Biophys 420 Exam 3

Topic 3, Methods of Structure Determination

What are the different sample conditions you can use NMR for

solution (micelles), solid state (powder, gel, crystal) cell extracts in vivo, perfused organs (MRI scale)

Why has NMR been used so frequently for membrane structure solving

solving structures is hard when you extract the protein outside of its native environment, ex hydrophobic effect is dependent upon it being in the membrane so removing it causes aggregation

How accurate is NMR structural information

structure determination of protieins and nucleic acids up to 40-50 kDa

How does NMR get structure determination of proteins and nucleic acids up to 40-50 kDA

measuring distances, torsion angles, H-bonds, alignment tensors

How does NMR get structure determination of proteins and nucleic acids above 50 kDA

rapid identification of ligand binding sites, associated conformation changes,

How is NMR useful for protein shuttling / acid base chem

can monitor the change of protons and the environment that they are in directly into the protein

How can you do selective observation of specific sites/components with NMR

isotopic labeling

What are all the purposes of NMR

diverse sample conditions, lots of structural information, selective obervation of sites, macromolecular dynamics on picosecond timescale, reaction kinetics

Frst step of NMR spectroscopy

generate EM radiation

Second step of NMR spectroscopy

send EM through sample, see if EM energy (E = hv) matches energy separation between states in molecules to see if EM will interact and achieve resonance

Third step of NMR spectroscopy

Detect sample induced changes in EM via absorbance, emission, polarization, etc

What is the bore of the NMR machine

cylindrical cavity inside of an NMR machine where the sample is placed

what is the superconducting coil of the NMR machine

uses power and cold temperatures to generate a force that makes metals stick to it

What are the two liquids in an NRM machine

nitrogen, helium

why does the NMR use liquid

cryogenic coolants, maintain the superconducting magnet at low temperatures

What are the shims in the NMR machine

electromagnets used to maintain uniformity/homogeneity of the main magnetic field across the entire sample

Why do superconductors at higher powers need fancier equipment

need to be colder to have the superconducting properties needed

Describe the necessary amount of concentration/sample volume for an NMR machine

high concentration, relatively low volume

What volume advantage does NMR have over x-ray crystallography

requires less sample

Which sample measurement technique requires the lowest sample amount

cryo-EM

How does NMR fix the signal-to-noise ratio problem

• repeats experiment multiple times, hundreds of pulses, average the experiment across this

• noise averages to 0, signal increases as it averages additively to measurements

What is the NMR formula for signal to noise (S/N) ratio

(number of scans) ^ (1/2)

• as # of scans increases, more signal and less noise

What is the change in energy /field ∆E that you’re measuring for NMR based on

quantum property of nuclear spin (I)

What is the purpose of nuclear spin for NMR

• Confers a tiny magnetic field (nuclear magnetic moment)

• Magnetic moments are randomly oriented without an external field

• With an externally applied magnetic field, moments align in specific energy states

• Low energy is aligned WITH field and high energy is aligned AGAINST/antiparallel

• ∆E between these states is proportional to field strength

What does it mean for NMR nuclear spin to be quantized

Nuclear momentum/orientation along the external magnetic field have discrete values ranging from 0-6 (in steps of ½ because of contributing electron spin)

Why can’t you use nuceli with an even number of both protons and neutrons for NMR

0 nuclear spin, can’t produce an NMR signal

How is NMR dependence on nuclear particle composition leveraged in studies

atoms that are not NMR active (0 spin state) can be made active by isotopic labeling, ex. C13

How are isotopic labeling methods used to boost signals in NMR

replace inactive nuclei with active nuclei or vice versa to control what is visible and invisible to the NMR

Why is proton NMR so powerful

ton of spin-half isotopes that exist in biological systems, H1 is super abundant. Most carbon is C12 and very little of it is C13.

What are all the types of labeling schemes for NMR

uniform labeling, amino acid specific, site-specific, subunit/domain specific

How does uniform labeling for NMR work

Label as many of the nitrogens or carbons inside a system as possible

How does amino acid/site/subunit specific labeling for NMR work

intein-based protein ligation

Describe the process of uniform labeling for NMR

• grow bacteria in regular media to desired OD600

• Switch to defined minimal media with a sole source of your desired nucleus ex. N15 ammonium chloride or C13 labeled glucose

• Induce expression, bacteria incorporates it into the protein, broadly labels the whole protein

Why isn’t the process of uniform labeling for NMR 100% efficient?

there is some turnover in the bacteria, bacteria degrades some of their own proteins to make new proteins

Describe the process of amino acid/site specific labeling for NMR

• load specific tRNAs with amino acids that are labeled

• use that in a system to make the protein

• more complicated than dumping in isotopically rich media

What is the nuclear magnetic moment formed by

nuclear spin (rotating charge), direction indicated by the right hand rule

What is the magnetic moment formula

μ = 𝛶 𝐼 ℎ

• μ = magnetic moment

• 𝛶 = gyromagnetic ratio

• 𝐼 = spin

• ℎ = Planck’s constant

What is the gyromagnetic ratio

ratio of a particle’s magnetic moment to its angular momentum, specific to each isotope

