Overview of Modern High Resolution NMR Spectroscopy High-resolution NMR spectroscopy provides atomic level three-dimensional structural information about proteins and other biomolecules in solution (high resolution NMR spectroscopy is also the basis for spatially-resolved NMR spectroscopy, otherwise known as magnetic resonance imaging or MRI; however the information these two techniques provide is completely different; high-resolution NMR spectroscopy yields signals from defined atoms in molecules; MRI yields an image of a organism or tissue, typically at a resolution of 0.1 micrometers). High-resolution NMR has emerged as an important tool in modern biomedical research because it provides an alternative technique to X-ray crystallography for three-dimensional structure determination and can be applied to proteins and other biomolecules in solution. The technique was initially (1970s to late 1980s) limited in terms of its applicability to relatively small proteins (about 10 kDa and smaller), although advances in spectroscopic techniques, NMR instrumentation, and methods for recombinant protein expression in E. coli (which facilitates the preparation of proteins and other biomolecules with appropriate isotope labeling patterns for NMR; see below) have extended the technique to biomolecules with molecular weights of 40 kDa, and larger
NMR Provides Information on an Atom-by-Atom Basis NMR is a spectroscopic technique where specific signals can be observed from each and every atom in a molecule (even high moleculear weight proteins). The extraction of structural and dynamic information by NMR consequently has two parts: (1) The first, called assignment, involves the identification of which atoms in a molecule give rise to which signals in the NMR spectrum, (2) The second involves measuring, in a site-specific manner, various physical phenomena that are dependent on either the structure or dynamics of the molecule of interest. The actual three-dimensional structure or dynamics of the molecule are then inferred indirectly from the measurement of the structure or dynamic-dependent physical parameters. This distinguishes NMR from X-ray crystallography as a technique for structure determination because multiple types of physical measurements (such as 1H-1H NOE interactions, scalar coupling constants, residual dipolar coulings; see below) may be used to determine the 3D structure, not just diffraction patterns alone, as in X-ray crystallography (this does not imply in any way that NMR structures are any better than those determined than by X-ray; it simply means that there are important differences in ways that NMR and X-ray structures are determined)
NMR Active Istopes and Sensitivity NMR signals arise from the magnetic properties of atomic nuclei (in particular the magnetic moment and angular momentum of the nuclear spin). Not all atomic nuclei possess a magnetic moment and angular momentum, and hence not all nuclei yield an observable NMR signal (general rule is that nuclei with an even nuclear charge and even nuclear mass do not possess nuclear spin). Atoms that do possess a nuclear spin are also are known to differ from one another due to differences in nuclear structure. Atoms that possess a so-called spin ½ nucleus (designated I = ½) are the simplest kind to study and the kind that are commonly used in high resolution NMR studies (spin ½ nuclei are characterized by an odd nuclear charge and an odd nuclear mass). Atoms that possess spin quantum numbers greater than ½ are also common, although they are generally not well-suited for high resolution NMR studies because by definition such nuclei also possess a nuclear property known as an electric quadropole moment, which causes these signals to be very broad, and thus difficult to study. A partial listing of the NMR properties of nuclei commonly encountered in biomolecules is shown below.
