Methods Flashcards

Question

What is nuclear magnetic resonance (NMR)?

Answer 1

* Certain nuclei (see footnote) change spin state in external magnetic field * Oriented nuclei are able to absorb certain frequencies of light in the magnetic field * Frequency of energy absorbed depends on the local magnetic field (**affected by the molecular structure**) ## Footnote **Spin active isotopes in proteins:** 13C, 15N, 1H (1H is the naturally occuring isotope of carbon, but 13C and 15N are not naturally occuring, they are the rare heavier isotopes - 12C and 14N are the common isotopes - 2H (deuterium) is the heavier isotope for hydrogen but it is not spin active)

Answer 2

* 1D: one frequency axis and one nuclei type * 2D: two frequency axes and one or two nuclei types (look for through-bond and through-space effects)

Answer 3

* **Through-bond** = correlation spectroscopy (COSY) - within 3 bonds * **Through-space** = nuclear overhauser enhancement spectroscopy (NOESY) - within 5 Å * Measure 13C, 15N, and 1H spectra * look for correlations and build structure model ## Footnote You can tell the difference between through-bond vs. through-space interactions.

Answer 4

* Isotope labelling protein (13C; 15N) * Must recombinantly produce the protein with microorganisms fed with isotope-labelled enriched carbon and nitrogen since these are not the naturally occuring isotopic forms * Not too floppy * Not too large (< 35,000 Da, <350 aa) - as you get larger, the spectra gets 'messier' and it is harder and harder to make correlations

Answer 5

* High-resolution atomic structures possible (<2Å) * in solution (no crystals; vary conditions) - might be more accurate because of this * + ligands * Instrumentation is highly accessible/available

Answer 6

The study of the interaction (absorption and/or emission) of electromagnetic radiation with matter

Answer 7

* Energy is **proportional** to frequency and wavenumber * Energy is **inversely** related to wavelength * Wavenumber = # of wavelengths per unit length * Frequency = # of occurences per unit time

Answer 8

proportional

Answer 9

of wavelengths per unit length

Answer 10

of occurences per unit time | e.g., cycles per second

Answer 11

* Visible spectrum is from 700 to 420 nm * Radio waves have very long wavelengths and very low energy * Gamma rays; x-rays etc... have very short wavelengths and very high energy

Answer 12

* A given transition corresponds to a certain energy * Need a photon of particular energy to cause a given transition

Answer 13

Electronic: UV-VIS (180 - 750 nm) Vibrational: IR (0.78 - 300 um) Rotational: Microwave (0.75 - 3.75 mm) ## Footnote E stands for energy (in Joules), v stands for frequency [in reciprocal seconds – written s-1 or Hertz (Hz)- 1Hz = 1 s-1), h is Planck’s constant. (6.626x10^-34 J.s)

Answer 14

n → π^star transition: source of nonbonding valence electrons → N, O, S, P π → π^star transition: source of π electrons → double, triple bonds ## Footnote It takes less energy (lower wavelength) to excite conjugated (pigments!) systems. This is why we can't see tryptophan with the naked eye, but you can see beta-carotene.

Answer 15

the distance between successive maxima

Answer 16

magnitude of the electric vector at the wave maxima

Answer 17

* 190 - 220 nm peaks are common to all proteins (backbone transitions) * Only proteins with Trp and Try have appreciable absorption around 280 nm * Phenylalanine has a very small absorption at 260 nm (shown on the graph it is multipled by a factor of 10 so it can be seen) ## Footnote Used mainly for detecting proteins and measuring protein concentration. Not used as much for protein structural determination anymore.

Answer 18

* Measure absorbance at 280 nm * Interference by aggregation (can cause scattering), DNA and other chromophores (e.g., bound groups that absorb at 280 nm) * Valid over Abs ~0.2-1 * Extinction coefficient, absorptivity (ε M-1cm-1) * Pathlength (l cm) * Concentration of absorbing species (c M) | A = ε l c ## Footnote Can determine extinction coefficient using bioinformatics (if the protein is known).

Answer 19

lower frequency and lower energy

Answer 20

Absorption spectroscopy with **circularly polarized light**

Answer 21

* Circular dichroism is due to the differential absorption of left- vs. right-handed circularly polarized light. * Induces ellipticity, θ

Answer 22

Optically active molecules: asymmetric in structure (e.g., glycine is not chiral, therefore not asymmetric in structure and not optically active)

Answer 23

Asymmetric in structure

Answer 24

Inversely proportional ## Footnote This can also be useful information for predicting tertiary and quaternary structure.

Answer 25

* Estimating secondary structure content (%)

Answer 26

* Industry: dairy; meat; leather; detergents * Medicine: malaria (plasmepsins); hypertension (rennin); Alzheimer's (beta-secretase)

Answer 27

Irreversibly denatured

Answer 28

* To elucidate why pepsin is irreversibly denatured at pH > 6, heat, or urea.

