1 Modern Approaches to Protein structure Determination 1. Introduction to NMR. 2. Solving Protein Structures by NMR - The features of a 1D spectrum - what.

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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.