This Thesis describes the application of conventional 13
C and 1
H high resolution Fourier Transform Nuclear Magnetic resonance (HR FT NMR) to Tobacco Mosaic Virus (TMV) and its protein oligo- and polymers and some other large
biological systems. The rod-like (TMV) consists of 2200 identical protein subunits protecting one RNA chain (molecular weight 42 x 10 6
). The most important protein oligo- and polymers are the trimer (molecular weight ≈50,000), rod-like polymer (molecular weight>10 6
) and the double disk-like oligomer (molecular weight ≈0.6 x 10 6
). This study could be carried out because these large biomolecules exhibit internal mobility. Apart from rotation of (TMV) and protein oligo- and polymers themselves several types of such internal mobility can be distinguished: rotational motions about carbon-carbon
bonds in the polypeptide chain (backbone and side chains) within the protein subunits characterized by a rotational diffusion correlation time τ g
< 5 x 10 -10
s, assumed to correspond to small-amplitude rotation extending over ≈40° ; translational and rotational motions of protein subunits within the protein oligomers about one or (more probably) two mutually perpendicular
axes with a correlation time τ p
< 4 x 10 -8
s, temperature dependent rotational motions over a full 2πangle of both backbone and side chain about at least two carbon-carbon bonds of the section 57 to 62 of the polypeptide chain in the virus and of the section 57 to 62 and in the section 90 to 120 in the double disk-like oligomer on a time scale < 10 -7
s, The section 90-120 is known to constitute the RNA binding site. From the effect of proton binding to the rod-like polymers on internal mobility, it turns out that at least one aspartic, one arginine and probably a glumatic acid are involved in the anomalous titration behaviour of (TMV) protein rod-like polymers. These amino acids probably belong to the so called carboxyl cage which, based in our
results, is expected to be hydrophobic. After the addition of the first proton the carboxyl cage is still incomplete, a result which follows from the fact that protein subunits with one bound proton, are still detectable.
Based on the finding that protein subunits are mobile, it is shown that the stability of the double disk-like oligomer solely arises from entropy increase upon shielding of hydrophobic protein surfaces from water during the protein polymerization process to double disks, no specific protein-protein interactions being present. The positive ΔH and ΔS for this polymerization process led us to conclude that, if enough water is removed from the double disk protein surface, the double disk destabilizes and dissociates. From this conclusion a model for the assembly of (TMV) from double disk and (TMV) RNA has evolved.
The first step in the assembly process, the formation of the initiation complex, is based on the double disk which specifically recognizes an RNA region. Summarizing, the model implies, that recognition takes place with an intact RNA hairpin; that the double disk dissociates at least at that surface which is approached by the RNA hairpin because of diminished water contact; that the heat released during double disk dissociation, because of the positive ΔH, is available for melting the RNA hairpin; that the initiation complex then can be completed and, finally, that the specificity arises from size and stability of the RNA hairpin, i.e. its secondary structure. It is reasonable to suppose that initiation is completed with both 3' and 5' end of RNA protruding from different sides of the initiation complex. The elongation process can be described similar as the formation of the initiation complex.
Also a simple model for TMV dissociation, under physiological conditions in protein and RNA upon entering the plant cell, is presented. It is suggested that (TMV) dissociates when passing the cytoplasmic menbrane.
In a small excursion to other large biological systems (plant viruses, phages, ribosomes) we show that the type of expirements described in this Thesis can be extended to many large biological systems. In this way proteins can be studied in their natural environment, close to the in vivo
situation. Finally this Thesis shows that it is relatively simple to enrich these systems with stable isotopes against low material costs. For 13
C NMR measurements (TMV) was enriched with 13
C up to 12-15%.