental time, 23.3 h). Red labels mark peaks arising from trans hydrogen-bond scalar couplings (donor/acceptor). Additionally observed peaks correspond to incompletely suppressed sequential correlations (15Ni/13C′i−1) or intraresidue two-bond correlations (15Ni/13C′i). b, One-dimensional traces through selected resonances of the two-dimensional spectrum with the associated h3JNC′ coupling constants. The excellent sensitivity of the experiment can be appreciated from the high signal-to-noise ratio of even very weak hydrogen-bond correlations.

  3. Pressure-induced variations of all detected hydrogen-bond correlations in ubiquitin.
    Figure 3: Pressure-induced variations of all detected hydrogen-bond correlations in ubiquitin.

    The h3JNC′ HBCs are quantified from the two-dimensional 1H/13C-HNCO-TROSY for 31 hydrogen bonds in ubiquitin as a function of pressure from 1 to 2,500 bar at 35 °C. The data points are the mean of repeated measurements, with error bars showing standard deviations. In many cases, error bars are smaller than the size of the individual data point symbols. The red line corresponds to the global fit of the entire h3JNC′(p, T) surface using all h3JNC′ coupling constants measured in the temperature range 5–50 °C and pressure range 1–2,500 bar (see Supplementary Fig. S1 for details).

  4. Combined pressure and temperature dependence of representative hydrogen-bond correlations in ubiquitin.
    Figure 4: Combined pressure and temperature dependence of representative hydrogen-bond correlations in ubiquitin.

    The stability of hydrogen bonds was assayed over a complete rectangle in pT space covering 1–2,500 bar and 5–50 °C. Different hydrogen bonds reveal a distinctive stability behaviour. a, h3JNC′ coupling constants of selected hydrogen bonds as a function of pressure and temperature. The red line indicates the global fit over the entire h3JNC′(p, T) surface (see Supplementary Fig. S1 for details). b, Three-dimensional representation of the pressure and temperature dependence of h3JNC′ and corresponding fitted surface.

  5. Pressure and temperature derivatives of stability of h3JNC′ as quantitative measures of hydrogen-bond stability.
    Figure 5: Pressure and temperature derivatives of stability of h3JNC′ as quantitative measures of hydrogen-bond stability.

    Derivatives of h3JNC′(p, T) with respect to pressure and temperature are used as a measure of individual hydrogen-bond thermodynamic stability. The stability is correlated to the sequence separation of hydrogen-bond donor and acceptor Δs. a, Derivatives ∂|h3JNC′|/∂p and ∂|h3JNC′|/∂T for all detected hydrogen bonds calculated from the fit of the two-dimensional h3JNC′(p, T) surface at 20 °C and 1 bar. Data points are labelled by the residue number of the hydrogen-bond donor. Low-contact-order hydrogen bonds (Δs < 20) are in black. High-contact-order hydrogen bonds (Δs ≥ 20) are in blue for small values in ∂|h3JNC′|/∂p and ∂|h3JNC′|/∂T corresponding to high stability, and in red for low stability (large values of ∂|h3JNC′|/∂p and ∂|h3JNC′|/∂T). b, Topology diagram of ubiquitin with backbone hydrogen bonds indicated by dotted lines between amide protons (filled circles) and carbonyl oxygen atoms (open circles). Hydrogen bonds are coloured as in a according to their contact order and stability.

  6. Immediate vicinity of high- and low-stability hydrogen bonds in β-sheet β3/β5.
    Figure 6: Immediate vicinity of high- and low-stability hydrogen bonds in β-sheet β3/β5.

    Whereas many high-contact-order hydrogen bonds have increased sensitivity to temperature and pressure perturbations, some high-contact-order hydrogen bonds are particularly stable and are located in the immediate vicinity of high-contact hydrogen bonds with low stability. a, Three-dimensional representation of the h3JNC′ pressure and temperature dependence for hydrogen bonds in β-sheet β3/β5, including the fitted h3JNC′(p,T) surface. b, View of the three-dimensional structure of ubiquitin (1D3Z; ref. 35) showing the stabilization of β-sheet 3/5 by hydrophobic and polar interactions involving residues V70 and R42.

