Studies of high molecular weight systems by NMR spectroscopy

Abstract

Proteins are essential components for all living cells. For better understanding of proteins and how they work, it is essential to know their 3D structure as well as how they interact with other molecules and their dynamics. Nuclear magnetic resonance (NMR) spectroscopy is a versatile technique to study biomolecules, most notably proteins, in solution. NMR provides structural information at atomic resolution and, compared to the other techniques with similar resolution, the measurements can be carried out under near-physiological conditions that are meaningful for the investigation of dynamics.

A great number of unnatural amino acids can now be incorporated into proteins both in vivo and by cell-free protein synthesis at genetically encoded positions. Isotopically labelled unnatural amino acids present outstanding probes for site-specific studies of proteins by NMR. In this thesis, the unnatural amino acid 13C-O-tert-butyltyrosine (13C-Tby) was incorporated as an NMR probe to study the proteins IMP-1 and the E. coli single-stranded DNA binding protein (SSB).

IMP-1 is a clinically important metallo-beta-lactamase which catalyzes the hydrolysis of almost all beta-lactam antibiotics. In order to develop inhibitors against this enzyme it is important to understand its structure and dynamics. Pseudocontact shifts (PCSs) provide long-range structural information on biological macromolecules. In Chapter 2, PCSs were measured to study the conformation of an active site loop of IMP-1 and its change upon binding of a ligand. This loop is important as it lines the substrate binding site and contributes to substrate specificity. The results suggest that the binding of an inhibitor induces two different conformations in the loop which are in slow equilibrium between each other.

E. coli SSB is a homotetramer of molecular mass 76 kDa. It binds single-stranded DNA (ssDNA) with high affinity and little sequence specificity in two main binding modes, named (SSB)65 and (SSB)35. Chapter 3 describes studies of ssDNA-SSB complexes at high and low salt concentrations by solution NMR spectroscopy. Furthermore, experiments were conducted to determine the binding polarity of ssDNA on SSB. The results obtained are in broad agreement with the unusual binding polarity reported by the crystal structure of a complex between E. coli SSB and a poly-deoxycytidine oligomer.

In addition to using the tert-butyl group of a Tby residue as an NMR probe, the present thesis introduced a novel chemical tag containing a trimethylsilyl (TMS) group. Chapter 4 demonstrates the ability of the tert-butyl and TMS groups to deliver site-specific information in high-molecular weight systems without any isotope labelling. The approach was illustrated by measuring intermolecular nuclear Overhauser effects (NOEs) in the 95 kDa complex between SSB and ssDNA. Show more