1H AND 31 P NMR STUDIES OF SMALL MOLECULES AND THEIR INTERACTION WITH PROTEINS

Hicham  H.  KHODR

The aims of this thesis are to investigate the use of solid‑state NMR spectroscopy for characterizing the interactions of small molecules with proteins, and to investigate the interaction of small  molecules  with  the iron‑storage protein  ferritin  using  solution‑state NMR spectroscopy.

³¹P NMR spectra of inorganic phosphate (Pi) in Chapter 4 were recorded with varying pH in solid and solution State to study the ionization constant of Pi. The outcome of this study shows that pK_a values obtained from the pH titration of Pi in the solid and solution states is approximately the same and this strengthens the case for employing solid‑state NM to investigate the properties of proteins.

Chemical shift anisotropies, CSA values, of phosphorus compounds, (see Chapter 5), calculated by using the Herzfeld Berger method, which uses side‑band intensities from magic‑angle spinning spectra to evaluate the CSA's, were used as input data to the SPIN program for simulating the experimental spectrum. The agreement between the experimental and theoretical spectra supports the tensor elements of these phosphorus compounds. CSA's obtained by this procedure were plotted against the geometrical parameters (e.g bond lengths (P‑O) and bond angles (O‑P‑O) of phosphorus compounds to establish whether or not a linear correlation exists between these geometrical parameters and the CSA values. The results show that there is no simple correlation.

The interaction between inositol hexaphosphate (1HP) and human haemoglobin (Hb) was examined in Chapter 6 by recording solution state ³¹P NMR spectra of mixed solutions. Broad spectra resulted from the strong interaction between 1HP and Hb. The line widths of these broad resonances were reduced in the solid‑state MAS spectrum. A further decrease in the NMR line width resulted from the application of resolution enhancement methods.

¹H and ³¹P NMR spectroscopy of small molecules has been carried out, in Chapter 7, to study their interaction with ferritin.

There are two general ways in which iron may be taken out of the ferritin core. Firstly a reductant, (e.g. thioglycollic acid, flavine mononucleotide dihydride (FMNH₂), flavine adenine dinucleotide dihydride (FADH₂), may enter the core and reduce Iron (III) at short range, perhaps at the same time chelating the Iron (II) produced. Secondly, a reductant may remain outside the protein and pass electrons into the core. This study shows that only small molecules e.g. (thioglycollic acid, methylamine, methanol) can enter the ferritin core, where iron (III) reduction may take place. Larger molecules, such as flavine mononucleotide (FMN), flavine adenine dinucleotide (FAD), IHP, quaternary n‑butyl‑, n‑propyl‑, ethyl‑, methyl‑amino compounds and tripolyphosphate (TPP), cannot pass through the protein shell. Thus the work supports the idea that long‑range electron transfer through the protein shell is coupled to iron release.