NMR Spectoscopy Flashcards
Dynamic processes which NMR helps to study
- Protein folding
- Protein hydration
- Ligand binding
- Enzyme kinetics
Protein-Ligand Interaction
- If there is binding of the ligand to the protein, some peaks will change, larger the change, closer the ligand is to the corresponding nucleus
in space
Basic Principle of NMR Spectroscopy
Nuclear Spin State (I)
Isotopically labelling, by growing proteins in culture with C13 and N15. Using probes
Modern NMR Spectrometers
Built to observe nuclear “spin
flips” – all consist of a magnet, a source of radiofrequency and a
probe to accommodate the sample
Basic Principle
Nuclei lined up and hit with 90 degree radiofrequency pulse
Acquisition Phase
tD = Recovery time
Example (CH3CH2OH)
What information does 1H NMR Spectroscopy give us?
1) Chemical Shift - Indicating nature of functional groups
2) How many protons present in each chemical environment => molecular symmetry
3) Peak Splitting - relationships of groups of protons to one another
Chemical Shift
Effect of greater shielding on position of resonance
Effect of less shielding on position of resonance
Shielding Effects
1H NMR Chemical Shifts in Alkenes (double bonds)
Chemical Shift Anisotropy
= Bonds create anisotropic chemical shifts in molecules
- Shielded and deshielded regions
Summary of Chemical Shifts
Integration
Peak Splitting / Coupling
2D NMR (COSY)
Require NMR experiments that consist of several pulses of radiofrequency radiation, together with variable delays between these pulses
Adv of NMR
- Generally applicable for obtaining a complete set of 3D- information (primary, secondary and tertiary structure) for most small/medium-sized proteins
- Uniquely able to directly study dynamic/functional aspects of
proteins (e.g. catalysis, receptor binding, folding etc. - Offers the possibility to study dynamic properties that occur in
solution, just as they would in the living cell
Comparison of Common Techniques used in Protein Structure Determination
Requirements for Protein
- 1-10 mg sample
- Close to 100% purity as possible
- Sample must be soluble in 90% H2O/10% D2O
- pH 7 or less to prevent Hs exchanging
- Stable over period of days
- Paramagnetic ions avoided
- Unlabelled proteins ~ 10kDa
- C13 N15 labelled proteins ~ 30 kDa
NMR of unlabelled proteins
TOCSY extends the reach of COSY into sidechains
Polypeptides/Protein from NMR viewpoint
20 AAs classified for NMR
NOESY
Detect whether protons are close to one another in space
- Determine the position of an amino acid in the sequence (primary
structure)
- Determine the three-dimensional shape of the protein (secondary and
tertiary structure)
- The size of the nOe can be calculated from a two-dimensional
volume integral
- Establishes the order of AAs in primary stucture
General conclusion for the applicability of of COSY/TOCSY & NOESY strategy for larger proteins
Likely to become less and less successful in assigning resonances in the backbone as proteins become larger. The more amino acids there
are in the molecule, the higher the probability of overlap between 1H
resonances in the protein backbone
3D NMR Techniques
Double-labelled proteins
High Field NMR Spectroscopy & Current uses
- Higher magnetic strength, better quality structures
- Macromolecular complexes and other ‘omics’