What happens to μ (magnetic moment) when there is no externally applied magnetic field

μ will be randomly oriented in space, Gaussian of a random species

What happens to μ when there is an external magnetic field applied

• Magnetic moment attempts to align to field

• Moment is quantized, can only adopt certain orientations

• Experiences torque that causes it to wobble about the magnetic field at the Larmor frequency

• Measure the vibration that occurs in the system around this

• External magnetic field applied around z axis

What is the larmor frequency

rate of wobbling of the magnetic moment around the magnetic field due to torque from the quantized aspect

What is the larmor frequency formula

w₀ = - 𝛶 * B₀

• w₀: frequency

• 𝛶: gyromagnetic ratio

• B₀: magnetic field strength, closest the magnetic moment can get to the magnetic field

How do nuclei align in the case of an I = ½ nucleus and an external magnetic field B₀

• nuclei align either up or down relative to the field

• up states slightly more populated than down states

Why are the “up” oriented nuclei more popular than the down states

• slamming a powerful magnetic field in a particular direction: align with mag field, hit with EM pulse to throw some species up, observe as they decay back down to native speces

• in spin you have positive or negative spin states that align with the magnetic field

• overall a slight delta in the positive direction because you’re applying a large external field the system

Why is the difference in population of nuclei in up and down states in NMR important

• wouldn’t be able to measure anything if they were equal

• population difference determines net signal and sensitivity

• For a signal to be detected, there must be more nuclei in the ground state than the excited state If the population ratio was 1:1, there would be no net absorption of energy, resulting in no signal.

What is the purpose of the radio frequency pulse addition to the populations of up and down states in NMR

• some of the population shifted from up to down state

• magnetization then relaxed/decayed back to original state

• Energy released during decay, measured by reciever

Why is it important that receiver measures in the perpendicular direction in NMR

Receiver is placed perpendicular to the magnetic field to measure perpendicularly

• spins are in the XY plane

• magnet B₀ in the z axis

• Receiver coil is perpendicular to transmitter to make sure sensitive receiver not overloaded by high power RF pulse

• Otherwise would only see pulse, no change due to magnetic spin about z-axis

Why was it important to standardize NMR?

• with more and more powerful magnets, you change the population of states in up versus down

• changes effective field strength, magnitude of quantization gets larger

• spectra on one instrument less powerful would have different chemical shifts

is the observed frequency in NMR the same as the larmor frequency

depends on local environment of individual nucleus (ex. shielding or exposing), differs slightly from Larmor

What is the chemical shift for NMR

difference in resonance frequencies due to local enviroment

Is the magnetic field at the nucleus the same as the applied magnetic field

no, due to chemical shift

In practice, the chemical shift is defined relative to?

the reference resonance signal

Do NMRs of the same strength have the same chemical shifts?

No, in raw values only have the same shifts when defined relative to a defined 0 point

what is the relation between chemical shift and magnetic field strength

independent of each other

What is an experimental limitation of NMR

relatively insensitive as a technique (super small diff between the populations of up and down magnetic spin states, need lots of signal in order to see anything out of random nucleic noise)

How can you get around NMR insensitivity through sample prep

High concentration, more volume than cryo-EM, relatively low salt

Why do NMR samples need lower salt

ionic strength of solution as you’re applying RF field through it induces currents the stronger the ionic strength is, obscures data by adding random noise and can cook the sample by generating heat with induced current

Why can high concentration of sample of protein be an issue for proteins

some proteins don’t like to be concentrated form aggregates

How can you get around NMR insensitivity through experimental changes

repeats the same experiment multiple times to improve signal to noise ratio

what is the relation between signal to noise ratio and number of scans

proportional to square root of # of scans

Explain NMR with handbell frequency

• Nucleus of each atom rings at different frequency depending on size, magnetic moment, overall field strength, applied RF pulse

• The ringing of external atoms connected together bleed into each other, can determine where things are in the system but signals can bounce between a lot of diff things

• Different ringing frequencies allow you to get different chemical shifts and see individual atoms, not just one massive broad peak

How is carbon frequency different than hydrogen frequency in NMR

Carbon rings at a lower frequency because it’s heavier, bigger, harder to spin. Hydrogen rings at a higher frequency because it’s smaller, easier to disturb

How are fourier transforms used in NMR signals

converts a time domain to a frequency signal (NMR signal/ringing to frequency)

What does the ringing of each atom’s nucleus depend on

its identity, identity of neighboring atoms, overall strength of magnetic field itself

What is the formula to convert frequency to ppm?

chemical shift (ppm) = (resonance frequency of atom - resonance frequency of standard (always 0 ppm) * 10^6 ppm / operating frequency of the magnet

Describe the ease of interpretation 1D NMR for proteins

crowded and overlapped, hard to interpret

Why is NMR harder for bigger molecular weight molecules

more proteins obscured in the center of a species, hard for them to be chemically distinct. wrapped them up, each nuclear spin is shielded and surrounded by diff aromatic residues. Affected by coulombic interactions, salts, hydrogen bonding

What solves the problem of 1D NMR being hard for large molecules?

