NMRFAM Experiments and Capabilities

The following experiments have been implemented and optimized for routine use at NMRFAM. If you are interested in other experiments or require specific forms of these experiments (such as gradient coherence selection, specific TROSY version, selective excitation, etc.), please contact our staff for more information. Some specialized experiments are only available on certain spectrometers or require staff assistance for setup and data acquisition.

We are happy to install and test published pulse sequences upon request. If you are developing novel pulse sequences and would like to test your experiments at NMRFAM, please contact our staff. We have a range of samples and instrument configurations that may be useful for testing and optimization and can assist with dissemination.

Experiments optimized for 13C,15N-labeled proteins of small/medium size:
Assignment
2D HSQC experiments (1H-13C and 1H-15N)
Triple resonance 3D experiments for backbone assignments
Triple resonance 3D experiments for aliphatic side chain assignments
2D and 3D experiments for aromatic side chain assignments

Structure
3D NOESY experiments (1H-13C-1H and 1H-15N-1H) for extracting proton-proton distance restraints
1H-13C and 1H-15N experiments for extracting residual dipolar couplings
2D/3D 13C and/or 15N filtered/edited NOESY experiments for extracting intermolecular contacts in protein complexes

Dynamics
2D 1H-15N-HSQC based experiments for 15N-relaxation studies (15N-T2, 15N-T1rho, 15N-T1, 15N-1H-NOE, 15N-CPMG relaxation dispersion)
2D/3D ZZ-exchange experiments (1H-13C-1H and 1H-15N-1H)
2D CEST experiments for measuring chemical exchange

Experiments optimized for 2H,13C,15N-labeled proteins of medium/large size:
Assignment
2D 1H-15N HSQC TROSY experiments
TROSY-based triple resonance 3D experiments for backbone assignments

Structure
3D 1H-15N-1H NOESY TROSY-HSQC experiments for extracting proton-proton distance restraints
2D TROSY-based 2D 1H-15N experiments for extracting residual dipolar couplings

Dynamics
2D 1H-15N-HSQC-TROSY based experiments for 15N-relaxation studies (15N-T1rho, 15N-T1, 15N-1H-NOE, 15N-CPMG relaxation dispersion)
TROSY-based 2D 1H-15N ZZ-exchange experiments
2D CEST experiments for measuring chemical exchange

Experiments optimized for methyl-labeled proteins of medium/large size:
Assignment & Structure
2D 1H-13C HMQC experiments
2D/3D COSY- or TOCSY-based experiments for methyl assignments
3D/4D NOESY HMQC experiments for extracting proton-proton distance restraints

Dynamics
2D HMQC-based methyl 1H and 13C relaxation experiments
2D/3D HMQC-based ZZ-exchange experiments
2D CEST experiments for measuring chemical exchange

The following experiments to study nucleic acids have been implemented at NMRFAM. Our staff has experience working with both isolated RNA and RNA/protein complexes. If you are interested in other experiments or require more detailed information, please contact our staff . We are happy to install and test published pulse sequences upon request.

If you are developing novel pulse sequences and would like to test your experiments at NMRFAM, please contact our staff. We have a range of samples and instrument configurations that may be useful for testing and optimization and can assist with dissemination.

1H, 13C, 15N and 31P NMR experiments optimized for nucleic acids available at NMRFAM include:

All standard 1D and 2D experiments for 1H
2D HSQC (1H-13C and 1H-15N)
3D NOESY (1H-13C-1H and 1H-15N-1H)
3D TOCSY and COSY (1H-13C-1H)
2D and 3D 13C and 15N filter/edit NOESY experiments

Through-hydrogen bond experiments (HNN-COSY and HNN-TOCSY)
1H-13C and 1H-15N RDCs
1H-13C-15N tiple resonance experiments (HCN)
1D, 2D and 3D 31P (H-P and HCP)

Metabolomics is the qualitative and quantitative study of small molecules (<1500 Da) in living systems. These types of experiments are used for establishing diagnostics, monitoring the effects of medical intervention, quality control and detection of food additives or alterations, and analysis of biochemical pathways and their perturbations resulting from mutations, aging, diet, exercise, drug treatment, change of lifestyle or other conditions. At NMRFAM we have expertise in analyzing a wide range of samples, including parmesan cheese extract, avian liver, cancer cells, and more.

