Nuclear Magnetic Resonance (NMR) Spectroscopy: Structure Determination of Proteins and Nucleic Acids

Nancy Greenbaum, Hunter College and the Graduate Center of the City University of New York, New York, USA
Ranajeet Ghose, The City College of New York and the Graduate Center of the City University of New York, New York, USA

Published online: March 2010

DOI: 10.1002/9780470015902.a0003100.pub2

Nuclear magnetic resonance (NMR) spectroscopy is a powerful biophysical technique that facilitates determination of the three-dimensional structure and interactions of proteins and nucleic acids in solution. A set of NMR experiments identifies spectral signatures corresponding to specific atoms on individual residues in the molecule and determines their order in the primary sequence. Distance and angular relationships are subsequently measured and utilized to supplement standard force-fields used in molecular dynamics (MD)-based protocols to obtain high-resolution structural models. Structure determination is aided by molecular biology and biochemistry to generate samples optimally labelled with NMR-active isotopes; by improvement in the design of NMR instrumentation to allow spectral signatures to be recorded with high sensitivity; by development of efficient techniques to manipulate nuclear spins at the quantum level; and by generation of advanced computer algorithms that allow rapid processing, manipulation and storage of large quantities of NMR data.

Key Concepts:

  • Solution NMR is an excellent spectroscopic technique for the determination of the three-dimensional structure of biomolecules at near-physiological conditions.
  • The abundance of chemical and structural information available from NMR spectra derives from the ability to selectively manipulate specific nuclei by customizable sequences of radio-frequency pulses in a large static magnetic field.
  • NMR signals report on the variety of chemical environments experienced by nuclei in biomolecules through unique spectral signatures.
  • The repertoire of information-rich NMR experiments has been expanded by the ability to enrich biomolecules with NMR-active nuclei taking advantage of advances in molecular biology and biochemistry.
  • Nuclear spins in biomolecules form magnetically coupled networks, with characteristic spectral profiles that can be manipulated to extract angular, orientational and distance information for calculation of three-dimensional structural models.

Keywords: NMR; proteins; nucleic acids; three-dimensional structure

Figure 1. Schematic representation of multidimensional NMR experiments. (a) A hypothetical molecule of two nuclei has a 1D NMR spectrum that is displayed along the diagonal of a 2D spectrum (cyan circles). Correlation of the resonance frequencies of the two atoms leads to off-diagonal cross-peaks (pink circles) that represent a connectivity between the nuclei. The connectivity can derive either from the spatial proximity of the two nuclei or the chemical bonding relationship between them, depending on the pulse sequence used. The two frequency axes, F1 and F2, are typically displayed in units of parts per million (ppm). (b) The transition from two to three dimensions can be appreciated when cross-peaks (green) in the 2D spectrum appear at the same frequency along one of the axes. In the example shown, the three cross-peaks are resolved along one frequency axis (F1), but two of them (shown in green) are degenerated along the second frequency axis (F2). Dispersion of the F2 frequencies along a third axis, F3, now resolves all three cross-peaks. Each cross-peak now has a unique F1–F2–F3 position in the 3D spectrum. (c) Two dimensional 1H–15N correlation spectrum (HSQC) of a protein displaying the protein backbone nitrogen and attached proton chemical shifts.
Figure 2. Two schemes for isotope labelling of nucleic acids. (a) Template-directed synthesis. The template (black lines) is a duplex RNA or DNA molecule with an extended overhang. The overhang sequence is complementary to the DNA or RNA sequence that is desired. The short vertical line represents the transition point between the promoter sequence and the template. During RNA synthesis, the enzyme (red oval) processively synthesizes the product RNA (red line) using the overhang as a template. During DNA synthesis, the enzyme is capable of making only one copy of the product per template. For DNA synthesis, the vertical transition point must be a single ribonucleotide to prime the synthesis. (b) PCR-based approach to isotope enrichment of DNA. A tandem repeat of the desired sequence is prepared with a restriction endonuclease site 5¢ and 3¢ to the desired sequence. The tandem repeat is then propagated by thermal amplification in the manner illustrated. Following the two-step synthesis, the product DNA is a tandem repeat of the desired sequence containing as many as 20 000 copies of the desired sequence. The product DNA is digested with the restriction endonuclease, dividing the product into the individual oligonucleotide duplexes.
Figure 3. Sequence-specific assignment of proteins. The HNCACB experiment (top) correlates the chemical shifts of the 13C and 13C nuclei of a (i) residue and those of the 13C and 13C nuclei of the previous (i–1) residue with the chemical shifts of its (i) 15N and 1HN nuclei. The CBCA(CO)NH (bottom) experiment correlates the chemical shifts of the 15N and 1HN nuclei of a given residue (i) with the 13C and 13C shifts of the preceding (i–1) residue. The nuclei observed in each experiment are shown in green and the J-couplings that allow transfer of magnetization are indicated by red lines – these include 1J(CH)~140 Hz, 1J(NHN)~92 Hz, 1J(CC)~35 Hz, 1J(CC¢)~55 Hz, 1J(NC¢)~15 Hz, 1J(NC)~7–11 Hz and 2J(NC)~4–9 Hz. The flow of magnetization is indicated by the red arrows and is bidirectional for the HNCACB experiment (a so-called out-and-back experiment) and unidirectional in the CBCA(CO)NH experiment (a so-called straight-through experiment).
Figure 4. Sequence-specific assignment of nucleic acids. (a) The sequential assignment of nucleic acids is accomplished by an NOESY experiment using through-space intra- and internucleotide connectivities involving H6/H8 and H1¢ protons in the sequence, as indicated. Intraresidue H6/H8 and H1¢ connectivities and sequential H1¢H6/H8 connectivities, assisted by interresidue H5/H6/H8H5/H6/H8 NOEs (‘basebase’ NOEs, not shown) often permit complete assignment of the nucleotide sequence within helical regions when combined with residue-type assignments indicated in Table 3. (b) Identification of Watson–Crick base pairs is achieved by assignment of characteristic NOE patterns between imino protons of guanosine (G) and uridine (U) and nearby protons of the opposing residues cytidine (C) and adenosine (A), respectively. Arrows depict the NOE connectivities seen.
Figure 5. Orientational and angular constraints from NMR experiments and iterative structure calculation from NMR data. (a) Representative Karplus curve relating measured 3J-couplings to the corresponding dihedral angle. The black curve represents the influence of the backbone dihedral angle on the 3J(HNH)-coupling constant and the red curve depicts the influence of the 1 side-chain dihedral angle on the 3J(HH)-coupling constant. The parametric equations representing the relationships are given by
  math

