Nuclear Magnetic Resonance Spectroscopy
This technique provides detailed solution structural information about (metallo)biomolecules, either para- or diamagnetic. For smaller proteins (less than ca. 30 kd), 2-dimensional NMR techniques may provide a complete solution structure (i.e., the position of all the atoms). For paramagnetic metallobiomolecules, identification of magnetic nuclei surrounding the paramagnetic metal site can be accomplished [Don Kurtz, UGa].
- Nuclear Magnetic Resonance Spectroscopy
- 1D NMR, one-dimensional NMR
- 2D NMR, two-dimensional NMR
- Nuclear Overhauser Effect Spectroscopy (through space)
- Correlation Spectroscopy (through bonds)
Measured physical quantities
- Structure of molecules in solution, including H-bonding interactions
- Dynamic properties of molecules in solution, including rates
- Sites of binding interactions
- Presence and, sometimes, location of metal centers and infrequently occurring nuclei (e.g., 31P)
Information NOT available, limitations
- Very large molecules cannot be studied in detail and sometimes not at all
- Information on electronic properties of molecules difficult to obtain
- Very floppy molecules provided very limited data (e.g., single strands of DNA, RNA, short peptides)
Examples of questions that can be answered
- How are proteins folded?
- Where are cofactors and substrates bound?
- Which Fe atoms in Fe-S clusters have particular oxidation states?
- Where are the sites of interaction of drugs on proteins and DNA?
- How do proteins and nucleic acids interact?
- What conformational changes occur on binding cofactors, substrates, nucleic acids, etc.
- Can simultaneously provide structural, dynamic, and thermodynamic information
- Applicable to almost all biomolecules of reasonable size
- Provides information across almost the entire molecule
- Probe nuclei can be selectively added or observed (e.g., 113Cd)
- Nuclei close to paramagnetic centers can be observed selectively in some cases
- Requires relatively high concentrations (mM)
- Generally all nuclei of a given type are observed (lacks selectivity for a 'chromophore')
- Not useful for poorly soluble species or very large molecules
- Some protons exchange too rapidly to be observed
- Sometimes long distances between nuclei lead to "blind spots" in structure
- Solvent/solution must be free of other species giving large signals
- Molecules need to be relatively small, <50 kDa and usually <15 kDa
- Salt concentration should be low
- Need ~0.5 mL
- Sequence of polymer (DNA, RNA, protein) should be known