|Isotope||Natural Abundance||(rad Hz T-1)||spin (I)|
|1H||99.985%||26.75 x 107||½|
|2H||0.02%||4.12 x 107||1|
|13C||1.1%||6.73 x 107||½|
|14N||99.63%||1.93 x 107||1|
|15N||0.37%||-2.71 x 107||½|
|17O||0.04%||-3.63 x 107||-5/2|
|31P||100%||10.83 x 107||½|
As shown, hydrogen (1H or proton) and phosphorous (31P) atoms both possess a nuclear spin suitable for NMR studies (I = ½) and are highly naturally abundant. On the other hand, the naturally abundant isotopes of nitrogen, carbon, and oxygen are not suitable for high resolution NMR studies (12C is NMR-inactive, 14N has spin 1, and 16O is NMR-inactive), and thus cannot be studied. The general solution to this problem now in widespread use is to prepare biomolecules that are enriched with isotopic variants with the desired NMR properties. Thus, in the case of nitrogen and carbon, samples are prepared with the stable (non-radioactive) spin ½ nuclei 15N and 13C, respectively. This is most readily accomplished by recombinant expression of the protein in E. coli cultured on minimal medium (typically M9 medium, or a variant thereof; see below) containing 15NH4Cl and 13C uniformly labeled D-glucose as the sole nitrogen and carbon sources, respectively. The substitution approach is not applicable to oxygen atoms since 17O, the other stable NMR-active oxygen isotope, possesses a spin greater than ½. In summary, the most commonly studied NMR isotopes in biomolecular NMR are 1H, 31P, 13C, and 15N although studies of 15N and 13C are really only practical when samples are isotopically enriched in these isotopes (some studies that employ naturally abundant 13C and 15N are performed and reported in the literature; these studies are limited however because the effective concentration with which these spins are observed is scaled down by their relative natural abundance; as an example, natural abudance 13C NMR studies of a 1 mM protein solution can in effect be thought of as being conducted with an effective concentration of 0.01 mM; this concentration is low, but adequate for some kinds of studies, but not for all, especially those, such as some triple-resonance experiments (see below), that require coherent labeling of 15N and 13C spins).
The other important point to be made about the different NMR active isotopes is that they differ in terms of the intrinsic sensitivity. This is due to the differences in the intrinsic properties of the nuclei, in particular by a physical constant for each of the different atom types, known as γ, which is simply the ratio of the magnetic moment to angular momemtum for a given atom type. The γ value for the atom types studied in biomolecular NMR (1H, 31P, 13C, and 15N) have relative values of approximately 1:0.41:0.25:0.1 (see table above). The larger the magnitude of γ, the greater the population difference will be between the low and high energy quantum states (it is this population difference that ultimately determines the signal intensity) and the higher the frequency with which a particular nucleus will be observed. This is the underlying reason why most modern multidimensional NMR experiments rely on the observation of a 1H signal preferentially over those of other atom types involved, such as 13C or 15N [Back To Top].
Assignment Methodology for Proteins The technique used by chemists to assign the one-dimensional NMR spectrum of small molecules typically includes analysis of the observed chemical shifts (i.e. the position in the spectrum where the signals fall) together with an analysis of the J-couplings (J-couplings can be thought of as weak interactions between NMR spins separated by up to 4 or so chemical bonds). This same technique obviously cannot be applied to proteins and other biomolecules because the number of atoms (spins) typically yields so much overlap in the one-dimensional spectra that only in special cases can assignments be made. The general solution to this problem, which has been emerged over the course of the past twenty years or so, has been to utilize multidimensional NMR methods whereby the signals of interest are spread out into two or more frequency dimensions (although an explanation of the details by which multidimensional spectra are obtained lies beyond the scope of this introduction, it is important to note that it is often the J-couplings between adjacent spins that underlies our ability to record multidimensional NMR spectra).
The concept of multidimensional NMR is illustrated in the figure below where the amide region (backbone NH protons) of a small protein(human ubiquitin, 76 residues) is considered. The one-dimensional NMR spectrum, as shown on the top of the figure, has considerable overlap and does not yield the expected number of discrete signals (one peak for each backbone amide in the protein). The overlap apparent in the 1D 1H spectrum can be overcome by recording a two-dimensional 1H-15N correlation spectrum. This type of spectrum yields a peak for each proton in the molecule directly bonded to a nitrogen atom (see below for a more thorough description of this point). The fact that the positions of the proton and nitrogen signals for a single amide group are uncorrelated enables the signals to be essentially completely resolved from one another. The specific demonstration of this is shown by the red vertical line toward the right hand side of the 2D spectrum. Here there are two peaks clearly separated from one another by virtue of their very distinct 15N chemical shifts (of about 104.5 and 120.5 ppm). This is to be contrasted by the 1D 1H spectrum, shown on the top panel, where a single peak at a 1H ppm value of 6.4 is observed. This peak evidently does arises not from a single amino acid, but two instead, one with a 15N chemical shift of about 104.5 ppm and another with a 15N chemical shift of about 120.5 ppm.
The methods currently used to obtain sequential resonance assignments for proteins and other biomolecules (in particular RNA oligomers) are achieved by [UNDER CONTRUCTION!] [Back To Top].
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