The second equation corresponds to the H, H1 coupling. (b) Measurement of residual dipolar couplings from coupled HSQC spectra. Two sets of coupled HSQC spectra are shown in isotropic solution (left) and in a medium that leads to weak alignment of the molecule (right). Residual dipolar couplings (D) of both positive and negative signs are seen, leading to increased or decreased splittings of the resonances (J+D). (c) Iterative refinement procedure for NMR structure calculation. Distance, angular and orientational restraints are used as inputs into a calculation protocol termed dynamical simulated annealing. The simulated annealing calculation uses a target function that models the potential energies of chemical bonds, nonbonded energies such as van der Waals forces and angular relationships, in addition to the experimental distance, angular and orientational constraints. Validation is performed at each step in a cycle to check for violations in experimental restraints and deviations from chemical bond lengths and angles commonly found in proteins and nucleic acids. The iterative cycle attempts to refine the structure by input of additional experimental constraints until all possible experimental data are utilized in the calculation.
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 Further Reading
    book Cavanagh J, Fairbrother WJ, Palmer AG, Rance M and Skelton JJ (2007) Protein NMR Spectroscopy: Principles and Practice, 2nd edn. New York: Academic Press.
    Fürtig B, Richter C, Wöhnert J and Schwalbe H (2003) NMR spectroscopy of RNA. ChemBioChem 4: 936–962.
    Güntert P (1997) Calculating protein structures from NMR data. Methods in Molecular Biology 60: 157–194.
    Habeck M, Rieping W, Linge JP and Nilges M (2004) NOE assignment with ARIA 2.0: the nuts and bolts. Methods in Molecular Biology 278: 379–402.
    Herrmann T, Güntert P and Wüthrich K (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics program DYANA. Journal of Molecular Biology 322: 773–784.
    book Krishna NR and Berliner LJ (eds) (1998) Biological Magnetic Resonance, Vol. 16–17, Modern Techniques in Protein NMR. New York: Kluwer Academic/Plenum.
    Prestegard J, Bougault CM and Kishore AI (2004) Residual dipolar couplings in structure determination of biomolecules. Chemical Reviews 104: 3519–3540.
    Sattler M, Schleucher J and Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed filed gradients. Progress in NMR Spectroscopy 34: 93–158.
    Scott LG and Hennig M (2008) RNA structure determination by NMR. Methods in Molecular Biology 452: 29–61.
    Sprangers R, Velyvis A and Kay LE (2007) Solution NMR of supramolecular complexes: providing new insights into function. Nature Methods 4: 697–703.
    Tzakos AG, Grace CR, Lukavsky PJ and Riek R (2006) NMR techniques for very large proteins and RNAs in solution. Annual Review of Biophysics and Biomolecular Structure 35: 319–342.
    Varani G, Aboul-ela F and Allain FH-T (1996) NMR investigation of RNA structure. Progress in NMR Spectroscopy 29: 51–127.
    book Wüthrich K (1986) NMR of Proteins and Nucleic Acids. New York: Wiley Interscience.
    Zidek L, Stefl R and Sklenár V (2001) NMR methodology for the study of nucleic acids. Current Opinion in Structural Biology 11: 275–281.

Related Sub-Topics:

Protein Structure | Nucleic Acid Structure | Nuclear Magnetic Resonance | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism | Nucleic Acids: Structure, Function, Metabolism
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Article Title: Nuclear Magnetic Resonance (NMR) Spectroscopy: Structure Determination of Proteins and Nucleic Acids
 
How to Cite close
Greenbaum, Nancy, and Ghose, Ranajeet(Mar 2010) Nuclear Magnetic Resonance (NMR) Spectroscopy: Structure Determination of Proteins and Nucleic Acids. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0003100.pub2]