NMR Flashcards

1
Q

NMR fundamentals

A

Nucleic has quantum number (I = 1/2) will give signal in NMR (1H, 13C, 15N, 31P) I = 1/2 gives to states

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2
Q

Coherence

A

Magnetization transfer between nuclear neighbors.

Coherence transfer gives information on which nuclei are covalently bonded.

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3
Q

COSY

A

Direct neighbors

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4
Q

TOCSY

A

Entire spin - coupled system ¨spin-systems¨

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5
Q

NOESY

A

The cross of a NOESY spectrum indicate which protons are close to each other in space.

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6
Q

Basic strategy for NMR assignment of proteins

A

1, identify spin system for each amino acid

2, put them in order: sequence specific assignment

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7
Q

Spinn relaxation (nOe)

A

Magnetization transfer through space - distance dependent.

Required for put the spin system in order.

Sequential NOE
Internal NOE

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8
Q

2D NMR NOESY spectrum

A

Depicts intermolecular distances between all protons in the biomolecule.

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9
Q

Magnetic labelling

A

Requires gene technology and bacterial over expression

15N labelling

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10
Q

HSQC

A

Each peak corresponds to one amino acid

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11
Q

3D NOESY-HSQC

A

HSQC experiment can be recorded and analyzed separately

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12
Q

13C - labelling

A

With this we can do the sequential assignment by through-bond connectivities ¨Backbone experiments¨

side chain assignments are required for structure determination by NoEs

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13
Q

Calculating an NMR structure requires

A

Basics
protein sequence, covalent structure
assignments of all relevant NMR resonances

Conformational constraints 
NoEs (between H)
Hydrogen bonds 
Dihedral angles 
Chemical shifts 
Residual dipolar couplings
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14
Q

Secondary structure elements can be identified by unique short distances

A

α-helix
(CαH)i - NHi+3
(CαH)i - (CβH) i+3

β-sheets
NH-NH across the sheet
CαH - CαH across the sheet
CαHi - NHi+1 along the chain

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15
Q

problem at higher molecular weights

A

Slow movement in solution makes signal relax faster poor signal-to-noise and line broadening.

interaction between relaxing nuclei increases relaxation

Overlap also in 2D and 3D spectra

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16
Q

Pros of NMR

A

High resolution structures

Accessible for non-crystallizable proteins

Rapid and specific mapping of ligand binding and protein interactions

Dynamics studies

Everything is observed

17
Q

Cons of NMR

A

Technically advanced

Specific sample requirements

current convenient max size limit around 35kDa