Energetics of Intermolecular Hydrogen Bonds in a Hydrophobic Protein Cavity

  • Lan Liu
  • Alyson Baergen
  • Klaus Michelsen
  • Elena N. Kitova
  • Paul D. Schnier
  • John S. Klassen
Research Article


This work explores the energetics of intermolecular H-bonds inside a hydrophobic protein cavity. Kinetic measurements were performed on the gaseous deprotonated ions (at the −7 charge state) of complexes of bovine β-lactoglobulin (Lg) and three monohydroxylated analogs of palmitic acid (PA): 3-hydroxypalmitic acid (3-OHPA), 7-hydroxypalmitic acid (7-OHPA), and 16-hydroxypalmitic acid (16-OHPA). From the increase in the activation energy for the dissociation of the (Lg + X-OHPA)7– ions, compared with that of the (Lg + PA)7– ion, it is concluded that the –OH groups of the X-OHPA ligands participate in strong (5 – 11 kcal mol–1) intermolecular H-bonds in the hydrophobic cavity of Lg. The results of molecular dynamics (MD) simulations suggest that the –OH groups of 3-OHPA and 16-OHPA act as H-bond donors and interact with backbone carbonyl oxygens, whereas the –OH group of 7-OHPA acts as both H-bond donor and acceptor with nearby side chains. The capacity for intermolecular H-bonds within the Lg cavity, as suggested by the gas-phase measurements, does not necessarily lead to enhanced binding in aqueous solution. The association constant (Ka) measured for 7-OHPA [(2.3 ± 0.2) × 105 M–1] is similar to the value for the PA [(3.8 ± 0.1) × 105 M–1]; Ka for 3-OHPA [(1.1 ± 0.3) × 106 M–1] is approximately three-times larger, whereas Ka for 16-OHPA [(2.3 ± 0.2) × 104 M–1] is an order of magnitude smaller. Taken together, the results of this study suggest that the energetic penalty to desolvating the ligand –OH groups, which is necessary for complex formation, is similar in magnitude to the energetic contribution of the intermolecular H-bonds.

Key words

Hydrogen bonds Protein–ligand complexes Hydrophobic Kinetics energetics 

Supplementary material

13361_2014_833_MOESM1_ESM.doc (380 kb)
Figure S1 Plots of the fractional abundance of Lg6− (Ab (Lg6−)/Ab total) versus reaction time measured for the dissociation of (Lg + 3-OHPA)7− ions at (a) 57 °C, (b) 65 °C and (c) 81 °C. Plots of the abundance ratio of Lg6− and Lg7− (Ab (Lg6−)/Ab (Lg7−)) versus reaction time measured for the dissociation of (Lg + 3-OHPA)7− ions at (d) 57 °C, (e) 65 °C and (f) 81 °C. (DOC 380 kb)
13361_2014_833_MOESM2_ESM.doc (298 kb)
Figure S2 Plots of the fractional abundance of Lg6− (Ab (Lg6−)/Ab total) versus reaction time measured for the dissociation of (Lg + 16-OHPA)7− ions at (a) 47 °C, (b) 57 °C, (c) 65 °C and (d) 76 °C. (DOC 298 kb)


