Thermodynamics of Highly Charged Protein Interactions

Abstract

The primary objective of this master's thesis was to systematically investigate how the net charge and charge distribution of disordered Histone H1.0 peptide variants influence the thermodynamics of their interaction with the fully disordered protein, Prothymosin-alpha (ProTα). This research aimed to provide new insights into the principles governing polyelectrolytic binding between two highly charged, intrinsically disordered proteins.

Using Isothermal Titration Calorimetry, the binding affinities and thermodynamic profiles of four different H1 variants, CV+11, CV+21, CV+32, and the globular domain GD+11 were successfully characterized in various ionic strength buffers. A central finding was the strong linear correlation between the net charge of the H1 variants for both the enthalpy change and the binding affinity. These results suggest that the binding is primarily influenced by electrostatic forces, with the increase in charge density likely leading to a greater release of counterions. The identity of these ions also suggests having a large influence on the observed enthalpy change.

The ITC data were complemented with 1H- 15N HSQC NMR titrations for the CV+11 and CV+21 variants. The NMR spectra showed similar movements in chemical shifts for both variants, providing complimentary insights into the binding site of the interactions.

Popular Abstract

Intrinsically Disordered Proteins (IDPs) are a significant and functionally important class of proteins that challenge traditional structural biology paradigms. They lack a stable, well-defined three- dimensional structure and exist as a dynamic ensemble of different shapes. This is contrasting to the typical view of proteins, which are often described as having one specific structure in the 3D-space. IDPs’ flexibility and dynamic nature make them challenging to study using traditional structural biology methods, like X-ray crystallography or Cryo-EM. These techniques typically rely on a single, rigid structure to produce a clear result and are therefore not suitable methods for studying IDPs. Instead, disordered proteins require solution-state biophysical methods that can capture their structural ensembles and dynamic behaviors.
Isothermal Titration Calorimetry (ITC) and Nuclear Magnetic Resonance (NMR) spectroscopy are two methods that study proteins in solution. ITC is a technique that measures the heat changes that occur during a binding event. This allows for the determination of the full thermodynamic profile, such as binding affinity (KD), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS), of an interaction. NMR is a powerful technique for studying proteins at an atomic level. The method can provide a residue-specific view of a protein's structure, dynamics, and binding interactions.
An example of IDPs is the two highly charged, intrinsically disordered proteins: linker Histone H1.0 (H1) and the nuclear protein Prothymosin-alpha (ProTα). Their interaction with each other is considered an extreme case, as both proteins remain disordered even after they bind to each other. H1 has a net positive charge of +53, while ProTα has a strong negative charge of -44. Their high charge densities, together with their relatively simple protein sequence, make them resemble charged polymers, and their interaction is a key example of polyelectrolytic binding. While H1 is known to bind to nucleosomes in chromatin condensation, the process of organizing DNA into a more compact form, ProTα can act as a chaperone to H1. Chaperones are a type of protein that helps other proteins, and by competitively binding to H1, ProTα helps to increase its mobility in the nucleus.
The thermodynamics of this interaction have proven complex, with a large favorable increase in entropy and unfavorable enthalpy. It is suggested that, just like for charged polyelectrolytes, the binding is driven by the release of ions into the surrounding solution. The ions, bound to the proteins through coulomb attraction, are effectively forced out of the binding interface when the two oppositely charged proteins approach each other. So far, it is not known how the net charge or charge distribution of the complex is influenced and how that changes the thermodynamics of binding.
In this study, ITC and NMR are utilized to investigate how the interaction of ProTα and smaller peptides of H1, with individual net charges, is affected. The central finding of the study was a strong linear correlation between the net charge of the H1 variants for both the enthalpy change and the binding affinity. An increase in the net charge of the H1 variant led to a stronger affinity and a more endothermic reaction, likely due to a greater release of ions upon binding. It was also suggested that the identity of these ions could further complicate the thermodynamic profile of the interaction. The NMR data complemented the study by giving insight into the specific residues involved in binding, with the most charge-dense region of ProTα being the most pronounced.
In conclusion, this research provides new insights into the fundamental principles that govern polyelectrolytic binding between two intrinsically disordered proteins, establishing a valuable basis for further investigation.

• author: Walse, Klara
• supervisor: Birthe Kragelund; Kristofer Modig
• organization: Biophysical Chemistry
• course: KFKM05 20251
• year: 2025
• type: H3 - Professional qualifications (4 Years - )
• subject: Biology and Life Sciences; Chemistry; Technology and Engineering
• keywords: Intrinsically Disordered Proteins, Polyelectrolytic binding, Isothermal Titration Calorimetry, NMR spectroscopy, Biophysical Chemistry
• language: English
• id: 9212254
• date added to LUP: 2025-09-17 10:34:51
• date last changed: 2025-09-17 10:34:51