Molecular recognition—the non-covalent association of one molecule with another—is centrally important to biology. It guides the folding of proteins, the translation of DNA, the catalytic transformation of cellular metabolites, and the propagation of information throughout the cell (and between cells). The fundamental molecular driving forces involved in interactions between molecules have been enumerated (ionic, electrostatic, hydrophobic, etc.), but are not well understood, and the role of water in these driving forces is commonly neglected. Building a fundamental understanding of these interactions will facilitate the rational design of biologically active molecules—from antibodies to ligands, which could serve as drug leads—, and enable the engineering of novel biologically-inspired, protein-based materials.

We study molecular recognition in the contexts of protein-ligand interactions, protein crystallization, and multivalent receptor-ligand association. We integrate tools of calorimetry and thermodynamic analysis, protein crystallography and structural analysis, nuclear magnetic (NMR) resonance spectroscopy, capillary electrophoresis, and molecular dynamics simulations with the goal of building structure-function relationships that will help us to understand the molecular-level origins that guide affinity and specificity in biomolecular association (Figure 2).

The role of water

Water is a strong solvent and must therefore be included in the equations defining the equilibria of biomolecular recognition (Figure 1). We are particularly interested in the role water in hydrophobic interactions (1-5) and ionic interactions (6) (particularly those involving protein concavities), the relationship between protein surface chemistry and the interactions among proteins in their crystals (7). Our studies are designed to investigate the often encountered phenomenon of enthalpy-entropy compensation (H-S compensation) and to better understand structure-thermodynamic relationships in water, which would help to eventually break H-S compensation by molecular design (2,3,5,8,9).

Charged protein surfaces

Many proteins contain charged amino acids in their active sites, where they interact electrostatically with substrates—for binding or catalysis (Figure 3). Charged groups occur inside the protein (commonly referred to as ‘salt bridges’), but also on the entire surface: Approx. 30% of the solvent-accessible surface of a globular protein (10).

Our interest focus on these solvent-exposed charges, and their relevance to folding, stability, solubility, and supramolecular aggregation in water (11-15). We use carbonic anhydrase as a model protein and charge ladders in capillary electrophoresis to study the average contributions of a particular charged group (e.g., NH3+ or COO- groups) on the exterior of the protein (Figure 4).


Multivalency—the interaction of a single molecule with another by multiple, separate connections—is a central concept in molecular recognition in biology (e.g. viruses, antibodies, lectins, immunological cell-cell interactions). It enables specific interactions, which collectively can be much stronger than the corresponding monovalent ones (16).

We are interested in designing multivalent interactions in model systems (Figure 5) with relevance to immunology (17,18) and in designing ultra-tight, non-covalent complexes, e.g., a complex stronger than biotin-avidin (19-21).

Figure 1. Simplified schematic for the binding of a small molecule (dark gray circle) to a cavity on a protein (gray area) in water. Both molecules are hydrated before they form a complex, and the water molecules in the hydration shell are free-energetically less favorable—either by enthalpy (red molecules) or entropy (yellow molecules), or both—than bulk water (colorless molecules). The final complex is also hydrated, and retains parts of the initial hydration shell.
Biophysics Fig2


Figure 2. A demonstration of the use of our model system human carbonic anhydrase II (HCAII) to examine the influence of differences in ligand structure—in the absence of differences in protein conformation—on binding. The ligands 1,3-thiazole-2-sulfonamide (TA) and benzo[d]thiazole-2-sulfonamide (BTA) differ by a benzene ring, but not in binding geometry and, thus, reveal the thermodynamic contribution of the benzene ring to binding (1).



Biophysics Fig3
Figure 3. A cartoon of a protein, showing three types of charges: in the active site, buried, and solvent-exposed (10).


Biophysics Fig4
Figure 4. Electropherograms of the charge ladders of bovine carbonic anhydrase II (BCA) produced by increasing acetylation of exposed lysines (i.e., neutralizing positive charges; top) or amidation of exposed carboxylates with hydroxylamine (i.e., neutralizing negative charges; bottom). A neutral marker (NM) is included to monitor electroosmotic flow (10).
Biophysics Fig5
Figure 5. The binding strength (‘affinity’ in monovalent interactions, ‘avidity’ in multivalent interactions) of a dimer of carbonic anhydrase to a divalent ligand depends on the length of the linker (18).

[1] Snyder, P.W., Mecinovic, J., Moustakas, D.T., Thomas, S.W., III, Harder, M., Mack, E.T., Lockett, M.R., Heroux, A., Sherman, W. & Whitesides, G.M.: Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase; Proc. Natl. Acad. Sci. U. S. A. 108, 17889-17894 (2011).

[2] Lockett, M.R., Lange, H., Breiten, B., Heroux, A., Sherman, W., Rappoport, D., Yau, P.O., Snyder, P.W. & Whitesides, G.M.: The Binding of Benzoarylsulfonamide Ligands to Human Carbonic Anhydrase is Insensitive to Formal Fluorination of the Ligand; Angew. Chem. Int. Ed. 52, 7714-7717 (2013).

[3] Breiten, B., Lockett, M.R., Sherman, W., Fujita, S., Al-Sayah, M., Lange, H., Bowers, C.M., Heroux, A., Krilov, G. & Whitesides, G.M.: Water Networks Contribute to Enthalpy/Entropy Compensation in Protein-Ligand Binding; J. Am. Chem. Soc. 135, 15579-15584 (2013).

