Revealing functional details of molecular machines by NMR spectroscopy

Abstract

Since the development of the nuclear magnetic resonance method, the capability to unravel the dynamical properties and underlying functions of proteins has improved. By studying proteins in solution and utilizing advanced relaxation experiments together with specific probes such as methyl labeling, I have, in this thesis, investigated various proteins and their interactional partners on a nuclear level. The mitochondrion is unique in eukaryotic cells since it contains its own genome. Because mitochondrial DNA lacks the protected environment of the cellular nucleus, it is arranged in small spherical structures termed nucleoids. This is achieved by the proteins TFAM in humans and Abf2 in yeast; these proteins regulate the packaging, stability, and replication of the mtDNA. They occur in abundance in the mitochondria and are capable of covering the entire genome, keeping it protected. The two proteins comprise primarily of two HMG boxes connected by a linker; the HMG boxes have the ability to bind DNA and together bend the DNA in a sharp 180° turn. The ratio of TFAM or Abf2 to mtDNA is vital for the function of the mitochondria and is maintained by the hexameric Lon protein in humans. Lon is an ATP- dependent protease and has three functional roles in the mitochondria: proteolytic regulation of vital proteins, such as TFAM, degradation of oxidized proteins, and co-chaperoning functions together with other proteins, which makes up the protein quality control machinery (PQC) in mitochondria. The PQC is essential to keep the proteasome in the cell under control, and in addition to analyzing the dynamical features of Lon, I´ve investigated the protein human mitochondrial protease HtrA2. In addition to its protease functions, it also functions as a pro- apoptotic agent and is part of the apoptotic pathway.