 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 Static Magnetic Field Generated by the NMR Spectrometer Nucleus of NMR Active atom  NMR spins adopt specific quantitized states; for spin ½ nuclei, the type of nuclei most commonly studied, there are two such states,  and .   corresponds to preferred low energy configuration, while  corresponding to the non-preferred high energy configuration. 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  to .  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. Nuclear Magnetic Resonance (NMR)

NMR Chemical Shifts  = physical constant for a given type of nucleus (ratio of magnetic moment and angular momentum) h = Planck ’ s constant B o = 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 ( 1 H, 13 C, 15 N, 31 P etc) would have exactly the same NMR frequency

Predictions Do Not Match Reality  = chemical shielding tensor Frequency Ile in D 2 O 11 22 11   HDO   11 22 11 (Acquisition time = 30 s)

Chemical Shielding Shielding arises from the various ways by which electrons “ shield ” the nuclear spin from the external magnetic field (B o ) 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.

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

Chemical Shielding Trends for Protons Functional Groups Proteins Frequency

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

Chemical Shielding & Chemical Shifts Recall B o field dependence of frequency makes comparison of spectra difficult from one instrument to another Hence, report relative ’ s, not absolute ’ s Chemical Shift (ppm) =  = peak = frequency of signal of interest ref = frequency of reference signal IUPAC-IUB Shift Standard for Proteins Sodium-2,2-dimethyl-2- silapentane-5-sufonate (DSS)

J-coupling Ha Hb

J-couplings in Ile Ile in D 2 O 11 22 11     11 22 11 11

NMR Active Nuclei in Biomolecules

Sensitivity of NMR  &  spin states will assume a Boltzman distribution Implications: Highest sensitivity w/ higher  & higher B o

1D 13 C Natural Abundance Spectrum of Ile Ile in D 2 O ( 1 H Decoupled) (Acquisition time = 4 hr)   11 22 11 13 C ppm   11 11 22 COCO