The University of Pittsburgh Biomolecular NMR Shared Resource
Mission Statement: The mission of Biomolecular NMR Shared Resource is to allow experts and non-experts alike to access the powerful capabilities of NMR spectroscopy to better understand the structures, dynamics, and ultimately functions of biological macromolecules in health and disease. For experts who are already trained in NMR spectroscopy, the Shared Resource provides access to state-of-the-art instruments that are well-equipped for solution and solid-state NMR studies of biological macromolecules. For users who are either still learning NMR, or lack expertise altogether, the Shared Resource also provides project consultation and expert assistance with data collection and data analysis, as well as user training.
Biomolecular NMR: Biomolecular NMR allows one to identify spectroscopic signals arising from individual atoms within biological macromolecules – these signals can be used in a variety of ways to probe macromolecular structure, interactions, and dynamics. Biomolecular NMR can be carried out with the biomolecules in solution, such as a soluble enzyme, or in the solid state, such as an amyloid fibril or a membrane protein. Importantly, the type of information that can be derived from NMR studies of biomolecules varies widely – it could range from determining whether a drug binds at a single site, or multiple sites on a protein, characterizing the equilibrium between different conformational states of a protein in the presence of different ligands that it binds, mapping interfaces in complexes (e.g. protein-protein, protein-nucleic acid, protein-ligand), or determining the structure of a protein or protein complex in solution or the solid state.
Requirements for Biomolecular NMR: There are two requirements for the detection of signals in biomolecular NMR – the first is that the signals must be resolvable in the spectrum recorded – the second is that the signals must be detected with a sufficient signal intensity compared to the background noise (signal-to-noise ratio, or SNR).
To resolve NMR signals, one typically relies on a combination of 1) isotopic labeling with nuclei that are readily detectable by NMR, such as 13C, 15N, and 19F, 2) high field NMR spectrometers, which spread the signals over a wider range of frequencies, and in many, but not all cases, 3) multi-dimensional NMR methods which spread the signals over multiple frequency dimensions. The procedures used for isotopic labeling, as well as recording multi-dimensional spectra, can range from a simple to a complex procedure. The staff of the biomolecular NMR Shared Resource have extensive experience with procedures for isotopic labeling of biomolecules and recording, processing, and analyzing multidimensional NMR spectra are available to assist users.
To achieve a sufficient SNR, preparation of a suitably concentrated NMR sample and an NMR spectrometer with high intrinsic sensitivity are required– as the intrinsic sensitivity of the spectrometer increases, this lowers the concentration of the sample that is required – because the intrinsic solubility of most biological macromolecules is limited and because it is typically costly and labor intensive to prepare large amounts of highly purified isotopically labeled biomolecules, it is clearly advantageous to operate spectrometers that offer the highest possible SNR for a given sample concentration. In consideration of this, the biomolecular NMR Shared Resource operates spectrometers that operate at high magnetic field – this increases the NMR spin polarization and enhances the SNR, regardless of whether one is performing solution or solid state NMR measurements. In addition, each of the solution-based spectrometers that are part of the University of Pittsburgh Biomolecular NMR Shared Resource are equipped with high-sensitivity “cryoprobes”. In a cryoprobe, the sample is still retained in a liquid state at the desired temperature, but the interior the probe, which includes the radiofrequency (RF) electronics for excitation and detection, is cryogenically cooled to approximately 10 K with cold helium gas. Cryo-cooling of the RF electronics significantly lowers the background noise, leading to a dramatic increase in SNR compared to a conventional probe where the RF electronics are at room temperature. The usage of cryoprobes has generally allowed the transition from working with samples with concentrations of 500 – 1000 micromolar to samples with concentrations of 20 – 50 micromolar, and in some cases even lower. This, combined with optimized sample tubes that allow for a working volume of 300 microliters in a typical 5 mm probe, allows for greatly reduced sample requirements compared to conventional probes.
The requirements for solid state NMR depend on the rotor dimensions, but as general guideline the sample amount needed is between 10-50 mg of solid (or wet pellet) material with 10-100 microliters of volume in a magic angle spinning (MAS) rotor. In general, solid state NMR is less automated and the measurements are technically more demanding – nonetheless, many types of analyses are possible with the MAS rotors that are available for use with the 600 wide bore (600-WB) and 750 wide bore (750 WB) solid state NMR spectrometers that are part of the facility.
University of Pittsburgh Biomolecular NMR Shared Resource: The University of Pittsburgh Biomolecular NMR Shared Resource includes four Bruker spectrometers equipped for solution NMR, two each at 600 MHz (600-1 and 600-2) and one each at 700 and 800 MHz (700 and 800, respectively). The Shared Resource has two Bruker spectrometers equipped for solid state NMR, one at 600 MHz (600-WB) and one at 750 MHz (750-WB). The technical capabilities of each of the spectrometers and their main applications can be found on the Resourcessection of this website.