Structure and dynamics of biomolecules: probing muscle regulation, prion protein unfolding, and drug insertion into DNA by nuclear magnetic resonance spectroscopy

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a powerful approach to study the structure and dynamics of macromolecules in a close-to-native solution environment. In the present thesis I present my investigation of protein and nucleic acid structure and dynamics in a wide variety of biological systems using NMR spectroscopy. The center of attention of the Sykes laboratory for the last 35 years has been the role of the Troponin complex in the regulation of muscle contraction. Accordingly, the main focus of this thesis is the study of this important nano-machine, and how its structure and dynamics regulate its biological function. In Chapter II, the perturbation of Troponin C’s structure and dynamics by the attachment of two different bifunctional rhodamine probes is investigated. In Chapter III, the dynamics and position of the bifunctional rhodamine probe when attached on the C helix of Troponin C is studied. In Chapter IV, the structure and dynamics of tryptophan mutants of Troponin C is reported. In Chapter V, the effect of the co-solvent trifluoroethanol on the tryptophan side chain position of mutant F77W of the N-domain of Troponin C is examined. In the following chapter, Chapter VI, the structure and dynamics of a Troponin C – Troponin I chimera is studied using NMR spectroscopy and molecular dynamics simulations to assess the presence or absence of an intrinsically disordered region in Troponin I, and to assess the validity of the flycasting mechanism proposed to regulate muscle contraction. In Chapter VII and VIII, a different topic is introduced. The structural changes occurring during the denaturation process of the bovine prion protein are monitored using NMR spectroscopy to gain insights into the protein misfolding process that causes diseases. In Chapter IX, the structural impact of inserting nucleoside phosphonates into DNA are examined by reporting the NMR structure of a DNA dodecamer duplex containing the modified nucleoside Cidofovir at position 7.