Saturation transfer double‐difference NMR spectroscopy using a dual solenoid microcoil difference probe

Abstract

An experiment designed to collect a saturation transfer double difference (STDD) NMR spectrum using a solenoid microcoil NMR difference probe is reported. STDD-NMR allows the investigation of ligand-biomolecule binding, with moderate concentration requirements for unlabeled molecular targets and the ability to discern binding events in the presence of non-binding ligands. The NMR difference probe acquires the signals from two different samples at once, and cancels common signals automatically through a mechanism of switching between parallel excitation and serial acquisition of the sample signals. STDD spectra were acquired on a system consisting of human serum albumin and two ligands, octanoic acid and glucose. The non-binding ligand, glucose, was cancelled internally through phase cycling, while the protein signal was subtracted automatically by the difference probe. The proton NMR resonance signal from octanoic acid remained in the double difference spectrum. This work demonstrates that the double difference can be performed both internally and automatically through the utilization of the solenoid microcoil NMR difference probe and STDD-NMR pulse sequence, resulting in a clean signal from the binding ligand with good protein background subtraction and an overall favorable result when compared to the conventional approach.

Saturation Transfer Difference in Characterization of Glycosaminoglycan-Protein Interactions

“…This method is called saturation transfer double-difference (STDD). 9,10 Although primarily conducted as a one-dimensional (1D) NMR experiment, STD can also be applied in two-dimensional (2D) NMR experiments. An example are the studies involving NMR in the heteronuclear single-quantum coherence (HSQC) spectrum 11 in events in which signal overlap of the complexed ligand is too difficult for interpretation through the usual 1D techniques.…”

“…Examples of these events are regeneration of both hard and soft tissues across multiple organ systems. 50–52 These regulatory functions of FGFs have been exploited for therapeutic applications toward certain diseases such as Parkinson’s disease and diabetes. 53 The 23 currently known FGFs exhibit a conserved amino acid sequence.…”

Advances in NMR-Based Metabolomics

“…Apart from the small coil volume, their solenoidal geometry Chapter 8 Advances in NMR-Based Metabolomics provides signal enhancement compared to the Helmholtz geometry coils typically employed in conventional, large sample-volume probes (56). Micro-coil probes with a variety of coil sizes and with single-or dual-volume cells have been developed and their performance tested for metabolite analysis (51,52,57,58). Using susceptibility-matched plugs and zero-susceptibility wire, volume efficiency and improved magnetic field homogeneity have been achieved (52,53).…”

Direct nuclear magnetic resonance observation of odorant binding to mouse odorant receptor MOR244-3

“…Furthermore, as long as the ligand signals do not overlap, this technique is well suited to measuring competitive binding. STD NMR techniques have detected ligand binding to GPCRs by employing a 2.4 x 10 4 molar excess of ligand to protein and only 512 scans [22][23][24][25] . These techniques have been applied to determine ligand/protein binding in platelets 24 and suspension cells 26 .…”