Slide 1: BSB 512
Nuclear Magnetic Resonance Spectroscopy
TTh 2:20 - 3:25 pm
Lecture notes available at http://sos.bio.sunysb.edu/bsb512
Lecture 1: Basics
Lecture 2-4: Protein structure determination
Lecture 5: Relaxation and dynamics
Lecture 6: Lab Session:
Sample preparation.
Pulse Programs
Probe selection.
Tuning.
Shimming
Pulse calibration.
Data collection.
Slide 2: References
http://www.cis.rit.edu/htbooks/nmr
Books
Wuthrich, K. NMR of Proteins and Nucleic Acids
Levitt, MH Spin Dynamics
Cavanagh J. et al. Protein NMR Spectroscopy
Ernst, R. et al. Principles of NMR in One and Two Dimensions
Bax, A Two dimensional NMR in Liquids
Slide 3: NMR Spectroscopy: Some history
1915 Einstein and de Hass - Correlation between magnetic moment and spin angular momentum
1922 Stern and Gerlach - Spins are quantized
1946 Bloch and Purcell - First NMR experiment
Richard Ernst - Fourier transformations
Jean Jeener - Two dimensional NMR - COSY
1976 Richard Ernst - First two dimensional NMR experiment
1986 Kurt Wuthrich - First independent NMR - X-ray comparison
Slide 4: High resolution solution NMR
of proteins
Observe protons (1H)
This differs from x-ray
diffraction where one
determines structure based
on the electron density from
the electron rich atoms
(C, N, O).
Protein is solubilized in water.
Slide 5: High resolution solution NMR
of proteins
Observe protons
Assign proton resonances to
indivdual amino acids. Proton
resonances are often resolved
by differences in chemical
shifts.
Measure intra-residue and
inter-residue proton to
proton distances through
dipolar couplings.
Measure torsion angles
through J-couplings.
Use distance and torsion angle
constraints to determine
secondary and tertiary
structure.
Slide 6: High resolution solution NMR
of proteins
Protons have a property called spin
angular momentum.
They behave like small bar magnets
and align with or against a magnetic
field.
These small magnets interact with
each other. Bo S N S N
Slide 7: 13C and 15N also have spin angular momentum and “interact” with 1H
Slide 8: Bo N H C H C C H H H Magnetization can be
transferred between 1H, 13C and
15N to establish connectivities Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space)
Slide 9: N H C H C C H H H Magnetization can be
transferred between 1H, 13C and
15N to establish connectivities
HNCA
HNCOCA
HNCOCACB etc
HSQC-TOCSY
They all use INEPT tranfers Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space) N H C H C C H H H N H C H C C H H H N H C H C C H H H 3D HSQC - NOESY for
Inter-residue contacts
Slide 10: Concept 1: Some nuclei have non-zero spin quantum numbers. Nuclei with odd mass numbers have half-integer spin quantum numbers.
i.e. 13C, 1H, 31P are spin I = 1/2
17O is spin I = 5/2
Nuclei with an even mass number and an even charge number have spin quantum
numbers of zero.
ie. 12C
Nuclei with an even mass number and an odd charge number have
integer spin quantum numbers.
i.e. 2H is spin I = 1
Electrons also have a spin quantum number of 1/2
Slide 11: e- e- Concept 2: Current passed through a coil induces a
magnetic field. e- e- Concept 3: A changing magnetic field in a coil
induces a current.
Slide 12: Mz Classical picture Concept 4: Placing nuclei with spin I = 1/2 into a magnetic field leads to
a net magnetization aligned along the magnetic field axis.
Slide 13: Bo Large external magnet Net magnetization
aligned along Z-axis
of the magnetic field
Slide 14: Bo B 1 Net magnetization
aligned along x-axis
of the magnetic field
after application of B1
field. The B1 field is produced by a
small coil in the NMR
probe which is placed
in the bore of the large
external magnet.
