NMR arises from the fact that certain atomic nuclei have a property called “ spin ”

1. Nuclear Magnetic Resonance (NMR) Nucleus of NMR Active atom Static Magnetic Field Generated by the NMR Spectrometer • NMR arises from the fact that certain atomic nuclei have a property called “spin” • “Spin” is caused by circulating nuclear charge and can be thought of as bar magnet that adopts a preferred orientation in the presence of a static magnetic field  NMR spins adopt specific quantitized states; for spin ½ nuclei, the type of nuclei most commonly studied, there are two such states, a and b.  a corresponds to preferred low energy configuration, while b corresponding to the non-preferred high energy configuration.

2. Nuclear Magnetic Resonance (NMR)  Energy can be applied by the NMR spectrometer in the form of an oscillating magnetic field at a precisely defined frequency (and hence energy) that induces transitions from a to b.  Oscillating magnetic field is applied through the probe, which basically consists of a coil surrounding the NMR tube attached to a user controlled oscillating frequency source on the spectrometer console.

3. NMR Chemical Shifts • = physical constant for a given type of nucleus (ratio of magnetic moment and angular momentum) h = Planck’s constant Bo = static magnetic field strength Prediction, based on the fact that all nuclei in the sample are placed in the same magnet, is that all nuclei of one type (1H, 13C, 15N, 31P etc) would have exactly the same NMR frequency

4. Ile in D2O Predictions Do Not Match Reality (Acquisition time = 30 s) a g2 d1 HDO b a g1 g2 g1 b d1 Frequency s = chemical shielding tensor

5. Chemical Shielding Shielding arises from the various ways by which electrons “shield” the nuclear spin from the external magnetic field (Bo) Physical mechanism relates to induced circulation of electrons that oppose static magnetic field (Lentz’ Law) Shielding (tensors) can be determined through ab initio calculations. This, however, is computationally expensive, and not routinely applied to large molecules, such as proteins.

6. Classic Approaches to Shielding Local electronic structure; electronegativity of attached groups, bond lengths, bond angles, and conformation (dihedral angles) Anisotropy of local groups (circulating electrons from aromatic rings for example) Hydrogen bonds Electric field effects that polarize bonds

7. Chemical Shielding Trends for Protons Functional Groups Frequency Proteins Frequency

8. Chemical Shifts Can Change Dramatically with Changes in Conformation 8 M Urea No Urea

9. Chemical Shielding & Chemical Shifts Recall Bo field dependence of frequency makes comparison of spectra difficult from one instrument to another Hence, report relative n’s, not absolute n’s Chemical Shift (ppm) = d = npeak = frequency of signal of interest nref = frequency of reference signal IUPAC-IUB Shift Standard for Proteins Sodium-2,2-dimethyl-2- silapentane-5-sufonate (DSS)

10. J-coupling Ha Hb

11. J-couplings in Ile Ile in D2O g2 a d1 b g1 g2 d1 a g1 g1 b

12. NMR Active Nuclei in Biomolecules

13. Sensitivity of NMR a & b spin states will assume a Boltzman distribution Implications: Highest sensitivity w/ higher g & higher Bo

14. 1D 13C Natural Abundance Spectrum of Ile a Ile in D2O (1H Decoupled) (Acquisition time = 4 hr) b g1 g2 d1 a g1 b g2 d1 CO 13C ppm 13C ppm 13C ppm