Answer 29

* Typically absorbed energy is dissipated as radiationless decay (e.g., energy is released as heat) * Molecules that fluoresce (like tryptophan) will lose some energy by radiationless decay (collisions with the solvent for example), and then will give off a photon (i.e., emit) at a lower energy level (higher wavelength)

Answer 30

* excitation (λEX) and emission (λEM) wavelengths corresponding to max intensity * Stokes shift (λEM − λEX) * Extinction coefficient * Quantum yield (conversion of absorption → emission) * Lifetime (duration of excited state)

Answer 31

* Conjugated systems (more conjugation = more stable excited state = more likely to emit light) * Tryptophan is a natural tag on proteins because it fluoresces (relying on tyrosine is not as useful because the signal-to-noise ratio is too high)

Answer 32

* By measuring fluorescence at different pH levels we can infer informatino about the structure based on level of fluorescence. * Tryptophan is a built-in fluoretic probe that can be used to study protein folding

Answer 33

* You can measure surface hydrophobicity with this method. * The dye will binding to the hydrophobic patches of the protein, and so you can use ANS binding to look at the protein under different conditions to get a measure of how different treatments affect the protein and it's surface hydrophobicity (an important feature for properties like emulsion ability etc.)

Answer 34

* This is often used to measure kinetics of amyloid formation under a given set of conditions through THT fluorescence

Answer 35

* Location of the detector is different. * Fluorescence - detector is 90degrees to the sample so that only emitted light is detected (this makes sensitivity orders of magnitude (thousands) greater than absorbance) * Absorbance - detector is right behind sample (light path is straight line)

Answer 36

A molecule can absorb IR radiation if it vibrates such that its charge distribution (therefore, its electric dipole moment) changes during vibration ## Footnote If the v of light matches v of vibrations = absorb photon (hv)

Answer 37

Unique to each group of atoms

Answer 38

* Different functional groups: * different bond strengths (related to *k*) * different masses (e.g., H, C, N, O) * thus, vibrate at different *frequencies* * thus, absorb different *energies*

Answer 39

Mid-IR (4000-650cm^-1) ## Footnote Wavenumber is inverse of wavelength (reported this way by convention).

Answer 40

Position & intensity of peaks is inversely proportional to secondary structure

Answer 41

* Used to measure liquids * High absorptivity, thus use small very small pathlengths (0.01-1.0 mm) - hard to load samples in practice due to the small path length * Can be used for solids if sample is ground and pelleted with potassium bromide, or dispersed in Nujol mineral oil

Answer 42

* measures total energy reflected from surface of sample in contact with IR transmitting crystal * used to measure solids, pastes, and viscous liquids (e.g., peanut butter) | Easier to load samples in this method as compared to transmission IR.

Answer 43

* Where the scattered particle ends up is proportional to the size and shape of the particle it collided with. | Neutrons are particles NOT electromagnetic radiation. ## Footnote The classical way of generating neutrons for this method is a nuclear reactor.

Answer 44

* Radius of gyration: approximates center of mass as a sphere.

Answer 45

* X-rays 'see' electrons; x-rays will 'see' electron density * Neutrons are totally different; they interact very well with hydrogens more than anything else, but they don't interact nearly as well with deuterium (heavy hydrogen) - so in this way we can make water invisible (use heavy water) to only see the hydrogens on the protein.

Answer 46

* Heat protein; it unfolds (protein is taking in energy to break these bonds); so the instrument has to apply more power to the sample than it does to the reference cell (usually water) to maintain the temperature * Tm is the denaturation midpoint * Change in heat capacity (baseline before and after unfolding) are related to exposing the buried hydrophobic amino acids - so a larger change in heat capacity would result in a greater delta-Cp.

Answer 47

Calorimetry ## Footnote This is the best method for determining thermodynamic stability.

Answer 48

* Notice how between the first and second scans the Tm is very different (i.e., pepsin does not fold back to native pepsin after being denatured) * 2nd scan Tm is at a higher temperature but is a much shallower peak.

Answer 49

* From pH 5 to pH 8 the tryptophan become exposed to solvent, and there is a loss of beta-sheets and increase in disordered structure - so it becomes expanded and partially unfolded * Native activity is lost * In refolding: Partial recovery of secondary structure, tryptophan becomes reburied * Refolded/denatured form may be more stable than native pepsin. ## Footnote Calorimetry was essential to piecing this together - observing that the refolded state is thermodynamically stable.

Answer 50

* Gives information about the thermodynamics of binding (e.g., proteins to proteins; proteins to ligands; etc...) * Each peak tells you direct information about the enthalpy of binding. * This method gives information about **stoichiometry** (how many ligands bind to each protein), **thermodynamics** (enthalpy), and **affinity** (binding/association constants - how tightly the molecules bind)

Answer 51

* Interested in different ligands that may prevent formation of prions (in this case, PcTS, which has metal 'M' in its aromatic structure) ## Footnote How are so many of these PcTS molcules binding the prion? Likely via pi-pi stacking interactions (see image on the other side of this card).

Answer 52

* DCS for **stability**. (This is the gold standard for characterizing thermodynamic stability of a protein. Disadvantage = slower than other methods like fluorescence (which can measure 96 samples at once); only one sample measured at once in DCS; also requires more sample per test) * ITC for **binding**. (This is the gold standard for characterizing binding affinity. Disadvantage = screening hundreds/thousands of molecules, this would not be a good method, since only one sample can be measured at one time.) ## Footnote There are faster methods, but these are the only methods that give the thermodynamis directly without use of a model.

Answer 53

* Mix sample with SDS buffer * *May include reducing agent* * Heat to denature * Proteins bind SDS in proportion to their Mw * All proteins have similar Mw/charge ratio * Migration distance is proportional to size and molecular weight.

Answer 54

* We expect protein hydrolysis under acid conditions * Run samples as a function of time to see how quickly this hydrolysis happens.

Answer 55

* **Advantage**: Very accessible, easy, simple, and affordable; cheapest thing we can do in the lab! * **Disadvantage**: Can be hard to resolve proteins in gels.

Methods Flashcards

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