Three-Dimensional Structure of Phoratoxin in Solution: Combined Use of Nuclear Magnetic Resonance, Distance Geometry, and Restrained Molecular Dynamics

The solution conformation of phoratoxin, a 46-residue plant protein, has been investigated by ¹H nuclear magnetic resonance (NMR) spectroscopy. The spectrum is assigned in a sequential manner by a combination of two-dimensional NMR techniques to demonstrate through-bond and through-space (>5 Å) connectivities. A set of 331 approximate interproton distance restraints and six backbone torsion angle restraints is derived from the two-dimensional nuclear Overhauser enhancement and double quantum filtered homonuclear correlated spectra, respectively. These restraints are used as the basis of a structure determination with a metric matrix distance geometry algorithm. A total of eight structures are computed in this manner and subjected to refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of square well effective potentials. The average atomic root mean square difference between the final eight structures and the mean structure obtained by averaging their coordinates is 1.7 ± 0.5 Å for the backbone atoms and 2.1 ± 0.5 Å for all atoms. The overall shape of phoratoxin is that of the capital letter L, similar to that of crambin and α₁-purothionin, with the longer arm comprising two α-helices at an angle of ~140° to each other and the shorter arm a mini-antiparallel β-sheet and a loop made up of two turns and a strand.