In-cell NMR Spectroscopy for the Investigation of the Conformation of Macromolecules

Abstract

The non-invasive character of NMR spectroscopy allows researchers to investigate the conformation and dynamics of biological macromolecules in their natural environment, for example the cytoplasm of cells. In the past we have used in-cell NMR investigations to study several proteins as well as the behavior of telomeric DNA in different cellular systems. We will show data on two different systems: The telomeric G-overhang is the 3’ single stranded protrusion of double stranded telomeres and consists of repeating d(TTAGGG)n elements. These elements form G-quadruplexes, which however under different in vitro conditions can adopt several different conformations. In order to investigate which of these conformations is the biologically relevant conformation we have injected quadruplexes of different length into Xenopus oocytes or investigated them in oocytes extracts. These investigations revealed that G4 units coexist in two conformations, the hybrid-2 and the 2-tetrad antiparallel basket. In addition, we have used in-cell NMR to investigate the behavior of Pin-1, a peptidyl-prolyl isomerase and show that the protein uses its WW domain to nonspecifically investigate other proteins as potential substrates. This non-specific interaction can be blocked by phosphorylation in the WW domain.