NMR 2

Question 1

What four phenomena involving magnetic nuclei make NMR useful

Answer 1

1. Gyromagnetic ratio values
   a) Constant for a particular isotope, but differ between isotopes
2. Chemical shift (nucleus-electron interaction)
   a) Effect of surrounding electrons on magnetic nuclei:
   b) peak positions in NMR spectra reflect different functional groups
3. Scalar coupling or J coupling (nucleus-nucleus interaction)
   a) Through bond interaction between magnetic nuclei
4. Dipolar coupling (nucleus-nucleus interaction)
   a) Through space interaction between magnetic nuclei

Question 2

What is HSQC

Answer 2

1. NMR experiments comprise a sequence of pulses of rf radiation
2. 1H-15N heteronuclear single quantum correlation (1H-15N HSQC) pulse sequence
3. Pulse intensity and timing must be highly accurate and consistent
4. 1H-15N HSQC spectrum of a protein

Question 3

What does Resonant frequency depends on

Answer 3

1. gyromagnetic ratio
2. magnetic field strength
3. chemical enivronment

Question 4

What part of chemical environment changes the resonant frequency

Answer 4

1. functional group type in an organic compound or protein;

2. 1°, 2°, 3° and 4° structure in proteins

Question 5

What is chemical shift

Answer 5

1. Chemical shift helps make NMR useful
2. means that different functional groups produce signals in different parts of the NMR spectrum
3. NMR spectroscopists express resonant frequency in terms of chemical shift (“units” of parts per million; ppm)

Question 6

How are proteins adjusted for NMR

Answer 6

1. NMR-active isotopes commonly incorporated into proteins
2. NMR commonly incorporated isotopes extend the range of peaks- artificially
3. C13, N15 etc
4. Rule of thumb:
5. < 25 kDa- need to incorporate 13C + 15N;
6. > 25 kDa- need to incorporate 13C + 15N + 2H
7. 2H incorporated to reduce peak widths

Question 7

How does chemical shift change with variation of functional group and binding

Answer 7

1. Signals from functional groups occur in particular regions of an NMR spectrum
2. Each amino acid type produces signals with characteristic chemical shifts
3. Chemical shift changes reflect intermolecular interactions; affinity can be quantified by fitting changes in chemical shift or peak intensity
4. Binding-induced chemical shift changes can be highlighted to map the binding site on the protein structure

Question 8

What else is chemical shift dependent on

Answer 8

1. conformation-dependent chemical shift dispersion
2. The protein fold results in conformation-dependent chemical shift dispersion
3. Consequence: can assign individual peaks in a spectrum to individual nuclei in a protein

Question 9

What is point in ppm scale

Answer 9

1. The ppm scale corrects for the fact that the raw frequencies (usually in MHz) scale with the size of the NMR magnet.

Question 10

What is required for a sample for NMR

Answer 10

1. 0.3-0.5 ml of 0.02 mM-0.5 mM protein as rule of thumb.
2. Homogeneous (90-95% pure) and monodisperse e.g. 100% monomer or 100% dimer etc – only one type
3. Buffer (almost any, particularly at low concentration (10-20 mM)); lower ionic strength better
4. pH: exchange rate of chemically labile 1H (e.g. backbone and sidechain amide 1H) increases logarithmically above pH 2.6;
5. once exchange rate high enough, signal from labile 1H merges with solvent signal and hence unobservable- so best to be below pH 8 as peaks disappear

Question 11

What can result in changes in chemical environment

Answer 11

1. changes in solution conditions
2. intrinsic dynamics
3. molecular interactions
4. folding/unfolding
5. mutation

Question 12

What can be inferred about a protein from a simple NMR experiment?

Answer 12

1. Simple 1D 1H and 2D 1H-15N HSQC NMR spectra
2. Data acquired in minutes, even seconds
3. Useful information is encoded in the number, distribution/pattern, intensity and width (sometimes called linewidth) of peaks
4. Useful information, e.g. relating to
5. structure
6. folding
7. dynamics
8. stability