Field of structural biology
Nuclear magnetic resonance spectroscopy of proteins (usually abbreviated protein NMR) is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, Angela Gronenborn at the NIH, and Gerhard Wagner at Harvard University, among others. Structure determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.
NMR involves the quantum-mechanical properties of the central core ("nucleus") of the atom. These properties depend on the local molecular environment, and their measurement provides a map of how the atoms are linked chemically, how close they are in space, and how rapidly they move with respect to each other. These properties are fundamentally the same as those used in the more familiar magnetic resonance imaging (MRI), but the molecular applications use a somewhat different approach, appropriate to the change of scale from millimeters (of interest to radiologists) to nanometers (bonded atoms are typically a fraction of a nanometer apart), a factor of a million. This change of scale requires much higher sensitivity of detection and stability for long term measurement. In contrast to MRI, structural biology studies do not directly generate an image, but rely on complex computer calculations to generate three-dimensional molecular models.
Currently most samples are examined in a solution in water, but methods are being developed to also work with solid samples. Data collection relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, and measuring the absorption of those signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will absorb different frequencies of radio signals. Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei. This information can be used to determine the distance between nuclei. These distances in turn can be used to determine the overall structure of the protein.
A typical study might involve how two proteins interact with each other, possibly with a view to developing small molecules that can be used to probe the normal biology of the interaction ("chemical biology") or to provide possible leads for pharmaceutical use (drug development). Frequently, the interacting pair of proteins may have been identified by studies of human genetics, indicating the interaction can be disrupted by unfavorable mutations, or they may play a key role in the normal biology of a "model" organism like the fruit fly, yeast, the worm "C. elegans", or mice. To prepare a sample, methods of molecular biology are typically used to make quantities by bacterial fermentation. This also permits changing the isotopic composition of the molecule, which is desirable because the isotopes behave differently and provide methods for identifying overlapping NMR signals.
Sample preparation.
Protein nuclear magnetic resonance is performed on aqueous samples of highly purified protein. Usually, the sample consists of between 300 and 600 microlitres with a protein concentration in the range 0.1 – 3 millimolar. The source of the protein can be either natural or produced in a production system using recombinant DNA techniques through genetic engineering. Recombinantly expressed proteins are usually easier to produce in sufficient quantity, and this method makes isotopic labeling possible.
The purified protein is usually dissolved in a buffer solution and adjusted to the desired solvent conditions. The NMR sample is prepared in a thin-walled glass tube.
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 electronic 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 that correlate the frequencies of distinct nuclei are performed. 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, the magnetic field of the spectrometer, and 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, with a 15N labelled protein, one signal is expected for each nitrogen atom in the back bone, with the exception of proline, which has no amide-hydrogen due to the cyclic nature of its backbone. Additional 15N-HSQC signals are contributed by each residue with a nitrogen-hydrogen bond in its side chain (W, N, Q, R, H, K). 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.
Resonance assignment.
In order to analyze the nuclear magnetic resonance data, it is important to get a resonance assignment for the protein, that is to find out which chemical shift corresponds to which atom. This is typically achieved by sequential walking using information derived from several different types of NMR experiment. The exact procedure depends on whether the protein is isotopically labelled or not, since a lot of the assignment experiments depend on carbon-13 and nitrogen-15.
Homonuclear nuclear magnetic resonance.
