Elucidating the folding mechanism of a multi domain protein by combining quench-flow hydrogen exchange methodology with multidimensional NMR spectroscopy

• Applicant: Professor Dr. Michael Kovermann
• Subject Area: Biophysics ; Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 501283364

Elucidating the folding mechanism of a protein represents one major goal in structural biology. The majority of experimental studies aiming to unravel the folding path of a protein have focused on single domain proteins so far. The fact that about 65 % of the procaryotic and about 80 % of the eucaryotic proteome comprise multi domain proteins corroborates the distinct interest in obtaining spatial- and time-dependent insights into the folding path of proteins possessing higher complexity. Here, a synergistic combination between quench-flow hydrogen exchange and multidimensional NMR spectroscopy will be applied to three domain Adenylate Kinase (AdK) to determine the folding process of this enzyme on a millisecond to second time scale at atomic resolution. Functionally, the catalytic activity of native AdK ensures the maintenance of cellular concentration of adenylate nucleotides and acts for this reason as an eminent model system to study the precise relation between folding and function. The proposed work will include natural ligands and an enzymatic inhibitor in the experimental setup enabling to probe binding features within the folding landscape of AdK. Thus, this project aims to make new contributions regarding the following questions: Does AdK refold in a domain dependent manner? Which lifetime do potential folding intermediates possess? How much native structure is needed to act as a functional enzyme? How does the formation of secondary and tertiary structure accounts for functionality of an enzyme? It can be assumed that the precise knowledge of the folding mechanism of three domain AdK in space and time prepares an excellent ground for a better understanding of folding and function of multi domain proteins in general as well as demanding applications e.g. in protein design can be envisioned.

• DFG Programme: Research Grants