Nuclear magnetic resonance (NMR) spectroscopy provides information regarding the structure and dynamics for protein, RNA and DNA at the atomic level. These biomolecules may be studied individually or in the presence of ligands or other biomolecules. Determination of the three-dimensional structure of macromolecules and their complexes is vital for rational drug design and expanding knowledge within the field of mechanistic biology. The term “biomolecular NMR” refers to the use of NMR to study biological compounds in vivo or under conditions which best mimic in vivo conditions. Although most cytosolic proteins are relatively easy to study, membrane proteins require lipophilic environments for stability and function and thus are typically studied in micelles, lipid bilayers, cellular membranes, and living cells.
NMR generally lacks the sensitivity to detect useful signals from unlabeled sample, therefore biomolecules are often required to be enriched in 13C and/or 15N for analyses. Deuterium is often incorporated to simplify spectra or to alter relaxation effects so that the necessary spectroscopic information may be acquired. Over the years, advances in isotope-labeling strategies have expanded the size of macromolecules and the types of detailed information available for study.
in vivo Protein Expression
Using isotopically labeled reagents and media for overexpressing protein using prokaryotic or eukaryotic cells remains the most efficient way to produce isotope-enriched protein suitable for NMR analysis.
Sparse Labeling for Protein NMR
Sparse labeling means the expressed protein does not contain adjacent 13C nuclei. This can be accomplished by using selectively 13C-labeled carbon sources for the protein expression, such as glucose, glycerol, and pyruvate.
The study of membrane proteins can be challenging. Incorporating detergents and phospholipids into sample preparation conditions for membrane proteins helps achieve proper solubilization and stability.
Methyl and Amino Acid-Type Labeling
Methyl labeling refers to methods that introduce either 13CH3 or 13CHD2 groups into an otherwise uniformly deuterated protein by incorporating amino acid(s) or precursors to cell culture medium prior to protein induction.
It not surprising that many developments in the field of magnetic resonance are aimed to improve sensitivity. One can blame NMR spectroscopy’s inherently poor sensitivity on the very small population differences between magnetically induced nuclear spin states.
CIL offers a wide array of compounds labeled with 13C, 15N, and 17O for magnetic resonance spectroscopy (MRS) research and magnetic resonance imaging (MRI).