With unlabelled protein the usual procedure is to record a set of two-dimensional homonuclear nuclear magnetic resonance experiments through correlation spectroscopy (COSY), of which several types include conventional correlation spectroscopy, "total correlation" spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY). A two-dimensional nuclear magnetic resonance experiment produces a two-dimensional spectrum. The units of both axes are chemical shifts. The COSY and TOCSY transfer magnetization through the chemical bonds between adjacent protons. The conventional correlation spectroscopy experiment is only able to transfer magnetization between protons on adjacent atoms, whereas in the total correlation spectroscopy experiment the protons are able to relay the magnetization, so it is transferred among all the protons that are connected by adjacent atoms. Thus in a conventional correlation spectroscopy, an alpha proton transfers magnetization to the beta protons, the beta protons transfers to the alpha and gamma protons, if any are present, then the gamma proton transfers to the beta and the delta protons, and the process continues. In total correlation spectroscopy, the alpha and all the other protons are able to transfer magnetization to the beta, gamma, delta, epsilon if they are connected by a continuous chain of protons. The continuous chain of protons are the sidechain of the individual amino acids. Thus these two experiments are used to build so called spin systems, that is build a list of resonances of the chemical shift of the peptide proton, the alpha protons and all the protons from each residue’s sidechain. Which chemical shifts corresponds to which nuclei in the spin system is determined by the conventional correlation spectroscopy connectivities and the fact that different types of protons have characteristic chemical shifts. To connect the different spinsystems in a sequential order, the nuclear Overhauser effect spectroscopy experiment has to be used. Because this experiment transfers magnetization through space, it will show crosspeaks for all protons that are close in space regardless of whether they are in the same spin system or not. The neighbouring residues are inherently close in space, so the assignments can be made by the peaks in the NOESY with other spin systems.
One important problem using homonuclear nuclear magnetic resonance is overlap between peaks. This occurs when different protons have the same or very similar chemical shifts. This problem becomes greater as the protein becomes larger, so homonuclear nuclear magnetic resonance is usually restricted to small proteins or peptides.
Nitrogen-15 nuclear magnetic resonance.
The most commonly performed 15N experiment is the 1H-15N HSQC. The experiment is highly sensitive and therefore can be performed relatively quickly. It is often used to check the suitability of a protein for structure determination using NMR, as well as for the optimization of the sample conditions. It is one of the standard suite of experiments used for the determination of the solution structure of protein. The HSQC can be further expanded into three- and four dimensional NMR experiments, such as 15N-TOCSY-HSQC and 15N-NOESY-HSQC.
Carbon-13 and nitrogen-15 nuclear magnetic resonance.
When the protein is labelled with carbon-13 and nitrogen-15 it is possible to record triple resonance experiments that transfer magnetisation over the peptide bond, and thus connect different spin systems through bonds. This is usually done using some of the following experiments, , }, , , and . All six experiments consist of a 1H-15N plane (similar to a HSQC spectrum) expanded with a carbon dimension. In the , each HN plane contains the peaks from the carbonyl carbon from its residue as well the preceding one in the sequence. The contains the carbonyl carbon chemical shift from only the preceding residue, but is much more sensitive than . These experiments allow each 1H-15N peak to be linked to the preceding carbonyl carbon, and sequential assignment can then be undertaken by matching the shifts of each spin system's own and previous carbons. The and works similarly, just with the alpha carbons (Cα) rather than the carbonyls, and the and the contains both the alpha carbon and the beta carbon (Cβ). Usually several of these experiments are required to resolve overlap in the carbon dimension. This procedure is usually less ambiguous than the NOESY-based method since it is based on through bond transfer. In the NOESY-based methods, additional peaks corresponding to atoms that are close in space but that do not belong to sequential residues will appear, confusing the assignment process. Following the initial sequential resonance assignment, it is usually possible to extend the assignment from the Cα and Cβ to the rest of the sidechain using experiments such as HCCH-TOCSY, which is basically a TOCSY experiment resolved in an additional carbon dimension.
Restraint generation.
In order to make structure calculations, a number of experimentally determined restraints have to be generated. These fall into different categories; the most widely used are distance restraints and angle restraints.
Distance restraints.
A crosspeak in a NOESY experiment signifies spatial proximity between the two nuclei in question. Thus each peak can be converted into a maximum distance between the nuclei, usually between 1.8 and 6 angstroms. The intensity of a NOESY peak is proportional to the distance to the minus 6th power, so the distance is determined according to the intensity of the peak. The intensity-distance relationship is not exact, so usually a distance range is used.
It is of great importance to assign the NOESY peaks to the correct nuclei based on the chemical shifts. If this task is performed manually it is usually very