Partial 13C isotopic enrichment of nucleoside monophosphates: useful reporters for NMR structural studies.

Analysis of the 13C isotopic labeling patterns of nucleoside monophosphates (NMPs) extracted from Escherichia coli grown in a mixture of C-1 and C-2 glucose is presented. By comparing our results to previous observations on amino acids grown in similar media, we have been able to rationalize the labeling pattern based on the well-known biochemistry of nucleotide biosynthesis. Except for a few notable absences of label (C4 in purines and C3' in ribose) and one highly enriched site (C1' in ribose), most carbons are randomly enriched at a low level (an average of 13%). These sparsely labeled NMPs give less complex NMR spectra than their fully isotopically labeled analogs due to the elimination of most 13C-13C scalar couplings. The spectral simplicity is particularly advantageous when working in ordered systems, as illustrated with guanosine diphosphate (GDP) bound to ADP ribosylation factor 1 (ARF1) aligned in a liquid crystalline medium. In this system, the absence of scalar couplings and additional long-range dipolar couplings significantly enhances signal to noise and resolution.

Molecular orientation and conformation of phosphatidylinositides in membrane mimetics using variable angle sample spinning (VASS) NMR.

For many biological molecules, determining their geometry as they exist in a membrane environment is a crucial step in understanding their function. Variable angle sample spinning (VASS) NMR provides a new route to obtaining geometry information on membrane-associating molecules; it has been used here to scale and separate anisotropic contributions to phosphorus chemical shifts in NMR spectra of phosphatidylinositol phosphates. The procedure allows spectral assignment via correlation with isotropic chemical shifts and determination of a family of probable headgroup orientations via interpretation of anisotropic shift contributions. The molecules studied include phosphtidylinositol-4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). A membrane-like environment is provided by a dispersion of alkyl-poly(ethylene) glycols and n-alcohols that forms a field-orienting liquid crystal with a director that can be manipulated by varying the sample spinning axis. The experiments presented indicate that the variable angle sample spinning method will provide a direct approach for assignment and extraction of structural information from membrane-associating biomolecules labeled with a wider variety of NMR active isotopes.