Indispensable for characterizing structures, interactions, and functional processes
Instruct has 5 centres offering Solution NMR across Europe. Navigate the map and click on the pins to discover centres near you.
Nuclear Magnetic Resonance (NMR) has evolved as the main technique to obtain structural information at atomic resolution in solution on macrobiomolecules such as proteins and nucleic acids. Nowadays solution NMR is an indispensable enabling technology for determining not only structures of such molecules but also their interactions, even weak and transient, as well as for characterizing functional processes in solution and also directly in living cells. The power of NMR resides in linking structural, dynamic, kinetic and thermodynamic information so as to make it a technique of choice in cutting-edge research in medicine and biology.
Solution NMR within INSTRUCT therefore provides a major approach to obtaining the molecular-level information needed to build networks of interactions responsible for vital cellular processes and to describe them at molecular level. Thanks to the recent hardware and software developments, its applicability ranges from supra-molecular structures to intrinsically unfolded proteins at almost physiological concentrations. Solution NMR offers unique possibilities to study dynamic processes at atomic resolution and over a very wide range of timescales, from picoseconds to hours, including folding mechanisms and transient formation of complexes. A computing grid is developing to help researchers to rapidly and efficiently perform structure calculations exploiting forefront approaches. A web portal, WeNMR (www.wenmr.eu), allows the scientists to have an easy interface where submitting all the data necessary for protein structure determination. With the WeNMR GRID computing any user can have access to state-of-the-art software tools and to extensively validated protocols for their use. In this way, scientists already involved in or wishing to make use of bio-NMR for the structural investigation of protein systems are free from the hassle of setting up, validating and updating their calculation protocols and, at the same time, can save on the cost of computational power.
To get structural information on proteins by solution NMR, isotopically labelled samples (usually 15N and/or 15N/13C) are required. For proteins with MW > 20 kDa, deuteration is also needed. In general, the degree of deuteration required is largely dependent on spectral quality, the size and modularity of the system studied and the types of experiment performed. However, very high levels of deuteration (>90 %) are usually required for proteins with molecular weights of the order of 35 kDa or greater.
The protein sample is usually dissolved in an aqueous buffer (typically ranging from 10 to 50 mM buffer concentration) to avoid pH variations in the biological samples, which can otherwise drastically change chemical shifts during NMR spectra acquisition. Buffer content plays a critical role in protein sample stability. Buffer optimization may be used to improve sample stability and avoid the following issues: slow precipitation; mixture of folded and unfolded protein; aggregation problems; degradation. A screening with several buffers (with different pHs and containing chemicals such as detergents, protease inhibitor cocktails, arginine etc.) is therefore recommended to optimize NMR samples. These buffers usually contain salts (i.e. NaCl or KCl) which often increase protein solubility. Samples high in salt or ionic strength may however yield reduced signal to noise when using cold NMR probes. The use of 3 mm tubes in this NMR probe, instead of the standard 5 mm tube, can alleviate this problem. When available, use of deuterated buffer is preferred.
Samples should be clear of precipitate and particulates and therefore need to be filtered or centrifugated in this circumstance. Samples require deuterium solvent for a lock signal and therefore water protein samples contain approximately 5-10% D2O.
Samples should be slowly transferred into and out of NMR tubes using long pipettes that reach the bottom of the NMR tube to avoid losing liquid to the walls of the tube or adding air bubbles to the sample which can negatively affect the shimming profile.
Standard NMR tubes are 5mm, which should contain at least 500 microliters of protein sample. If a small volume is required, Shigemi tubes, 5mm, use volumes of 270-300 microliters and are made of glass that is matched to the magnetic susceptibility of the solvent to be used. In the high-field cold probes, Shigemi tubes should also be used for more efficient water suppression.
A concentrated solution of a biological macromolecule at high temperature and around neutral pH represents an ideal growth medium for bacteria. Sodium azide and fluoride at less than 50 mM can be good growth inhibitors. Metal chelators (EDTA or EGTA) can also be good suppressor of microbial growth.
Structure determination by NMR typically requires a protein concentration of 0.5 mM or greater, stable for several days at the desired temperature. NMR studies for ligand binding or protein-protein interaction studies require concentrations of at least 0.05 mM.
The acquisition of standard NMR spectra takes from a few minutes (1D spectra) to a maximum of 3/4 days (3D or 4D spectra) for each experiment. A full set of experiments for protein structure determination usually takes about 15-20 days. In the case of relatively small proteins, recent advanced multidimensional data acquisition schemes have been successfully used to reduce experimental acquisition time by up to an order of magnitude or more.
The first NMR experiments acquired on site will be focused on the investigation of protein folding state of user’s sample as well as its aggregation state at the selected NMR protein concentration, acquiring 1D 1H and/or 2D 15N HSQC spectra and 15N backbone relaxation properties. This will take about 5-6 days. These preliminary data will allow to evaluate the time and the experimental conditions (i.e. kind of labelled nuclei, optimal protein, buffer concentration and pH) needed to obtain a high resolution structural determination on the user’s protein. A large number of pulse sequences is routinely available for assignment and structural characterization, and local and global dynamics can be easily estimated.