Lecture 7 Methods of Studying Protein Structure I

Question 1

What does NMR reveal/what is it used for?

Answer 1

• Nuclear Magnetic Resonance (NMR) can reveal the
  atomic structure of macromolecules
• NMR is unique in giving the structure in chemical shifts

Question 2

How is intrinsic magnetism used to study atomic structure?

Answer 2

• Many nuclei of atoms have an intrinsic magnetism
• The most significant for biological systems are:
• 1H 13C 15N 31P
• These isotopes can be used in NMR to study macromolecular structures
  -1H is useful for biological studies and is also the most commonly used in NMR experiments (its the most sensitive nucleus)
  + Extensively found in biological systems
  -31P is useful for organic compounds and coordination complexes ( chemical compounds with Posphorus)
  + Remember - (NOT found in proteins)
• The magnetism in these nuclei comes from one property: the SPIN of their protons
  + Example: 1H has a spin of 1/2

Question 3

What are the two orientations H nuclei can adopt in a magnetic field?

Answer 3

• The spin of 1H generates a magnetic moment
• In an applied magnetic field this adopts one of two
  orientations
• α = orientated with the field
• β = orientated against the field

Question 4

How can 1H nuclei be excited to a higher state and what is the formula used to calculate this?

Answer 4

-1H nuclei in the α state can be excited into the β state
- This requires a pulse of radio-frequency energy
- υo = γHo /2π
where:
- υo is the radio-frequency - Important (so the pulse)
- γ is the magnetogyric ratio for a given nucleus (ratio of its magnetic moment to its angular momentum)

Question 5

What is a chemical environment, how is it created and give examples.

Answer 5

• electrons around 1H alter the magnetism that the nucleus experiences (different amount of e-)
• this Creates a local chemical environment
• Examples:
• A methyl group (CH3) is one chemical environment
• An aromatic ring (C6H6) would be a different chemical environment
• Electrons shield protons from the applied field
  + Ho = Bo ( 1 - σ )
• Ho is the nuclear magnetic field strength
• σ is the shielding factor
  -1H nuclei in different chemical environments will have
  different υo values
• Differences in υo values are very small
• Scaled as a term, δ
• Called the chemical shift
• Measured as parts per million (ppm)

Question 6

Give some examples of regions of chemical shifts.

Answer 6

-1H nuclei in different chemical environments have specific regions of chemical shift
\+ Examples:
- Methyl groups:
- δ = 0 = 1.5 ppm
 - Aromatic groups:
- δ = 6 = 7.5 ppm

Question 7

How can the local magnetic field be further altered?

Answer 7

• 1H proton bonds to neighbouring nuclei with magnetic moments
• Nuclei affect each other =This is called spin-spin coupling
  Requirements for observing spin-spin coupling:
  + Neighbouring nuclei must be in different chemical
  environments
• Note: True even for two seemingly identical groups
  + Nuclei must usually be two bonds apart
• Otherwise the effect is too small to be seen

Question 8

What is relaxation of nuclei?

Answer 8

• Nuclei excited to the β state must lose energy to return to the α state
• This is called relaxation
• Occurs via an interaction with other near neighbour magnetic nuclei
• An NMR spectrum is the observation of β state protons falling back to the α state
• Two forms of relaxation occur:
  + Relaxation through bonds
  + Relaxation through space
• Relaxation can also be observed by 2-D NMR
  Spectroscopy

Question 9

What is 2-D NMR Spectroscopy?

Answer 9

• provides General Information
• Spectra are drawn as a 2-D ‘contour’ map of peaks
• Peaks on the diagonal are the 1-D NMR spectra
• Peaks off the diagonal show where relaxation
  interactions have occurred

Question 10

What types of 2-D NMR Spectroscopy exist?

Answer 10

• 2-D Correlation Spectroscopy (COSY)
• 2-D Nuclear Overhauser Enhancement Spectroscopy (NOESY)
  (remember cozy and nosy)

Question 11

What information does 2-D Correlation Spectroscopy (COSY) provide?

Answer 11

• Provides information on the through bond connections between residues
• Identifies the correlation (In it’s most used form this allows us to see which proton resonances are mutually coupled. )

Question 12

What information does 2-D Nuclear Overhauser Enhancement Spectroscopy (NOESY) reveal?

Answer 12

• Reveals information about through space relaxation
  interactions
• Uses Nuclear Overhauser Effect (NOE) data
• This interaction is from nuclei separated in space by < 5Å (so closer than 5A)

Question 13

Describe how NOESY works.

Answer 13

• The peaks on the diagonal of a NOESY spectrum are a normal 1-D spectrum
• Used as markers to locate the off-diagonal peaks
  -1H nuclei close in space to each other will ‘connect’ on an off-diagonal peak
• Off-diagonal peaks are symmetric in appearance
  -Off-diagonal peaks show where relaxation has occurred between 1H nuclei through space
• A large number of off-diagonal peaks are present in a 2-D spectrum
• All represent nuclei in close proximity to each other
• For atomic structure determination all the NOESY data must be satisfied
  + All 1H nuclei shown by NOESY to be close together
  must be close together in the structure
• Together with additional data obtainable from NMR a SET of solution conformation of a protein can be obtained

Question 14

What potential problems exist with NOESY?

Answer 14

• No SINGLE STRUCTURE exists for a protein
  + Because NOESY data are for a range of lengths
• Methyl groups often dealt with as a SINGLE atom