Abstract
Since the functions of biological molecules are closely related to their structures, obtaining the three-dimensional structures is of great importance to the understanding of biology. Currently, the only two methods that can be used for protein structure determination at atomic resolution are X-ray crystallography and NMR spectroscopy.
In this thesis the three-dimensional solution structure of the 1:1 complex between the synthetic peptide ZF-l and zinc determined by IH NMR spectroscopy is presented. The peptide, initially isolated from pig intestines, is identical in sequence to the 30 N-terminal amino acid residues of the human protein Lasp 1 belonging to the LIM domain protein family and coordinates zinc in a Cys Cys-His-Cys motif. LIM domains consist of two sequential zinc-binding modules and the NMR structure of the ZF-l-zinc complex is the first example of a structure of an isolated module. It is found that a single zinc-binding motif exists in LIM domains and establishes the N-terminal half of a LIM domain as an independent folding unit. The structure of the ZF-I-zinc complex is also compared to other zinc-binding proteins.
NMR is an inherently insensitive technique due to the very small difference in population of the stationary states. Novel experimental schemes are presented that improve the sensitivity and resolution in one- and multi-dimensional lH NMR spectra. Using semi-selective inversion pulses during the evolution period the sensitivity and spectral resolution in COSY spectra of peptides and proteins are enhanced by removing the passive couplings to the B-protons from the multiplet fine-structure of the aH-NH crosspeaks. During the detection period increased resolution can be obtained by applying homonuclear semi-selective acquisition modulated (SESAM) decoupling. The SESAM decoupling scheme consists of a train of semi-selective inversion pulses which are applied in a time shared mode during the acquisition of the FIDs. The origin of unwanted axial peaks artifacts in a heteronuclear single quantum coherence (HSQC) experiment is analysed and a method to shift these peaks to the side of the spectrum without extending the experimental time is also presented.
