We study general principles in proteomics and protein biophysics using carbonic anhydrase as our model protein. Our studies are directed towards understanding such issues as the role of surface charge in protein folding and surfactant denaturation, and the nature of enthalpy/entropy compensation in protein-ligand binding.

Electrostatic Effects in Proteins

All proteins contain charged amino acids, both in the interior and on the surface, but the role(s) of these charges is not well described. We focus on the role surface charges, and study how the chemical modification of these residues affects the behavior of the protein. We have recently shown [1] that eliminating the 18 positive charges from the surface of carbonic anhydrase does not affect its folding characteristics. This highly-charge derivative of the protein is more stable to SDS, but less stable to heat, urea, and guanidinium, than is the native protein. We are currently using modeling and simulations to investigate the molecular details behind the reduced stability of the charged derivatives relative to the native protein.

Our other approach to investigating the roles of charged residues on the surfaces of proteins is via protein charge ladders - derivatives of a protein with incremental changes in charge. We use capillary electrophoresis to separate mixtures of charged proteins into peaks of mixtures of regioisomers with equal charge. Using charge ladders, we can study the effects of charge on ligand binding,[2] proton binding, [3] and stability.

Protein-Surfactant Interactions

SDS-PAGE is one of the most ubiquitous tools in proteomics and biochemistry, but the molecular mechanism of the interaction between SDS and proteins is incompletely understood. The relative importance of electrostatics and hydrophobicity, the final structure(s) of the protein-SDS complex, and the reasons behind the fact that nearly all proteins bind SDS in the ratio of ~1 SDS molecule per 2 amino acids are not known. Our current focus is aimed at understanding importance of electrostatics and hydrophobicity of SDS binding to carbonic anhydrase - a model globular protein. We chemically modify the surface lysine residues of CA; each modification removes the charge from the lysine group and adds variable degree of hydrophobicity. We then study the behavior of denaturation of these derivatives in solutions of SDS. We find that removing the positive charge from the lysine groups makes the derivatives more stable to SDS up to some critical number of modifications; above the critical number each additional modification makes the derivatives less stable. Modifications with more hydrophobic groups render the protein less stable to SDS than less hydrophobic groups.

Enthalpy / Entropy Compensation

We have used the combination of carbonic anhydrase and benzenesulfonamides as a model system for understanding principles of drug design.[6-9] Our current interests are to use this well-characterized system to probe the nature of enthalpy/entropy compensation [10,11] in protein-ligand interactions, that is, the off-setting (often, perfectly) changes in binding enthalpy and entropy that accompany alterations in ligand structure. We are using a series of systematically varied sulfonamides and isothermal titration calorimetry for these studies.

Select Publications

1. Gudiksen et al "Eliminating Positively Charged Lysine-NH3+ Groups on the Surface of Carbonic Anhydrase Has No Significant Influence on Its Folding from Sodium Dodecyl Sulfate" J. Am. Chem. Soc., 127 (13), 4707 -4714, 2005.

2. Gitlin et al "Significance of Charge Regulation in the Analysis of Protein Charge Ladders" J. Phys. Chem. B, 107 (6), 1466 -1472, 2003.

Figure 1

Figure 2