[4] Snyder, P.W., Lockett, M.R., Moustakas, D.T. & Whitesides, G.M.: Is it the shape of the cavity, or the shape of the water in the cavity?; Eur. Phys. J. Special Topics 223, 853-891 (2014).

[5] Fox, J.M., Kang, K., Sastry, M., Sherman, W., Sankaran, B., Zwart, P.H. & Whitesides, G.M.: Water-Restructuring Mutations Can Reverse the Thermodynamic Signature of Ligand Binding to Human Carbonic Anhydrase; Angew. Chem. Int. Ed. 56, 3833-3837 (2017).

[6] Fox, J.M., Kang, K., Sherman, W., Heroux, A., Sastry, G.M., Baghbanzadeh, M., Lockett, M.R. & Whitesides, G.M.: Interactions between Hofmeister Anions and the Binding Pocket of a Protein; J. Am. Chem. Soc. 137, 3859-3866 (2015).

[7] Kang, K., Choi, J.-M., Fox, J.M., Snyder, P.W., Moustakas, D.T. & Whitesides, G.M.: Acetylation of Surface Lysine Groups of a Protein Alters the Organization and Composition of Its Crystal Contacts; The Journal of Physical Chemistry B 120, 6461-6468 (2016).

[8] Krishnamurthy, V.M., Bohall, B.R., Semetey, V. & Whitesides, G.M.: The Paradoxical Thermodynamic Basis for the Interaction of Ethylene Glycol, Glycine, and Sarcosine Chains with Bovine Carbonic Anhydrase II: An Unexpected Manifestation of Enthalpy/Entropy Compensation; J. Am. Chem. Soc. 128, 5802-5812 (2006).

[9] Fox, J.M., Zhao, M., Fink, M.J., Kang, K. & Whitesides, G.M.: The Molecular Origin of Enthalpy/Entropy Compensation in Biomolecular Recognition; Annual Review of Biophysics 47, 1-28 (2018).

[10] Gitlin, I., Carbeck, J.D. & Whitesides, G.M.: Why Are Proteins Charged? Networks of Charge–Charge Interactions in Proteins Measured by Charge Ladders and Capillary Electrophoresis; Angew. Chem. Int. Ed. 45, 3022-3060 (2006).

[11] Gitlin, I., Mayer, M. & Whitesides, G.M.: Significance of Charge Regulation in the Analysis of Protein Charge Ladders; The Journal of Physical Chemistry B 107, 1466-1472 (2003).

[12] Gudiksen, K.L., Gitlin, I., Yang, J., Urbach, A.R., Moustakas, D.T. & Whitesides, G.M.: Eliminating Positively Charged Lysine ε-NH3+ Groups on the Surface of Carbonic Anhydrase Has No Significant Influence on Its Folding from Sodium Dodecyl Sulfate; J. Am. Chem. Soc. 127, 4707-4714 (2005).

[13] Gudiksen, K.L., Gitlin, I., Moustakas, D.T. & Whitesides, G.M.: Increasing the Net Charge and Decreasing the Hydrophobicity of Bovine Carbonic Anhydrase Decreases the Rate of Denaturation with Sodium Dodecyl Sulfate; Biophys. J. 91, 298-310 (2006).

[14] Gitlin, I., Gudiksen, K.L. & Whitesides, G.M.: Peracetylated Bovine Carbonic Anhydrase (BCA-Ac18) Is Kinetically More Stable than Native BCA to Sodium Dodecyl Sulfate; The Journal of Physical Chemistry B 110, 2372-2377 (2006).

[15] Gitlin, I., Gudiksen, K.L. & Whitesides, G.M.: Effects of Surface Charge on Denaturation of Bovine Carbonic Anhydrase; ChemBioChem 7, 1241-1250 (2006).

[16] Mammen, M., Chio, S.-K. & Whitesides, G.M.: Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors; Angew. Chem., Int. Ed. 37, 2754-2794 (1998).

[17] Mack, E.T., Snyder, P.W., Perez-Castillejos, R. & Whitesides, G.M.: Using Covalent Dimers of Human Carbonic Anhydrase II To Model Bivalency in Immunoglobulins; J. Am. Chem. Soc. 133, 11701-11715 (2011).

[18] Mack, E.T., Snyder, P.W., Perez-Castillejos, R., Bilgicer, B., Moustakas, D.T., Butte, M.J. & Whitesides, G.M.: Dependence of Avidity on Linker Length for a Bivalent Ligand-Bivalent Receptor Model System; J. Am. Chem. Soc. 134, 333-345 (2012).

[19] Rao, J.H. & Whitesides, G.M.: Tight binding of a dimeric derivative of vancomycin with dimeric L-Lys-D-Ala-D-Ala; J. Am. Chem. Soc. 119, 10286-10290 (1997).

[20] Rao, J., Lahiri, J., Isaacs, L., Weis, R.M. & Whitesides, G.M.: A trivalent system from vancomycin·D-Ala-D-Ala with higher affinity than avidin·biotin; Science (Washington, D. C.) 280, 708-711 (1998).

[21] Rao, J., Lahiri, J., Weis, R.M. & Whitesides, G.M.: Design, Synthesis, and Characterization of a High-Affinity Trivalent System Derived from Vancomycin and L-Lys-D-Ala-D-Ala; J. Am. Chem. Soc. 122, 2698-2710 (2000).