Protein structure and dynamics by NMR: synergy between biochemistry and pulse sequence design

PhD ceremony: Mr. R. Otten, 11.00 uur, Academiegebouw, Broerstraat 5, Groningen

Title: Protein structure and dynamics by NMR: synergy between biochemistry and pulse sequence design

Promotor(s): prof. B. Poolman

Faculty: Mathematics and Natural Sciences

 

Proteins are the molecular machines of a cell and are involved in virtual all cellular processes. To understand the function of these biological macromolecules, it is of critical importance to complement a high-resolution 3-dimensional structure with a detailed insight in its molecular dynamics.

The work described in this thesis focuses on the development of nuclear magnetic resonance (NMR) methods to study protein structure and dynamics, using an approach where novel NMR pulse sequences are designed in concert with specific biochemical labeling patterns. In particular, methyl-containing amino acids are targeted because they are important reporters of protein structure and dynamics. Methodology is described for the residue-specific assignment of methyl-groups and their use in investigating protein dynamics on the micr micro sec time scale. The experiments have been used to study the 34 kDa periplasmic binding protein FepB and the transcriptional activator NtrC. Our results indicate that these methods allow for a comprehensive and cost-effective study of protein structure and dynamics for proteins up to ~40 kDa. Furthermore, we demonstrate the strength of NMR spectroscopy to characterize an important class of proteins, the so-called intrinsically disordered proteins. Their structural characterization is challenging due to the inherent flexibility and NMR is, therefore, the most appropriate technique to study them. A novel experiment to probe the local polypeptide backbone structure is described together with its application to human neuroligin-3. The synergy between developments in biochemistry and NMR methodology are shown to be crucial for the advancement of NMR spectroscopy in the field of structural biology.