Lecture #6 (The NOE)

Transcription

1 Lecture #6 (The OE) 2/24/17 Clubb Determining Protein tructures by MR: Measure thousands of shorter inter-hydrogen atom distances. Use these to restrain the structure of protein computationally. Distances are derived The uclear Overhauser Effect (OE) Distance restraint Macromolecular structure Reading: Claridge

2 Remember.. ignal amplitude is proportional to the population difference between the alpha and beta states 2

3 The uclear Overhauser Effect (OE): a reduction or gain in intensity of a resonance (MR signal/peak) when another resonance which is near in space is selectively perturbed (either saturated or altered by the application of RF ). The OE is caused by dipolar relaxation between the nuclei The magnitude of the OE depends on the distance (r ) between hydrogen atoms and therefore provides structural information about the macromolecule. OE 1 r 6 distance between hydrogen atoms and 3

4 1D teady tate OE Experiment (not frequently used but, conceptually easier to understand) Expt. (1) FT o o Expt. (2) FT irradiated spin Pre-irradiate spin with a long, low power pulse on resonance to saturate. ubtract (1) from (2) -o < 0 OE i (s) = (an example of a negative OE. positive OEs are also possible depending on molecular size) Fractional change in intensity of peak results if the 2 protons are dipolar coupled (i.e. near in space). The intensity change depends on interproton distance. 4

5 Two spins at equilibrium: (not J-coupled) Transition probabilities () () transitions: - - W 0 W 2 transitions: - - () () W 0 W forbidden, i.e. not excited by RF pulse or directly observed. 5

6 Two spins at equilibrium: (not J-coupled) W 0 - W 1 W 2 W 1 W 1 transitions: - - transitions: - - Population difference W W 2-2 Total spin population = 4 Population of () 6

7 1D Experiment Expt. (1) 90 o (A) (B) (A) (B) - + Total spin population = 4 Before pulse Total spin population = 4 After pulse 7

8 1D teady tate OE Experiment (not frequently used but, conceptually easier to understand) Expt. (1) FT o o Expt. (2) FT irradiated spin Pre-irradiate spin with a long, low power pulse on resonance to saturate. OE i (s) = ubtract (1) from (2) -o (a negative OE) Fractional change in intensity of peak results if the 2 protons are dipolar coupled (i.e. near in space). The intensity change depends on interproton distance. 8

9 Expt. (2) (no noe) (a) (b) (a) (b) - - 1/2-1/2 + 1/ /2 Total spin population = 4 Prior to saturation During saturation 9

10 Expt. (2) (with noe) (a) (b) (a) - - 1/2 = - 1/2 = W 0 + 1/ /2 Total spin population = 4 Prior to saturation During saturation (WTH OE effect) 10

11 Expt. (2) (with noe) (a) (b) (a) - - 1/2 transitions = 2-1/2 = 2 W 2 + 1/2 + transitions + 1/2 Total spin population = 4 Prior to saturation During saturation (WTH OE effect) 11

12 OE effect (+ or -) depends whether Wo or W2 dominates - 1/2 transitions - 1/2 W 2 + 1/2 transitions W 0 + 1/2 12

13 egative OE Wo dominates Positive OE W2 dominates 13

14 W 2 transitions E = [E E ] = h h = h () Fast tumbling small molecules Varying magnetic field at atom at the appropriate frequency causes transitions across different energy states. Fluctuating fields at will cause transitions (~ 1 GHz on a 500 MHz spectrometer) - 1/2-1/2 W 2 + 1/2 W 0 + 1/2 low tumbling large molecules W 0 transitions E ~ h () ~ 0 ~ 0 Hz Low frequency fluctuating fields at will cause transitions 14

15 Physical origin of oscillating magnetic fields that gives rise to the OE: Dipole-dipole (dipolar) interactions Each atom produces its own magnetic field Tumbling of protein Tumbling of protein causes magnetic field from proximal atoms to vary. 15

16 ign of maximum observable OE depends on field strength and molecular size W2 dominates OE goes thru zero when at c 0 = 1.12 c 1/ 0, when c 0.3 nanoseconds. (macromolecules with MW ~1000 daltons) Large molecules: W 0 dominates c

17 Transient OEs are used to determine distances between atoms: elective inversion of spins (like a selective T1 experiment) Determine effect on nucleus intensity (similar to steady state OE) 1D variant selective inversion Pulse ( spin) 90 pulse tm (a) (b) (c) variable delay (tm) (a) - (b) tm Population change during tm (via Wo and W2) Gives rise to OE elective 180 pulse on nucleus inverts populations 17

18 Transient OEs are used to determine distances between atoms: elective inversion of spins (like a selective T1 experiment) Determine effect on nucleus intensity (similar to steady state OE) 90 pulse (1) FT o o (2) selective inversion Pulse ( spin) tm 90 pulse FT variable delay (tm) ubtract (1) from (2)

19 Time it takes to develop transient OE during tm period is dependent on distances separation between the nuclei (r -6 ) Distance r r Large maller Measured OE effect Observe OEs between atoms separated by < ~5Å 1Å = 0.5 nanometers Experiment performed at tm 50ms 19

20 A variant of the 1D Transient OE experiment is used to measure interhydrogen distances (Multi-dimensional OEY Experiments) 1D Transient OE Expt. selective inversion Pulse ( spin) tm 90 pulse 2D OEY: (uclear Overhauser Enhancement pectroscopy): variable delay (tm) More on this later. 20

21 aturation Transfer Difference (TD) MR Experiment (protein-small molecule interactions) Application of the teady-tate OE Expt. n Drug Discovery 21

22 Protein hydrogen atoms (only) <5Å <5Å mall molecule <5Å pin-diffusion. ntensities of, A, C and D nuclei reduced Atoms in drug farthest away from protein show the smallest effect. Protein Pre-irradiate RF aturation of Protein ignals pin-diffusion: Process in which original OE to hydrogen atom is relayed to other hydrogen atoms that are proximal to atom

23 aturation Transfer Difference (TD) (implified) 20 M Protein MLigand mall molecule ligand ignals (protein too dilute to observe) H 2 O ignal Expt. (1) Expt. (2) Pre-irradiate Protein only On protein signal only ubtract (2) from (1) mall molecule interacts with protein (no signal would be observed if there was no interaction) caled 10X