Protein folding studies at equilibrium by NMR spectroscopy

Characterizing the mechanisms by which proteins fold to their native structures is of fundamental interest to biological sciences. Proteins are synthesized as chains of amino acids that need to acquire defined three- dimensional structures in order to generate biological activity. In addition, folding and unfolding processes of various proteins are directly coupled to important biological processes such as regulation of cellular growth and differentiation as well as to targeting of proteins for different cellular locations and degradation. Moreover, the failure of proteins to fold correctly and aggregation of misfolded proteins have been linked to numerous prevalent diseases, including Alzheimer`s disease and type II diabetes. In this project, we will investigate folding of the three-helix bundle protein KIX, a domain of the transcriptional co-activator protein CBP. KIX folds in a three-state manner via a partially folded intermediate, in which an isolated helix is formed while the hydrophobic core of the domain and the remainder of the protein backbone are disordered and devoid of specific interactions. We will focus on nuclear magnetic resonance (NMR) relaxation techniques that can be performed under equilibrium conditions and do not require a perturbation of the folding equilibrium of the protein. These experiments provide site-resolved kinetic information about protein folding processes along with structural information in the form of chemical shifts. To complement the NMR spectroscopic data and provide further insights into the folding mechanism, a variety of mass spectrometric techniques will be employed, as well as protein mutagenesis and other spectroscopic methods. The integration of diverse sets of experimental data will enable a detailed kinetic and structural description of the consolidation of structure along the folding pathway of KIX and provide a comprehensive picture of the folding mechanism of this protein.

In this project of the Austrian Science Fund FWF we characterized the molecular mechanism by which a model protein folds into its native three-dimensional structure. Proteins are the workhorse molecules of cells, as they perform most of the chemical reactions that form the basis of life. Initially, proteins are synthesized as chains of amino acids that need to acquire defined three-dimensional structures in order to generate biological activity. Characterizing the mechanisms by which proteins fold into their native structures thus is of fundamental interest to biological sciences, in particular in light of the fact that the failure of proteins to fold correctly has been linked to numerous prevalent diseases. Using nuclear magnetic resonance (NMR) relaxation techniques in solution we were able to show that our model protein, the KIX domain of the transcriptional co-activator CBP, acquires its fully folded structure via an intermediate state in which part of the native structure is formed. Combination of complementary experimental approaches enabled us to quantitate the amount of residual structure that is present within this folding intermediate. We showed that two (out of three) structural segments of the KIX domain are partially formed in the folding intermediate, while the hydrophobic core of the domain and the remainder of the protein backbone are disordered and devoid of specific interactions. Of note, the experiments that we used do not require the perturbation of the folding equilibrium, which excludes various possible error sources and therefore enables a genuine description of protein folding mechanisms under native conditions. In addition, we characterized the three-dimensional structure of the KIX domain using mass spectrometry. Our experiments showed that in the gas phase, and in the absence of hydration water, structural properties of the KIX domain are preserved; in particular, the order of stabilities of the structural segments of KIX that has been found in solution and in the gas phase are identical. The mass spectrometric data provide direct evidence for the preservation of the native three-dimensional structure of KIX in the gas phase and point towards the structural properties of the folding intermediate as an inherent thermodynamic feature of this protein.