NMR and mass spectrometry (MS) metabolomics studies are highly complementary. MS metabolomics has higher sensitivity and can thus detect a larger number of small molecule metabolites, while NMR has advantages in sample preparation and inherent quantitation. NMR chemical shifts provide information on functional groups and is exquisitely sensitive to molecular structure. This makes NMR data useful for resolving the identity of compounds with identical masses, including those with different isotopomer distributions. NMR is non-destructive and metabolomics studies can be performed using either solution or solid-state NMR instrumentation, enabling observation and quantification of the more abundant compounds present in biological fluids, cell extracts, and tissues without the need for elaborate sample preparation or extraction. NMR also offers advantages for compounds that are difficult to ionize or require derivatization for MS.

One-dimensional 1H NMR spectra are particularly useful for metabolomic studies. This technique is easily automatable, reliable, and relatively fast. The chemical information contained in a single proton spectrum can often be enough identify and quantify more than 50 metabolites at a time. The additional resolution gained by 2D NMR (1H-1H and 1H-13C, most often) can be used to overcome the problem of overlapping resonances by spreading the peaks into a second dimension.

Molecular interactions can be studied by NMR for a variety of systems, including interactions of protein or RNA with other macromolecules, lipids, small molecules, or ions. These interactions can be monitored by observing resonances from the macromolecule – in this case the protein or RNA is isotopically labeled and assigned first and addition of ligand leads to chemical shift perturbations that can be used to locate the binding site and determine the affinity. Unassigned macromolecules can still be used to monitor ligand interaction and determine whether binding sites for different ligands overlap, but assignments are required to locate the binding site. Standard 2D or 3D experiments are used to monitor chemical shift perturbation upon binding of ligands to protein or RNA molecules.

NMR is uniquely able to directly detect proton binding due to the change in electrostatic environment and consequent chemical shift perturbation in the vicinity of the residue that is protonated or deprotonated. NMR-monitored pH titrations can determine pKa values for individual residues independently of any coupled binding events or pH-dependent activity, allowing detailed investigation of the microscopic steps in proton-coupled biological processes.

Macromolecular interactions can also be monitored from the small molecule perspective. When small molecule based ligand-detect approaches are used there is no need for isotopic labeling or assignment of the macromolecule. These methods can detect binding through the impact on small molecule relaxation properties or saturation-transfer based approaches. While the exact binding site on the macromolecule is not determined, some of these methods can detect whether multiple small molecules binders compete for binding in the same location.

In NMR titrations, the accuracy of K_d determination depends on the on- and off-rates, with NMR ideally suited for studying weak binding interactions (µM to mM) where on- and off-rates are fast. This makes NMR highly complementary to other methods, most of which are better suited for detecting and quantifying tighter binding interactions. The ability of NMR to detect and locate binding sites for very weak interactions led to the development of fragment-based drug discovery, where a library of small molecular fragments are screened against a target of interest and then medicinal chemistry approaches are used to extend, merge, and modify hit fragments to develop higher affinity binders.

Common 1D small molecule ligand-observe experiments implemented at NMRFAM
1D Proton
1D WaterLOGSY
Saturation-Transfer Difference (STD) NMR

Solid state NMR (SSNMR) spectroscopy is used for studying wide range of biomolecular systems such as crystalline proteins, fibrillar proteins, membrane proteins, as well as concentrated soluble and sedimented protein samples. For SSNMR analysis, isotopically labeled (13C and 15N) protein samples are centrifugated to obtain hydrated pellets that are packed into MAS (magic angle spinning) rotors. The sample in the rotor is spun at magic angle axis, i.e., 54.7o with respect to Bo field (z-axis). The maximum spinning frequency is inversely proportional to the probe rotor diameter, and therefore requires distinct MAS probe for achieving different range of spinning speeds. Protein structural data is obtained by using 13C and/or 1H detected experiments. For 13C detected experiments, typical MAS rotor diameter sizes are 1.6 mm and 3.2 mm with respective sample volumes,10 and 25 ml. Under these conditions 13C detected experiments are acquired with MAS rates of 10 to 35 kHz. On the other hand, 1H-detected experiments require ultrafast and very fast MAS probes with smaller rotor diameters, such as 1.3 mm and 0.7 mm with respective samples volumes, 3 and 1 ml, and MAS rates of 60 to 100 kHz are used. For 1H-diluted or perdeuterated protein samples, 1H detected experiments are acquired using fast MAS (1.6 mm) or ultrafast (1.3 mm) MAS probes using MAS rates of 30 to 60 kHz.

More coming soon!