Multidimensional NMR, to separate out the peaks

How does multidimensional NMR connect 1D peaks

• Interaction between nuclei observed in the 1D spectra, ex. J-coupling or NOE

• atoms close to each other have similar properties when hit with an EM pulse, can be correlated

in HSQC, what does the 2D NMR take advantage of

takes advantage of coupling between atoms because they are connected by chemical bonds and shared within space

in 2D NMR, magnetization only happens if:

1. two atoms are connected via covalent bond (J-coupling). Probing one to ring transfers ringing to the other atom to make it ring too

2. close in space (<5 A)

What is coupling in NMR

when 2 nuclei are coupled, the activity of one nucleus affects the activity of the other nucleus

How is 2D NMR visualized

stacked plots and contour plots

Describe what a stacked plot looks like

2D surface with peaks emerging from the plane, wherever there is a peak, there is a correlation between those two atoms

Why is there a bunch of signals along the center diagonal in stacked NMR plots

that represents atoms that are physically the same, always going to be correlated with each other

what is a key advantage of a stacked plot

easy to see intensity differences between peaks, strengths of the interactions between atoms

What is the most common way to visualize 2D NMR data

contour plots

Why are contour plots more commonly used than stacked plots

stacked plots are complicated and hard to interpet

What does a contour plot look like

topographic map, different rings indicate steepness of the peak

How does weak peak visualization differ between stacked and contour plots

You can see more of the weaker peaks in a contour plot, whereas they’re hidden behind larger peaks in the stacked plot

Why is HSQC (proton nitrogen) the most common experiment for protein structure determination

relatively fast to acquire, 10 mins for 500 micromolar, cheap (15-N labeling)

What does HSQC (proton nitrogen) experiment allow for

fingerprinting, comparing where positions of amide/peptide bond and nitrogen containing side chains.

Why is HSQC called fingerprinting

you can get overall relative position of a lot of the atoms in a species

What are the benefits of 15-N labeling in HSQC

• Eliminate aliphatic aromatic protein peaks

• Only observe 15N1H pairs

• 15N chemical shift provides a second dimension that spreads out peaks with the same proton shifts that would otherwise overlap, side chains are further spread apart in spectra than if you just took two proton spectra and correlated them

What are the applications of H-15N HSQC experiment

rapid identification of folded proteins and monitoring ligand binding

How is HSQC NMR used for identifying protein folding

Chemical shift dispersion or range of many protons is much worse in unfolded proteins than in folded proteins, can qualitatively assess folding of recombinant protein fragments. Useful for intrinsically disorderd regions, transcription factors, or other instances with lots of structural flexibility.

Why is there a broadening of ppm shifts in unfolded proteins

most of the protons and nitrogens will see the same environment, surrounded by a large bulk of solvent with the same forces and interactions

Why is the ppm shifts in folded proteins more narrow

specific hydrogen bonds, things become more specific in a system

If you have a large broad peak, the protein is likely to be ?

unfolded

If you have distinct peaks that are more spread out, the protein is likely to be ?

folded

Why is being able to rapidly identify folded proteins so helpful for intrinsically disordered regions

you can monitor the folding process, can quantify the peaks going from very broad to sharpening up to individual peaks that start to separate out better than initial broad peaks

How is HSQC NMR used for monitoring ligand binding

• monitoring physical position of atoms in your ligand assuming ligand or protein is labeled in some fashion

• titrate ligand into 15-N labeled protein, monitor chemical shift cahnges

• measures Kd by plotting shift vs ligand concentration

• can map which residues bind to ligand if you know what resonance belongs to what residue

• timescales important

How does the timescale analysis component of ligand binding monitoring work for HSQC NMR

• P L complex is in rapid exchange with P + L

• As it comes together you can tell whether it’s diffusing together or snapping together

What dictates the PL complex’s exchange with P + L

∆w

What happens if K_ex chemical exchange rate is > ∆w chemical shift difference

avg 𝛿 is observed, one averaged peak

What happens if K_ex chemical exchange rate is < ∆w chemical shift difference

two resonances are observed, not averaged

How can you figure out what resonance belongs to what residue for HSQC NMR?

• Make a mutation and look for what resonance disappeared/moved. But super time consuming and not feasible.

• Use a third frequency as a third dimension, needs uniformly labeled protein with both a 15N and 13C atom. Time consuming, needs to be repeated to assign peaks

What is 3D NMR

Triple resonance-based assignments, based on comparing data acquired from pairs of 3D NMR experiments

What do all 2D or higher spectra require for the nuclei they link?

Scalar coupling or NOE interaction between the nuclei that they link, in order to identify peaks

in 3D NMR, what is each peak associated with

3 chemical shifts (amide hydrogen, nitrogen, carbonyl)