  1. 1.
    Pace, C.N., Shirley, B.A., McNutt, M., Gajiwala, K.: Forces contributing to the conformational stability of proteins. FASEB J. 10, 75–83 (1996)Google Scholar
  2. 2.
    Efremov, R.G., Chugunov, A.O., Pyrkov, T.V., Priestle, J.P., Arseniev, A.S., Jacoby, E.: Molecular lipophilicity in protein modeling and drug design. Curr. Med. Chem. 14, 393–415 (2007)CrossRefGoogle Scholar
  3. 3.
    Meyer, E.E., Rosenberg, K.J., Israelachvili, J.: Recent progress in understanding hydrophobic interactions. Proc. Natl. Acad. Sci. U. S. A. 103, 15739–15746 (2006)CrossRefGoogle Scholar
  4. 4.
    Wang, R., Lu, Y., Fang, X., Wang, S.: An extensive test of 14 scoring functions using the PDB bind refined set of 800 protein-ligand complexes. J. Chem. Inf. Comp. Sci. 44, 2114–2125 (2004)CrossRefGoogle Scholar
  5. 5.
    Patil, R., Das, S., Stanley, A., Yadav, L., Sudhakar, A., Varma, A.K.: Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing. PLoS One 45, e12029 (2010)CrossRefGoogle Scholar
  6. 6.
    Friesner, R.A., Murphy, R.B., Repasky, M.P., Frye, L.L., Greenwood, J.R., Halgren, T.A., Sanschagrin, P.C., Mainz, D.T.: Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein–ligand complexes. J. Med. Chem. 49, 6177–6196 (2006)CrossRefGoogle Scholar
  7. 7.
    Young, T., Abel, R., Kim, B., Berne, B., Friesner, R.A.: Motifs for molecular recognition exploiting hydrophobic enclosure in protein-ligand binding. Proc. Natl. Acad. Sci. U. S. A. 104, 808–813 (2007)CrossRefGoogle Scholar
  8. 8.
    Kitova, E.N., Bundle, D.R., Klassen, J.S.: Partitioning of solvent effects and intrinsic interactions in biological recognition. Angew. Chem. Int. Ed. 43, 4183–4186 (2004)CrossRefGoogle Scholar
  9. 9.
    Kitova, E.N., Seo, M., Roy, P.-N., Klassen, J.S.: Elucidating the intermolecular interactions within a desolvated protein–ligand complex. An experimental and computational study. J. Am. Chem. Soc. 130, 1214–1226 (2008)CrossRefGoogle Scholar
  10. 10.
    Liu, L., Bagal, D., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Hydrophobic protein–ligand interactions preserved in the gas phase. J. Am. Chem. Soc. 131, 15980–15981 (2009)CrossRefGoogle Scholar
  11. 11.
    Liu, L., Michelsen, K., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Evidence that water can reduce the kinetic stability of protein-hydrophobic ligand interaction. J. Am. Chem. Soc. 132, 17658–17660 (2010)CrossRefGoogle Scholar
  12. 12.
    Liu, L., Michelsen, K., Kitova, E.N., Schnier, P.D., Klassen, J.S.: Energetics of lipid binding in a hydrophobic protein cavity. J. Am. Chem. Soc. 134, 3054–3060 (2012)CrossRefGoogle Scholar
  13. 13.
    Deng, L., Broom, A., Kitova, E.N., Richards, M.R., Zheng, R.B., Shoemaker, G.K., Meiering, E.M., Klassen, J.S.: Kinetic stability of the streptavidin–biotin interaction enhanced in the gas phase. J. Am. Chem. Soc. 134, 16586–16596 (2012)CrossRefGoogle Scholar
  14. 14.
    Deng, L., Kitova, E.N., Klassen, J.S.: Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis. J. Am. Soc. Mass Spectrom. 24, 49–56 (2013)CrossRefGoogle Scholar
  15. 15.
    Kontopidis, G., Holt, C., Sawyer, L.: Invited Review: β-lactoglobulin: binding properties, structure, and function. J. Dairy Sci. 87, 785–796 (2004)CrossRefGoogle Scholar
  16. 16.
    Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, H.M., Baker, E.N., Jameson, G.B.: Structure basis of the Tanford transition of bovine β-lactoglobulin. Biochemistry 37, 14014–14023 (1998)CrossRefGoogle Scholar
  17. 17.
    Qvist, J., Davidovic, M., Hamelberg, D., Halle, B.: A dry ligand-binding cavity in a solvated protein. Proc. Natl. Acad. Sci. U. S. A. 105, 6296–6301 (2008)CrossRefGoogle Scholar
  18. 18.
    Liu, L., Kitova, E.N., Klassen, J.S.: Quantifying protein-fatty acid interactions using electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 310–318 (2011)CrossRefGoogle Scholar
  19. 19.
    Dunbar, R.C., McMahon, T.B.: Activation of unimolecular reactions by ambient blackbody radiation. Science 279, 194–197 (1998)CrossRefGoogle Scholar
  20. 20.
    Price, W.D., Schnier, P.D., Jockusch, R.A., Strittmatter, E.R., Williams, E.R.: Unimulecular reaction kinetics in the high-pressure limit without collisoins. J. Am. Chem. Soc. 118, 10640–10644 (1996)CrossRefGoogle Scholar
  21. 21.
    Sun, J., Kitova, E.N., Klassen, J.S.: Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry. Anal. Chem. 79, 416–425 (2007)CrossRefGoogle Scholar
  22. 22.
    Bagal, D., Kitova, E.N., Liu, L., El-Haweit, A., Schnier, P.D., Klassen, J.S.: Gas phase stabilization of noncovalent protein complexes formed by electrospray ionization. Anal. Chem. 81, 7801–7806 (2009)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

  • Lan Liu
    • 1
  • Alyson Baergen
    • 1
  • Klaus Michelsen
    • 2
  • Elena N. Kitova
    • 1
  • Paul D. Schnier
    • 3
  • John S. Klassen
    • 1
  1. 1.Alberta Glycomics Centre and Department of ChemistryUniversity of AlbertaEdmontonCanada
  2. 2.Molecular Structure, AmgenCambridgeUSA
  3. 3.Molecular Structure, AmgenSouth San FranciscoUSA

Personalised recommendations