Protein Interactions: Electrostatics and Ligand Binding

Research output: ThesisDoctoral Thesis (compilation)


This thesis deals with Ca2+ binding to proteins, electrostatic interactions in and between proteins as well as inter- and intramolecular interactions. A computer program was developed to determine Ca2+ binding constants from experimental titration data of proteins. We further studied the effect of deamidation in the Ca2+ binding site of Calbindin D9k on the Ca2+ binding and cooperativity and conclude that introduction of b-peptide linkage leads to a reduction of both binding and cooperativity. The binding of Ca2+ to the individual EF-hands of Calbindin D28k and the coupling of Ca2+ binding to oligomerization was studied by analysis of Ca2+ binding at different protein concentrations and computer fitting of data to a 4- or 6-state model. Three different modes of coupling between Ca2+ binding and oligomerization emerge for the studied EF-hands, which allows for interpretation of their position within the intact protein.

The importance of electrostatic interaction for the binding of a highly positively charged peptide to the negatively charged calmodulin was studied. Charge mutations and variations in protein and peptide, together with changes in pH and salt content allow us to experimentally verify the importance of electrostatic interactions. Experimental results were coupled to simulations and it was concluded that the system is surprisingly invariant to changes in the charge state of protein and peptide. This insensitivity is caused by the high charge of the system, which behaves as it is "saturated" with charge, and a charge regulation at lower pH. Furthermore, we find that binding is optimal at physiological conditions and that binding affinity of two oppositely charged binding partners can be increased by screening of electrostatic interactions. We propose a model to explain the data.

Electrostatic interactions can be studied by determining pKa values of titrating groups in proteins. A heteronuclear NMR method was developed to study the charge state of arginine and lysine residues in proteins and to determine pKa values. The method was applied to apo-calmodulin where site-specific pKa values of lysine residues were determined. In another project the pKa values of acidic groups in a variant of protein GB1 in low and high ionic strength was determined using NMR. This allowed for the pH dependence of protein stability to be determined. From this data it can be concluded that the unfolded state contains residual structure. Many of the proton titration curves are significantly non-ideal and to analyze this data we propose new methods to visualize and analyze cooperativity of proton binding.

The conformation of C4BP-binding region of M proteins derived from streptococcus pyogenes was investigated in isolation and in complex with C4BPa-12 using NMR. We conclude that this region adopts a coiled-coil and that it binds in an extended shape in the complex.

The importance of tertiary contacts on secondary structure formation was studied by searching for fragments in the PDB database which can adopt different secondary structure depending on depending on the protein framework. We conclude that secondary structure switching is rare and that there is a strong inherent propensity in structured segments to form only one type of secondary structure.


Research areas and keywords

Subject classification (UKÄ)

  • Physical Chemistry


  • Molecular biophysics, Molekylär biofysik, secondary structure formation, protein GB1, pKa values, pH stability, NMR, electrostatic interactions, M proteins, calbindin D9k, calcium binding, cooperativity, calmodulin, calbindin D28k
Original languageEnglish
Awarding Institution
Supervisors/Assistant supervisor
Award date2005 Sep 23
  • Department of Biophysical Chemistry, Lund University
Publication statusPublished - 2005
Publication categoryResearch

Bibliographic note

Defence details Date: 2005-09-23 Time: 10:30 Place: Centre for Chemistry and Chemical Engineering, room B, Lund Institute of Technology External reviewer(s) Name: Baldwin, Robert Title: Professor Affiliation: Stanford University School of Medicine, USA. ---