Frueh_NMR Flashcards
pros to NMR
non-destructive, can be used in near physiological conditions, labile modifications, time-scale reporting and transient binding events
cons to NMR
can’t do big proteins, need a ton of material
4 molecular properties of NMR spectra
signal position, relative intensities, splitting, line broadening
Ampere’s Law/Maxwell equation (4 parts)
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.
Faraday’s law
a magnetic field that varies in time creates an electric field
What property of nuclei does NMR exploit?
Magnetic property of spin, think of nuclei as mini magnets with poles
T/F. In the absence of a magnetic field, there is a preferential orientation in nuclear spins.
False, there is no preferential orientation and all the vectors would cancel each other out
T/F. The amount of magnetization (sensitivity) will be proportional to the ratio of the gyromagnetic radii.
True, this is what gives protons the highest sensitivity
Why proton NMR most common?
Highest sensitivity, and are also found at natural abundance
why do carbon or nitrogen NMR?
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
how do you detect magnetization?
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
how is frequency of oscillation turned into NMR signal?
fourier transform of frequency gives you PPM position
T/F. NMR spectra signals report on concentration
Yes, so they are proportional to the number of molecules detected in that volume
chemical shift is a measure of…
environment. it differentiates diff protons, gives signature of AA, and is sensitive to binding and slow dynamics
why do we care about electron density?
because protons with a higher electron density are more shielded, giving smaller frequencies.
T/F. ppm on X axis is expressed in terms of reference frequency
True. For an XXX Mhz spectrophotometer, 1 ppm = 600 Hz
Three elements that pull e- away from carbons
Nitrogen, oxygen and sulfur. All other atoms take e- away from protons
T/F. You can overlay spectra at 600 MHz with those at 800 MHz
True
how to tell rate of reaction from NMR
slow/intermediate/fast exchanges will give diff chemical shifts as environment changes around the nuclei involved in the interaction
n+1 rule
how to tell how many split peaks to expect, based on n neighbors (coupling partners)
T/F. Labile proteins don’t show up on the spectra, as they are rapidly exchanging with environment
Truth
how do dipolar couplings affect spectra?
line broadening, not split peaks because the coupled nuclei change orientation randomly and molecular tumbling
scalar couplings
neighbor effects
what causes scalar couplings to happen
effect mediated by electrons, and whether electron spin state of neighbor is up or down
what property of the spectra does scalar couplings lead to
splitting
why does dipolar coupling need two time constants?
T1 and T2, the trajectory going back to equilibrium is complex and experiences relaxation along two axes
which time constant relates to longitudinal relaxation?
T1, the time we need to wait before repeating the experiment
which time constant relates to transverse relaxation
T2, dictates the amount of signal we detect, its width and height.
T/F. Small T2 = weaker and broader signals
True
T/F. A larger T1 has a higher sensitivity
False, larger T1 is lower sensitivity
T/F. The slower a molecule tumbles, the less efficiently its signals relax
False, the slower a molecule tumbles, the more efficient its relaxation. Large proteins have large, weak signals
T/F. For proteins, T1 increases as size increases
True
How can you determine folding status of a protein by looking at its NMR spectra?
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
How can you assign secondary structures by looking at correlation maps?
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
What are three things you can tell from an NMR spectra without having to do atom assignment?
Folding status, secondary structure, binding
How do you assign backbone resonances in protein NMR?
- 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
How do you know if AA residues identified in a spectra are sequential?
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
What are 7 advantages of NMR?
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)
What are data collection times for 1D, 2D, and 3D spectra?
1D - seconds - minutes
2D - minutes - hours
3D - many hours - days
HSQC spectrum
correlation map with one axis representing proton chemical shift, and the other the N or C chemical shift
HNCO spectrum
triple resonance, 3D spectra