References

  1. Raman, S. et al. NMR structure determination for larger proteins using backbone-only data. Science 327, 10141018 (2010).
  2. Kang, Y. Which functional form is appropriate for hydrogen bond of amides? J. Phys. Chem. B 104, 83218326 (2000).
  3. Desiraju, G. R. A bond by any other name. Angew. Chem. Int. Ed. 50, 5259 (2011).
  4. Dingley, A. & Grzesiek, S. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J. Am. Chem. Soc. 120, 82938297 (1998).
  5. Shenderovich, I. et al. Nuclear magnetic resonance of hydrogen bonded clusters between F and (HF)n: experiment and theory. Ber. Bunsen Phys. Chem. 102, 422428 (1998).
  6. Cordier, F. & Grzesiek, S. Direct observation of hydrogen bonds in proteins by interresidue 3hJNC′ scalar couplings. J. Am. Chem. Soc. 121, 16011602 (1999).
  7. Cornilescu, G., Hu, J. & Bax, A. Identification of the hydrogen bonding network in a protein by scalar couplings. J. Am. Chem. Soc. 121, 29492950 (1999).
  8. Grzesiek, S., Cordier, F., Jaravine, V. & Barfield, M. Insights into biomolecular hydrogen bonds from hydrogen bond scalar couplings. Prog. Nucl. Magn. Res. Spectrosc. 45, 275300 (2004).
  9. Cornilescu, G. et al. Correlation between 3hJNC′ and hydrogen bond length in proteins. J. Am. Chem. Soc. 121, 62756279 (1999).
  10. Scheurer, C. & Bruschweiler, R. Quantum-chemical characterization of nuclear spin–spin couplings across hydrogen bonds. J. Am. Chem. Soc. 121, 86618662 (1999).
  11. 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, 41584168 (2002).
  12. 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, 58985903 (2007).
  13. 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, 11901191 (2002).
  14. 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, 32653273 (2003).
  15. Cordier, F. & Grzesiek, S. Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR. J. Mol. Biol. 317, 739752 (2002).
  16. Hawley, S. A. Reversible pressure–temperature denaturation of chymotrypsinogen. Biochemistry 10, 24362442 (1971).
  17. Privalov, P. L., Griko, Y. V., Venyaminov, S. Y. & Kutyshenko, V. P. Cold denaturation of myoglobin. J. Mol. Biol. 190, 487498 (1986).
  18. Smeller, L. Pressure-temperature phase diagrams of biomolecules. Biochim. Biophys. Acta 1595, 1129 (2002).
  19. Kalbitzer, H. R. et al. 15N and 1H NMR study of histidine containing protein (HPr) from Staphylococcus carnosus at high pressure. Protein Sci 9, 693703 (2000).
  20. 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, 311319 (2002).
  21. Kitahara, R. & Akasaka, K. Close identity of a pressure-stabilized intermediate with a kinetic intermediate in protein folding. Proc. Natl Acad. Sci. USA 100, 31673172 (2003).
  22. Kitahara, R., Yokoyama, S. & Akasaka, K. NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar–3 kbar. J. Mol. Biol. 347, 277285 (2005).
  23. 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, 14321440 (2008).
  24. Inoue, K. et al. Pressure-induced local unfolding of the Ras binding domain of RalGDS. Nature Struct. Biol. 7, 547550 (2000).
  25. Li, H., Yamada, H., Akasaka, K. & Gronenborn, A. M. Pressure alters electronic orbital overlap in hydrogen bonds. J. Biomol. NMR 18, 207216 (2000).
  26. 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, 235241 (2008).
  27. 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, 943950 (2001).
  28. Baldwin, R. L. Temperature dependence of the hydrophobic interaction in protein folding. Proc. Natl Acad. Sci. USA 83, 80698072 (1986).
  29. Kauzmann, W. Some factors in the interpretation of protein denaturation. Adv. Protein Chem. 14, 163 (1959).
  30. Tanford, C. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J. Am. Chem. Soc. 84, 42404247 (1962).
  31. Kauzmann, W. Protein stabilization—thermodynamics of unfolding. Nature 325, 763764 (1987).
  32. 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, 15521555 (1998).
  33. Grigera, J. R. & McCarthy, A. N. The behavior of the hydrophobic effect under pressure and protein denaturation. Biophys. J. 98, 16261631 (2010).
  34. Vijay-Kumar, S., Bugg, C. E. & Cook, W. J. Structure of ubiquitin refined at 1.8 Å resolution. J. Mol. Biol. 194, 531544 (1987).
  35. 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, 68366837 (1998).
  36. 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, 195215 (1994).
  37. 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, 77097719 (2010).
  38. Kiel, C. & Serrano, L. The ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes. J. Mol. Biol. 355, 821844 (2006).
  39. Makhatadze, G. I., Lopez, M. M., Richardson, J. M. & Thomas, S. T. Anion binding to the ubiquitin molecule. Protein Sci. 7, 689697 (1998).
  40. Hicke, L., Schubert, H. L. & Hill, C. P. Ubiquitin-binding domains. Nature Rev. Mol. Cell. Biol. 6, 610621 (2005).
  41. Lange, O. F. et al. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320, 14711475 (2008).
  42. Herberhold, H. & Winter, R. Temperature- and pressure-induced unfolding and refolding of ubiquitin: a static and kinetic Fourier transform infrared spectroscopy study. Biochemistry 41, 23962401 (2002).
  43. Day, R. & García, A. E. Water penetration in the low and high pressure native states of ubiquitin. Proteins 70, 11751184 (2007).
  44. Bai, Y., Sosnick, T. R., Mayne, L. & Englander, S. W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192197 (1995).
  45. 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, 193205 (1996).
  46. 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, 352357 (2004).
  47. Fitzkee, N. C. et al. Are proteins made from a limited parts list? Trends Biochem. Sci. 30, 7380 (2005).
  48. Quinlan, R. J. & Reinhart, G. D. Baroresistant buffer mixtures for biochemical analyses. Anal. Biochem. 341, 6976 (2005).
  49. Delaglio, F. et al. NMRPipe—a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277293 (1995).
  50. 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, 214220 (1991).

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Affiliations

  1. Division of Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland

    • Lydia Nisius &
    • Stephan Grzesiek

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L.N. and S.G. performed the experiments, analysed the data and wrote the Article.

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The authors declare no competing financial interests.

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