Slide 15: Bo B 1 NMR probe NMR magnet. e- e-
Slide 16: Concept 5: When the B1 field is turned on, the net magnetization
rotates down into the XY plane Bo z x y
Slide 17: Concept 6: When the B1 field is turned off, the net magnetization
relaxes back to the Z axis with the time constant T1 T1 is the “longitudinal” relaxation time constant
which results from “spin-lattice” relaxation
Slide 18: Exponential Functions y = e -x/t y x y = 1- e -x/t y x Mz = Mo (1- e -t/T1 ) Mz t
Slide 19: z x y Bo Concept 7: Individual spins precess about the magnetic field axis. Precession frequency = Larmor frequency
wo = -g Bo (MHz)
Slide 20: Concept 8: After magnetization is rotated into the xy plane by the B1 field
produced from a pulse through the coil, it will precess in the xy plane. Bo z x y x y
Slide 21: Concept 9: The individual magnetization vectors whirling around in the xy
plane represent a changing magnetic field and will induce a current in the
sample coil which has its axis along the x-axis. y x y
Slide 22: Concept 10: NMR signal is a Fourier transform of the oscillating
current induced in the sample coil y y x - y - x time frequency
Slide 23: Chemical Shifts
Slide 24: Concept 12: Nuclear spins produce small magnetic fields
Slide 25: Concept 13: Electrons are spin I =1/2 particles. They produce small magnetic fields
which oppose the external magnetic field. 1H has a small chemical shift range (15 ppm).
113Cd has a large chemical shift range (300 ppm).
Slide 26: What is a ppm? Ppm = part per million 1H has a small chemical shift range (15 ppm). 1H 13C 15N 400 MHz 100 MHz 30 MHz 1 ppm = 400 Hz
15 ppm = 6000 Hz 15 ppm
Slide 27: a b CH3 C-OH Concept 14: The surrounding electrons shield the nuclear spins from the
larger external Bo field. This results in a reduction in the energy spacing
of the two energy levels and a lower Larmor frequency. This is the
chemical shift. CH3 C-OH frequency
Slide 28: Concept 11: In a frame of reference that ROTATES at the Larmor (precession)
frequency, magnetization that is placed along the x-axis does not move. (It
simply relaxes back to the z-axis via T1 processes.)
Slide 29: a b a b CH3 C-OH 100,010,000 Hz 100,000,000 Hz Reference or carrier = 100,005,000 Hz
Slide 30: Concept 15: The nuclei with different chemical shifts and Larmor frequencies
will rotate around the z-axis at different speeds. T2 is the time constant for
the magnetization vectors to "dephase" in the xy plane. CH3 C-OH frequency reference frequency
Slide 31: N H C H C C H H H Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space) T1 relaxation
T2 relaxation Structure Dynamics
Slide 32: Acquire General One Dimensional Experiment Fourier Transform
t1 -> f1 f1 t1
Slide 33: Acquire General One Dimensional Experiment Fourier Transform
t1 -> f1 f1 t1
Slide 34: Acquire General One Dimensional Experiment Fourier Transform
t1 -> f1 f1 t1 Fourier Transformation
resolves multiple frequencies
that overlap in the time domain
Slide 35: Acquire General Two Dimensional Experiment Fourier Transform
t1 -> f1 and t2 -> f2 f2 t1 t2 f1
Slide 36: Acquire General Two Dimensional Experiment t1 t2 Acquire t1 t2 Acquire t1 t2 Vary t1
Collect a series of 1D spectra
Slide 37: General Two Dimensional Experiment t1 f2 Vary t1
Collect a series of 1D spectra Here, the intensities of and do not
change as a function of the t1 evolution time
Slide 38: General Two Dimensional Experiment t1 f2 Vary t1
Collect a series of 1D spectra Whereas here, the intensity of is modulated as
a function of the t1 evolution time
Slide 39: General Two Dimensional Experiment t1 f2 Transpose and then
Fourier transform in t1 dimension
Slide 40: General Two Dimensional Experiment t1 f2 Transpose and then
Fourier transform in t1 dimension
Slide 41: General Two Dimensional Experiment f1 f2 Projection on f2 gives
original chemical shifts
Slide 42: General Two Dimensional Experiment f1 f2 Projection on f1 yields
new information
Slide 43: General Two Dimensional Experiment 1H chemical shift J coupling 1H chemical shift Dipolar
coupling 1H chemical shift 13C chemical
shift 1H chemical
shift 1H chemical shift