1 modern approaches to protein structure determination (6 lectures) dr matthew crump
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Modern Approaches toProtein structureDetermination(6 lectures)
Dr Matthew Crump
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Two types of angular momentum
• “Normal” or “extrinsic” angular momentum (due to rotational or orbital motion)
• “Intrinsic” or “spin angular momentum” (a property of fundamental particles -- cannot be visualized).
use your right hand to figure out the way the angular
momentum vector points
the direction of the spin angular momentum is indicated by an arrow.
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• The gyromagnetic ratio determines the ratio of the nuclear magnetic moment to the nuclear spin.
• It is a fundamental property of each nuclear isotope
• Fundamental symmetry theorems predict that spin and magnetic moment are co-linear
Gyromagnetic ratio (1)
The gyromagnetic ratio is also
known as the magnetogyric
ratio
=IThis equation tells
us how much magnetism we get for a given spin.
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Quantum Angular Momentum
• If we specify an I value, quantum mechanics restricts us as well to specifying the projection of this vector along only one of the three Cartesian components of I. By convention the z-axis is chosen and Iz is given by
• where m is a second quantum number which can take values m=-I,-I+1,-I+2,..,I. Therefore I z has 2I+1 values.
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ITOT = I(I +1)[ ]1/ 2
h
• In quantum mechanics, angular momentum is quantized.
• The total angular momentum of particles with spin takes the values of the form
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Iz = mh
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• the energy of the state with quantum number Iz is given by
Zeeman splitting
Energy
ground state;
no field
ground state; with field
Zeeman splitting h B/2π
Planck constant
gyromagnetic ratio
• Energy of interaction is given by E=-.B in a magnetic field B. The dot product tells us the energy depends on the size and relative orientation of B and .
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E z = −γhIzBz
• We take B to be along the Z axis, so the dot product becomes E=-zBz (I.e. xBz and yBz = 0
m=-1/2
m=+1/2
m=-1
m=+1
m= 0
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€
E z = −γhIzBz = −γh1
2Bz
m=-1/2
m=+1/2
I=1/2m=-1
m=+1
I=1
m= 0
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E z = −γhIzBz = γh1
2Bz
The Zeeman splitting is therefore
€
hBz
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The gyromagnetic ratio determines how rapidly the Zeeman splitting increases when the magnetic field is increased.
Gryomagnetic ratio (2)
1H 15N 27Al
Note the ordering of the energy levels ( is negative
for 15N)
Note the ordering of the energy levels ( is positive
for 1H)
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Spins I and gyromagnetic ratios for some common nuclear isotopes:
Gyromagnetic ratio (3)
isotope naturalabundance
spin gyromagneticratio /rads–1T-1
1H 99.98% 1/2 267.5×106
2H 0.015% 1 41.1×106
10B 19.9% 3 28.7×106
12C 98.9% 0 -13C 1.1% 1/2 67.2×106
14N 99.6% 1 19.3×106
15N 0.37% 1/2 -27.1×106
16O 99.96% 0 -17O 0.04% 5/2 -36.3×106
19F 100% 1/2 251.8×106
23Na 100% 3/2 70.8×106
27Al 100% 5/2 69.8×106
31P 100% 1/2 108.4×106
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A compass in a magnetic field
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A nuclear spin precesses in a magnetic field
the circulating motion of the spin angular momentum is
called precession
Nuclear spins precess because:• they are magnetic•they have angular momentum
this arrow denotes the direction of the spin angular
momentum
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Precession frequency = Larmor frequency
0 = - Bz/2π
Larmor frequency in Hz (= cycles per second)
gyromagnetic ratio in rad s–
1 T–1
magnetic field inTesla (T)
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hBz = γh
2πBz = hv o
Compare with Zeeman Splitting
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Larmor frequency and Zeeman splitting
Zeeman splittingE = h 0
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Positive negative precession
Negative positive precession
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Precession frequencies for different isotopes
isotope naturalabundance
spin gyro-magnetic
ratio/rads–1T-1
Larmorfrequency(MHz)inafieldB0=11.7433T
1H 99.98% 1/2 267.5×106 -500.002H 0.015% 1 41.1×106 -76.7510B 19.9% 3 28.7×106 -53.7212C 98.9% 0 - -13C 1.1% 1/2 67.2×106 -125.7214N 99.6% 1 19.3×106 -36.1315N 0.37% 1/2 -27.1×106 +50.6816O 99.96% 0 - -17O 0.04% 5/2 -36.3×106 +67.7819F 100% 1/2 251.8×106 -470.47
23Na 100% 3/2 70.8×106 -132.2627Al 100% 5/2 69.8×106 -130.2931P 100% 1/2 108.4×106 -202.61
the Larmor frequency is proportional to the field
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Generation of the NMR spectrum
Fourier transform
The NMR spectrum
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The sense of the frequency axis
more rapid precession
increasing | |
less rapid precession
the sense of the precession is
ignored
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Chemical Shifts
The molecular environment distorts the magnetic field on a microscopic scale
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Mechanism of Chemical Shift
The magnetic field causes the electrons to circulate
The circulating electrons generate an additional
magnetic field which is sensed by the nuclei.This is
called the induced field. It is proportional to the applied
field.
The electrons in a molecule cause the localmagnetic fields to vary on a submolecular distancescale
2 steps…
1 2
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B jloc = Bo + B j
induced
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Proton Chemical Shifts
Chemical shifts correlate wellwith molecular structure and functional groups
“shielding” : magnetic field at nucleus reduced by
molecular environment
“deshielding” : magnetic field at nucleus enhanced by molecular environment
chemical shift
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This definition is used because it is field-independent
Definition of Chemical Shift
chemical shift
δj =|ν j |−|νref |
|νref |
chemical shift of site jLarmor frequency of site
j, ignoring the sign
Larmor frequency of spins in a reference compound,
ignoring the sign
By convention the spectrum is plotted with increasing from right to
left.
The result is usually quoted in units of ppm
(parts per million), where 1 ppm = 10-6
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A common reference compound: TMS
(Tetramethylsilane)
chemical shift
chemical shift of TMS protons
0
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Ethanol proton spectrum
chemical shift of TMS protons = 0
CH2 protons; = 3.7 ppm
OH proton; = 2.6 ppm
CH3 protons; = 1.2 ppm
chemical shift
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Cholesterol proton spectrum
chemical shift of TMS protons = 0
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Chemical equivalence
Two spins are chemically equivalent if • there is a molecular symmetry operation that exchanges their positions, or• there is a dynamic process between two or more energetically equivalent conformations, in which the positions of the two nuclei are exchanged.
Chemically equivalent spins have the same chemical shift.
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Examples of chemical equivalence
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An example of chemical inequivalence
chiral centre
the rotation around the C-C bond exchanges the
protons but the onformations are not equivalent (different
energies and different chemical shifts)
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Chemical inequivalence in amino acids:
L-phenylalaninechiral centre
chemically inequivalent CH2 protons
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Spin-spin couplings
Direct DD coupling (averages to zero in ordinary
liquids)
Indirect DD coupling or J–coupling (doesn’t average to
zero in ordinary liquids)electrons
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J-couplings cause splittings
chemical shift
ethanol proton spectrum
multiplet structure caused by J-couplings
multiplet structures caused by homonuclear J-couplings
between protons
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J-multiplets
J-coupling to N magnetically equivalent spins-1/2 splits the spectrum into N+1
multiplet components
1 coupling partner:doublet
2 coupling partners:triplet
3 coupling partners:quartet