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].

Acronyms, synonyms

• 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

• Nuclear spin transitions

Information available

• Structure of molecules in solution, including H-bonding interactions
• Dynamic properties of molecules in solution, including rates
• Sites of binding interactions
• Equilibria
• 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.

Major advantages

• 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

Major disadvantages

• 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

Sample constraints

• 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