Accurate estimation of inter-atomic distances in large proteins by NMR.

Recently a method was proposed which permits the extraction of the exact interatomic distance information from the measurement of the evolution of a single cross-peak relative to the mixing time in a NOESY experiment. This is performed through a careful multi-exponential analysis allowing the extraction of the relaxation parameter, and, consequently, the inter-proton distance. We investigate in the present paper whether this technique, already evaluated theoretically, can be used in a real experimental case. We have recorded and analyzed a set of 56 NOESY experiments on a lysozyme sample. Some 81 nOe build-up curves obtained from these data were analyzed in terms of distance. It is shown that the correlation between the measured distances and the reference distances obtained from crystallographic studies, is quite good. An accuracy of the order of 10% is obtained.

Proton resonance assignments and secondary structure of bovine angiogenin.

Angiogenins are 14-kDa proteins able to induce blood vessel growth in various preparations and are thus thought to be involved in the development of solid tumors. Angiogenins have significant similarities with extracellular ribonuclease and possess a characteristic nuclease activity against large RNA molecules. These proteins are also able to induce second-messenger pathways. We have undertaken the determination of the three-dimensional structure of bovine angiogenin by using nuclear magnetic resonance (NMR) spectroscopy. Since this protein was directly purified from cow milk, it was not possible to enrich angiogenin with 13C or 15N isotopes. However, extensive use of two-dimensional and three-dimensional proton NMR experiments enabled us to identify all but four spin systems and to assign all corresponding proton resonances. Identification of most backbone-backbone nuclear Overhauser enhancements led to the characterization of the secondary structure elements of the protein. Comparison with the structure of ribonuclease A and analysis of the location of conserved residues confirmed that the two molecules have very similar structures.