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1
Modern Approaches to Protein structureDetermination
1. Introduction to NMR. 2. Solving Protein Structures by NMR -
The features of a 1D spectrum - what can we tell? The need for 2D
3. 2D NMR - How NMR works through space not just bonds - we need this to solve structures.
The move to the third dimension
4-5. Modern methods for structure determination 6. Comparison of techniques and New developments
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Why study protein structure?
•The more we understand about a protein and its function, the more we can do with it. It can be used for a new specific purpose or even be redesigned too carry out new useful functions (biotechnology & industry).
•We can use this knowledge to help understand the basis of diseases and to design new drugs (medicine & drug design).
•The more knowledge we have how proteins behave in general, the more we can apply it to others (protein families etc)
Structure determination of biomacromolecules by NMR-no crystal needed, native like conditions -bandshift assays -Dynamics-Size limitations
Complex, couldbe the active form
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Nuclear Spin
Atomic nuclei are composed of protons and neutrons which have a spin
Protons spin neutrons spin nuclear spinEven even 0Even odd 1/2Odd even 1/2 Odd odd n
NMR properties of selected nuclei
Nucleus I s)-1 rad rel Natural Abundance (%)
1H 1/2 2.6752 x 108 1.00 99.982H 1 4.107 x 107 0.15 0.0213C 1/2 6.728 x 107 0.25 1.1114N 1 1.934 x 107 99.6415N 1/2 -2.712 x 107 0.1 0.3617O 5/2 -3.628 x 107 0.0419F 1/2 2.5181x107 10023Na 3/2 7.080 x 107 10031P 1/2 1.0841 x 108 0.41 100113Cd 1/2 5.934 x 107 12.26
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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
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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• the energy of the state with quantum number Iz is given by
Zeeman splitting
Planck constantgyromagnetic 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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Ez = −μ z Bo = −γIz Bo = −γmhBo
• We take Bo to be along the Z axis, so the dot product becomes E=-zBz(o) (i.e. xBz and yBz = 0)
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(Iz = mh where m = +1
2, -
1
2 for I = 1
2
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Ez = −γhmBo = −γh1
2Bo
m=-1/2
m=+1/2
I=1/2m=-1
m=+1
I=1
m= 0
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Ez = −γhmBo = γh1
2Bo
The Zeeman splitting is therefore
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hBo
ground state; no
field
ground state; with field
Zeeman splitting
Energy
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hBo
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rad s-1 rad s-1 T-1.T
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ωo
2π= vo
vo =γBo
2π
s-1 (Hz)
Larmor Frequency
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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 frequency0 = - Bo/2π Larmor frequency in Hz
(= cycles per second)
gyromagnetic ratio in rad s–
1 T–1
magnetic field inTesla (T)
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ΔE = γhBo
ΔE = hωo
ωo = γBo
Compare with Zeeman Splitting
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Since, ωo = 2πvo
ωo = −γBo Note – ignore sign difference – thisarises from convention and the signof the precession.
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Nα
Nβ
= eΔE
kT = eγhBo
kT =1.0001
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For protons, 4.69 T, 293K.
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http://www.chm.bris.ac.uk/polyketide/nmr.htm
Will have Lecture 1 (overheads)
Plus Notes on Basic NMR.