Nuclear Magnetic Resonance Spectroscopy of Proteins - Data Collection

Data Collection

Protein NMR utilizes multidimensional nuclear magnetic resonance experiments to obtain information about the protein. Ideally, each distinct nucleus in the molecule experiences a distinct chemical environment and thus has a distinct chemical shift by which it can be recognized. However, in large molecules such as proteins the number of resonances can typically be several thousand and a one-dimensional spectrum inevitably has incidental overlaps. Therefore multidimensional experiments are performed which correlate the frequencies of distinct nuclei. The additional dimensions decrease the chance of overlap and have a larger information content since they correlate signals from nuclei within a specific part of the molecule. Magnetization is transferred into the sample using pulses of electromagnetic (radiofrequency) energy and between nuclei using delays; the process is described with so-called pulse sequences. Pulse sequences allow the experimenter to investigate and select specific types of connections between nuclei. The array of nuclear magnetic resonance experiments used on proteins fall in two main categories — one where magnetization is transferred through the chemical bonds, and one where the transfer is through space, irrespective of the bonding structure. The first category is used to assign the different chemical shifts to a specific nucleus, and the second is primarily used to generate the distance restraints used in the structure calculation, and in the assignment with unlabelled protein.

Depending on the concentration of the sample, on the magnetic field of the spectrometer, and on the type of experiment, a single multidimensional nuclear magnetic resonance experiment on a protein sample may take hours or even several days to obtain suitable signal-to-noise ratio through signal averaging, and to allow for sufficient evolution of magnetization transfer through the various dimensions of the experiment. Other things being equal, higher-dimensional experiments will take longer than lower-dimensional experiments.

Typically the first experiment to be measured with an isotope-labelled protein is a 2D heteronuclear single quantum correlation (HSQC) spectrum where "heteronuclear" refers to nuclei other than 1H. In theory the heteronuclear single quantum correlation has one peak for each H bound to a heteronucleus. Thus in the 15N-HSQC one signal is expected for each amino acid residue with the exception of proline which has no amide-hydrogen due to the cyclic nature of its backbone. Tryptophan and certain other residues with N-containing sidechains also give rise to additional signals. The 15N-HSQC is often referred to as the fingerprint of a protein because each protein has a unique pattern of signal positions. Analysis of the 15N-HSQC allows researchers to evaluate whether the expected number of peaks is present and thus to identify possible problems due to multiple conformations or sample heterogeneity. The relatively quick heteronuclear single quantum correlation experiment helps determine the feasibility of doing subsequent longer, more expensive, and more elaborate experiments. It is not possible to assign peaks to specific atoms from the heteronuclear single quantum correlation alone.