Solid-state NMR determination of intra- and intermolecular phoshorus-31-carbon-13 distances for shikimate 3-phosphate and [1-13C]glyphosate bound to enolpyruvylshikimate-3-phosphate synthase

Abstract

Rotational-echo, double-resonance (REDOR) 31P NMR was used to obtain internuclear distances for shikimate 3-phosphate (S3P) and N-(phosphonomethyl)-[1-13C]glycine (glyphosate) bound to 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a 46-kDa enzyme essential for the synthesis of aromatic amino acids in plants and microorganisms. An intermolecular 31P-13C distance of 7.2 A was measured between the phosphate of S3P and the labeled carbon of glyphosate. This means that S3P and glyphosate are in proximity in the binding site of the enzyme. An intramolecular 31P-13C distance of 5.6 A was measured between the phosphonate 31P and the labeled carbon of glyphosate. This distance can be achieved only if glyphosate is completely extended when bound to EPSP synthase.

High-resolution solid-state MAS NMR of proteins—Crh as an example

“…In the latter case, protein-protein complexes, oligomerization during protein folding, or ligand binding to membranes or membrane receptors exemplify conditions where such interactions can occur in a noncrystalline and insoluble environment. As demonstrated in complexes between enzymes and their substrates, 68 membrane peptides, 69 -71 and amyloid fibrils, 72,73 intermolecular contacts can be readily probed by solid-state NMR methods. In these studies, the combined use of mutagenesis, X-ray/NMR structures, or other biophysical parameters were used to place specific isotope labels in the molecular region of interest.…”

REDOR NMR of Biological Solids: From Protein Binding Sites to Bacterial Cell Walls

“…Formation of the glass inhibits crystallization of ice within the binding site and prevents aggregation of the protein. Homogeneity of charge-stabilized binding sites of protein complexes embedded in ionic glasses has been observed in our laboratory for EPSP synthase (Christensen and Schaefer 1993), E. coli glutamine binding protein (Hing et al 1994), and ribulose 1,5-bisphosphate carboxylase (Mueller et al 1995). Distances resulting from inter-and intraligand EPSPS REDOR measurements helped to define the geometry of the two substrates relative to one another.…”

Structural and dynamic studies of proteins by high-resolution solid-state NMR

“…In the latter case, protein-protein complexes, oligomerization during protein folding or ligand binding to membranes or membrane receptors exemplify conditions where such interactions can occur in a non-crystalline and insoluble environment. As demonstrated in enzyme-substrate complexes [52], membrane peptides [53][54][55], and amyloid fibrils [56,57], intermolecular contacts can be readily probed by SSNMR methods. In these studies, mutagenesis, X-ray/NMR structures or other biophysical parameters were used to place specific isotope labels in the molecular region of interest.…”