Development of solid NMR methods for structural determination of a peptide

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The aim of this research is to establish a solid-NMR method, which enables us to determine a 3D structure of a fully ^<13>C-labeled biomolecule. In the first year, we developed a new method and modified our NMR machine as follows:(1) Development of ^1H-^<13>C dipolar assisted rotational resonance (DARR)We developed a method enabling us to determine a large number of distances by one 2D experiment. Such method has long been desired but not yet been realized. Since DARR deos not apply rf irradiation to the observed spins, the mixing time for distance measurement is limited by generally long spin-lattice relaxation times. This enables us to measure longer distances. Further, we showed that DARR has many features suitable for application to a multiply isotope-labeled sample, such as, observation is done unedr high-resolition conditions.(2) Installation of low-temperature observation apparatusFor acculate determination of a molecular structure, suppression of local molecular motion by lower … More ing the sample temperature is prerequisite. We bought a recently developed low temperature NMR observation system and also a liquid-nitrogen recondensation system. These systems were combined with our NMR system to realize safe and stable long low-temperature experiments.In the second year, we applied the DARR method to determine the structure of the eledoisin-related peptide which is a neurotransmitter. A fully ^<13>C and ^<15>N labeled peptide sample conposed of six residues (K-F-I-G-L-M), which is the common C-terminal sequence of the tachykinin family, was synthesized by using a solid synthesis procedure. The structural determination was done as follows, 81 relevant C-C distances were obtaned from the 2D ^<13>C-^<13>C distance-correlation experiment with DARR. These distances were fed into the X-Plor program developed at Yale University and a 3D structure was successfully obtaned based on the 81 distance constraines. Further, the four φ dihedral angles of the peptide main chain were determined and fed also into the X-Plor program as angle constrains. It was shown that the precision of the structure becomes better by adding these dihedral angle constraints. To summarize, in this project, we can establish the solid-NMR method enabling us to determine a 3D structure of a peptide molecule with reasonable acculacy by a few numbers of 2D solid-NMR experiments using one multiply labeled peptide sample. Less