Investigation of protein folding and refolding pathways using optical tweezers
The formation of protein structures is still a puzzling question. The first theories were developed 55 years ago, when Anfinsen et al. demonstrated that proteins can fold by themselves . Additionally, Levinthal and coworkers suggested that an amino acid chain can adapt a high number of possible folds and random searching through the many possible conformations could never happen in a reasonable folding time . Proteins are indeed able to fold on a time scale of milliseconds, which led Levinthal and coworkers to the conclusion that proteins follow a programmed structure formation pathway.
The current theory of the folding funnel describes folding based on an energy landscape model. An unfolded protein chain folds to the native state (global energy minimum) by passing local minima on the folding pathway. These local minima are called intermediates. High-resolution optical tweezers is a well-suited method to shed light on the folding processes of proteins. Using this method, the molecule of interest can be unfolded and refolded several times by successively stretching and relaxing the protein. Unfolding of a protein reveals on the one hand the stability of the protein determined by the unfolding force; on the other hand, unfolding intermediates which show stable intermediate structures on the unfolding pathway can be detected. In the refolding process of a completely stretched amino acid chain, refolding intermediates can be populated and be further investigated in the terms of kinetic and structural characterization. This information gives great insights into the folding pathway in terms of folding seeds and misfolded structures, which can accelerate or delay protein folding, respectively.
The protein with which we are currently investigating in terms of unfolding and refolding ability and pathways is the nucleotide binding domain of bacterial Hsp70 (NBD of DnaK). NBD is composed of two lobes, lobe I (blue) and lobe II (red). These two lobes are connected with a c-terminal helix (gray). Adenosine nucleotides (yellow) can be bound in an active site located mainly in lobe II. We can show that NBD can unfold and refold readily in the experimentally accessible time. The unfolding pathway of NBD follows a hierarchic path and can be modulated by the binding of adenosine nucleotides into the binding pocket of NBD. The modulation results in a shift of the unfolding hierarchy . The refolding of NBD is not dependent on the presence of nucleotide and follows always the same pathway to the natively folded protein.
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3. Bauer, D. et al. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK. Proc. Natl. Acad. Sci. U.S.A. 112, 10389–10394 (2015).