Frueh_NMR

Question 1

pros to NMR

Answer 1

non-destructive, can be used in near physiological conditions, labile modifications, time-scale reporting and transient binding events

Question 2

cons to NMR

Answer 2

can’t do big proteins, need a ton of material

Question 3

4 molecular properties of NMR spectra

Answer 3

signal position, relative intensities, splitting, line broadening

Question 4

Ampere’s Law/Maxwell equation (4 parts)

Answer 4

an electric field varies in time, generating a magnetic field. A current generates a magnetic field. A circular current will make a field perpendicular to plane of current, a current means electrons in motion.

Question 5

Faraday’s law

Answer 5

a magnetic field that varies in time creates an electric field

Question 6

What property of nuclei does NMR exploit?

Answer 6

Magnetic property of spin, think of nuclei as mini magnets with poles

Question 7

T/F. In the absence of a magnetic field, there is a preferential orientation in nuclear spins.

Answer 7

False, there is no preferential orientation and all the vectors would cancel each other out

Question 8

T/F. The amount of magnetization (sensitivity) will be proportional to the ratio of the gyromagnetic radii.

Answer 8

True, this is what gives protons the highest sensitivity

Question 9

Why proton NMR most common?

Answer 9

Highest sensitivity, and are also found at natural abundance

Question 10

why do carbon or nitrogen NMR?

Answer 10

although they give weaker signals, and you have to specially produce the protein, you can monitor your labeled protein specifically in a pool of others

Question 11

how do you detect magnetization?

Answer 11

apply a current (precessing magnet), use two torques to detect oscillating signal along the y axis. as you relax mag you get decay of signal, and that oscillating signal is your data

Question 12

how is frequency of oscillation turned into NMR signal?

Answer 12

fourier transform of frequency gives you PPM position

Question 13

T/F. NMR spectra signals report on concentration

Answer 13

Yes, so they are proportional to the number of molecules detected in that volume

Question 14

chemical shift is a measure of…

Answer 14

environment. it differentiates diff protons, gives signature of AA, and is sensitive to binding and slow dynamics

Question 15

why do we care about electron density?

Answer 15

because protons with a higher electron density are more shielded, giving smaller frequencies.

Question 16

T/F. ppm on X axis is expressed in terms of reference frequency

Answer 16

True. For an XXX Mhz spectrophotometer, 1 ppm = 600 Hz

Question 17

Three elements that pull e- away from carbons

Answer 17

Nitrogen, oxygen and sulfur. All other atoms take e- away from protons

Question 18

T/F. You can overlay spectra at 600 MHz with those at 800 MHz

Answer 18

True

Question 19

how to tell rate of reaction from NMR

Answer 19

slow/intermediate/fast exchanges will give diff chemical shifts as environment changes around the nuclei involved in the interaction

Question 20

n+1 rule

Answer 20

how to tell how many split peaks to expect, based on n neighbors (coupling partners)

Question 21

T/F. Labile proteins don’t show up on the spectra, as they are rapidly exchanging with environment

Answer 21

Truth

Question 22

how do dipolar couplings affect spectra?

Answer 22

line broadening, not split peaks because the coupled nuclei change orientation randomly and molecular tumbling

Question 23

scalar couplings

Answer 23

neighbor effects

Question 24

what causes scalar couplings to happen

Answer 24

effect mediated by electrons, and whether electron spin state of neighbor is up or down

Question 25

what property of the spectra does scalar couplings lead to

Answer 25

splitting

Question 26

why does dipolar coupling need two time constants?

Answer 26

T1 and T2, the trajectory going back to equilibrium is complex and experiences relaxation along two axes

Question 27

which time constant relates to longitudinal relaxation?

Answer 27

T1, the time we need to wait before repeating the experiment

Question 28

which time constant relates to transverse relaxation

Answer 28

T2, dictates the amount of signal we detect, its width and height.

Question 29

T/F. Small T2 = weaker and broader signals

Answer 29

True

Question 30

T/F. A larger T1 has a higher sensitivity

Answer 30

False, larger T1 is lower sensitivity

Question 31

T/F. The slower a molecule tumbles, the less efficiently its signals relax

Answer 31

False, the slower a molecule tumbles, the more efficient its relaxation. Large proteins have large, weak signals

Question 32

T/F. For proteins, T1 increases as size increases

Answer 32

True

Question 33

How can you determine folding status of a protein by looking at its NMR spectra?

Answer 33

Yes, even a 1D spectra can give you this info - you want to look for the relative intensity of the amide groups in the high PPM ranges - if you have more amides interacting with the environment the same way, you’ll get a single (or close to single)peak, whereas the folded will be split

Question 34

How can you assign secondary structures by looking at correlation maps?

Answer 34

Look for conserved patterns indicative of secondary structures - for example, alpha helices have a repeating pattern with amides in the core and side chains on outside. Beta sheets have “exquisite dispersions” with amides exposed to side chains above and below strand

Question 35

What are three things you can tell from an NMR spectra without having to do atom assignment?

Answer 35

Folding status, secondary structure, binding

Question 36

How do you assign backbone resonances in protein NMR?

Answer 36

1. Identify H,N correlations belonging to sequential residues, and 2) figure out the AA associated with these correlations. Amide groups used to anchor bc you get one HN correlation per group and these signals are well dispersed

Question 37

How do you know if AA residues identified in a spectra are sequential?

Answer 37

Have to tell by the similarity of the scalar coupling bw N and C-alpha within and between residues. Sequential residues have a common signal

Question 38

What are 7 advantages of NMR?

Answer 38

1) reliable and easy for proteins < 150 aa.
2) don’t have to crystallize
3) can get details on molecular motions
4) can quantify solution behavior, such as binding of ligands
5) can be performed in native conditions, or in cells
6) non destructive, sample can be recovered for other measurements
7) live systems can be measured (ie post translational modifications)

Question 39

What are data collection times for 1D, 2D, and 3D spectra?

Answer 39

1D - seconds - minutes
2D - minutes - hours
3D - many hours - days

Question 40

HSQC spectrum

Answer 40

correlation map with one axis representing proton chemical shift, and the other the N or C chemical shift

Question 41

HNCO spectrum

Answer 41

triple resonance, 3D spectra