Intro to NMR for CHEM 645 we also visited the website: The Basics of NMR by Joseph P. Hornak, Ph.D. The Basics of NMR Brian Bahnson Department of Chemistry & Biochemistry University of Delaware

A Spinning Nucleus Gives a Magnetic Moment unpaired electron spin + Spinning nucleus Natural abundance =  applied magnetic field gyromagnetic ratio photo frequency Nuclei Unpaired ProtonsUnpaired NeutronsNet Spin  (MHz/T) 1H1H 101/ H2H P 101/ Na 123/ N 011/ C 011/ F 101/240.1 Tesla

For I = ½ nuclei have two spin states M = -1/2 M = +1/2 The energy gap between states is small and corresponds to radio frequency waves E = h Planck’s constant

The energy gap gives rise to a slight excess of the +1/2 state - N S direction of applied static magnetic field +  E = h -1/2 +1/2 E N upper - /N lower + = e -E/kT Boltzmann Distribution

The Net Magnetization Vector N S A 90º pulse places the net vector along the y-axis

The spin-lattice relaxation time – T1 The return to equilibrium along the z-axis is the T1 For a clearer description click: The Basics of NMRThe Basics of NMR and follow the links – spin physics – T1 processes

The spin-spin relaxation time – T2 Following a 90º pulse The de-phasing of this precession is mediated by the spin-spin relaxation T2 For a clearer description click: The Basics of NMRThe Basics of NMR and follow the links – spin physics – T2 processes

What is actually measured in NMR experiment? FT But due to the T1 relaxation:

Definition of Chemical Shift  = ( - REF ) x10 6 / REF units of ppm

Spin-Spin Coupling is Through Bonds a.k.a. scalar-coupling or J-coupling C A C B HAHA HBHB B0B0 Looking at only H A deshielded shielded  ppm  If H B not there -1/2+1/2 -1/2 J A-B

NOE Effects are Through Space CC CC C C NOE  1/r 6. f (  c ) Correlation time is a combination of rotational motions, molecule size + shape dependent, and intra- molecular motions NOEs are highly distant dependent, seen from 2-5Å

A Protein’s 1 H-NMR Spectra is Complex To assign peaks of proteins, this needs to be spread into a 2 nd or 3 rd dimension.

2-D and 3-D Separates Out Spectra

Two Papers to Read for next Thursday’s Class Ikura et al.Ikura et al., (1989) “A Novel Approach for Sequential Assignment of 1H, 13C, and 15N Spectra of Larger Proteins: Heteronuclear Triple- Resonance Three-Dimensional NMR Spectroscopy. Application to Calmodulin” Biochemistry 29, WuthrichWuthrich, (1989) “Protein Structure Determination in Solution by Nuclear Magnetic Resonance Spectroscopy” Science 243,