NMR SPECTROSCOPY@ BINOD JENA

8/8/2019 NMR SPECTROSCOPY@ BINOD JENA

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PRESENTRED BY:

SATYAJIT PANIGRAHI

0701106331

7TH SEM ,BIOTECHNOLOGY

CET,BBSR



NMR and Structural Biology



Measures nuclear magnetismor changes in nuclear

magnetism in a molecule

NMR spectroscopy measures

the absorption of light (radio

waves) due to changes in

nuclear spin orientation

NMR only occurs when a

sample is in a strong magnetic

field Different nuclei absorb at

different energies

(frequencies)

PRINCIPLES



Atomic nucleus with odd mass number

or odd atomic number or both posses quantizedspin and magnetic moment

In presence of an applied magnetic

field, the spin states are not of

equivalent energy





13C NMR Spectrum of a Large Molecule



NMR TerminologyChemical Shift & Linewidth

Since the precession frequency is directly related

to the strength of the magnetic field at the

nucleus, even subtle changes in this field will have

detectable effects on the resonance frequency of

the spin. This is called the chemical shift .

Things that effect chemical shift

field strength

attached nuclei H-bonding



Resonance Assignment

The key attribute: use the scalar and dipolar couplings to

match the set of signals with the molecular structure



Scalar and Dipolar Coupling

1

1 1

1 2 1

1 3 3 1

1 4 6 4 1

1 5 10 10 5 1

Spin Coupling Intensities



Exposing sample to the RF

Causes nuclear energy transitions

Photon emitted when nucleus drops back to lowerenergy level and NMR recorded.

2-D NMR: COSY COSY(correlation spectroscopy)/Jcoupling)

Protons that are bonded to each other can be directlyspin-coupled; can ³track´ one atom to the next.

2-D NMR: NOESY

NOESY(nuclear Overhauser effect spectroscopy)/NOE coupling )

Spatial determination

STEPS IN NMR



Derive experimentalconstraints (NOE,scalar coupling,

dipolar coupling data)

Computer finds bestmodels of target

protein with

accordance toexperimental data

Result is a set of estimates of distancesbetween specific pairs

of atoms called

³restraints´

Get an ensemble of models and the

position of the modelsare averaged

Result is a bundle of superimposed

conformers STRUCTUREDETERMINATION

BY NMR



Can be performed inaqueous solutions

under in vivo

conditions

Does not rely onspecific reporter

groups or artificiallyattached dyes

Don¶t need tocrystallize the protein

Large array of parameters can be

extracted from theresonance lines

Advantages



NMR

Approximately ¼ of all structure in thePDB have been generated by NMR

Proteins as large as 700 residues havebeen analyzed/solved by NMR

Excellent method for studying proteinstructure in solution, probingdynamics, flexibility, stability, kineticsand rate processes

NMR compliments X-ray work

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Documents

NMR SPECTROSCOPY@ BINOD JENA