Methods of studying macromolecular structure & interactions Flashcards
How is the backbone well suited for its function? 2 mainly
Why is the 2’OH in RNA significant?
Which direction are B form DNA & A form RNA helices in?
How do their structures differ? (distance to axis & grooves)
What is the difference between major & minor grooves?
Diverse RNA structures with 2’ OH
6 degrees of freedom in backbone- more complex structures (7 total)
Undergo hydrogen bonding - so more reactive & more diversity of structures
Right-handed
B form DNA = centre close to axis & deep major groove
A form RNA = centre shifted from axis (more angle), shallow major groove
Major = more important side chains
What is significant about the canonical base pairing/stacking?
What is the main energetic contribution to helix formation?
How is the folding of a nucleic acid chain driven? How does it begin & progress?
List 4 other secondary structures of NAs:
What is RNA tertiary structure dependent on? What is an example?
How do very large RNA structures fold?
How are RNA metabolism/regulatory roles mediates?
Distance between the 2 backbones is constant
Base stacking between neighbouring base pairs
Base stacking interactions- very fast & end up with helix. Starts with nucleation (few bp) then speeds up
Hairpin loops, asymmetric loops, bulges & junctions
Concentration of Mg2+ (tRNA)
With protein-nucleic assemblies
Binding to proteins in protein-RNA complexes- part of protein sequence & global regulatory role
What are the 3 main forces in macromolecular folding? What else do they mediate?
In which direction do the side chains point in an alpha helix?
What residue does the carbonyl i form a hydrogen bond with NH?
What bonds does the fold rely on?
Where are the side chains in a beta sheet?
Hydrophobic (protected from water), electrostatic (opposing charge), hydrogen bonds (3D structures)
Protein interactions
Outwards/outside
i + 4
Hydrogen, side chain interactions
Above or below plane/sheet
What is the basic RNA folding pathway?
What happens to energy and entropy during this?
What are the tertiary interactions
What are the types of interactions between proteins and nucleic acids? If NA-protein binding has pI>7, what amino acids are most involved in binding?
unfolded, molten globule, native state
entropy decreases and so becomes more stable, lower energy state
Transient interactions between other proteins & macromolecules (some stable complexes are formed but less commonly)
Electrostatic: negative envelope NA and positive protein. Lysine and Arginine as has 1+ charge at pH 7
How is the structure of a macromolecule represented?
What are the key principles for assessing the quality of a macromolecule structure?
How do the outputs of an NMR structure & X-ray crystal structure differ?
What can surface representation help with in a less defined region of a protein? What display is the macromolecule in?
Coordinates x y z in arbitrary system
Geometry (bonds), ramachandran, side chain packing, dynamics, and if it fits to the data
NMR = several structures over eachother (family) that fit data & geometries- less defined X-ray = a single structure with positions of metal ions & water
Highlights its nature of the interactions on its surface (e.g hydrophobicity, charge)- can see which surfaces are exposed to what environment. Cartoon
What are the 3 benefits of X-ray crystallography?
What are the 2 disadvantages? How could you resolve the 2nd?
What is an X-rays wavelength and to what does this correspond to?
What does the interference of 2 waves give information on?
What is the resolution of 2 magnetic waves comparable to?
What form of X-ray scattering does it use?
Fast, provide atomic resolution structural info, accessible
Have to crystallise the molecule, can’t really study dynamic proteins (use variety of techniques)
1Å- covalent bond
Distance travelled or their starting points
The distance between them
Elastic
Electron absorbs X-rays & energy is transferred to vibrational energy. It’s then released. What is the difference between elastic & inelastic scattering (with energy, direction and damage)
When these rays are detected, how is an image/diffraction pattern built?
What is the problem of only using 1 macromolecule? How can you resolve this?
How are crystal structures different?
Why is irradiating a crystal better than a single macromolecule? (3)
Elastic = in different directions, frequency & wavelength does not change & does not damage macromolecule, no permanent energy transfer Inelastic = different directions, don't have same wavelength so permanent energy transfer, damage
Phase of X-rays depends on their origins- build up image of structure
Weak intensity of diffracted radiation. Use a crystal structure
1 crystal unit is an n-repeated macromolecule in different directions
Only damages a small subset. Signals in phase are stronger & rays not in phase cancel (Braggs Law). Get a diffraction pattern with discrete angles of X rays
What are the 2 steps involved in growing your crystal for X-ray crystallography?
How is the crystallisation process aided?
What are the steps involved in attaining a diffraction pattern?
What is the phase problem? How can this be resolved?
What are the steps after getting a diffraction pattern?
How do we know when our structure is reliable?
Why is high resolution useful for biotechnology? (3)
- Use hanging drop method to precipitate water out of the protein over time to reach metastable zone
- Remove crystal from drop with loop & freeze with liquid nitrogen
Salt & additives
- Mount crystal on goniometer & direct X rays on crystal
- Slowly rotate crystal to collect diffraction pattern at each angle
Only wave intensities are recorded & phases are lost- reobtain with different methods
- Convert to electron density map with Fourier transform (get phases)
- Fit electron density map with protein/atomic model
- Refine the model by going back to diffraction pattern & do it again
Fits data not used in procedure, complies with ramachandran plot, high resolution to fit model (observe individual atoms)
Design antibiotics, plan pandemics, control properties of insulin (different oligomeric states and switch between different ones for function e.g shelf life, activation time)
What’s the difference between SEM (scanning) and T/cryoEM (transmission)?
What is the difference between phases in X-ray and TEM?
What are the advantages/disadvantages of negative stain?
What are the advantages/disadvantages of cryoEM?
What can cyroEM be used to study? Why is its image better?
SEM = electrons reflect off, TEM = electrons go through
X-ray = phase information lost so computer used to build up an electron density map
TEM = beams focused with lenses for magnified object for phase information
+ simple prepare, quick to check, high contrast
- dye distorts sample & low resolution
+ sample in native state, high resolution, freeze intermediates
- low contrast, complex, long time to check
Macromolecular assemblies. Averaging improves signal to noise, image all possible orientations & in future all possible conformations
How do you attain a 3D image with cyroEM? What are the 3 steps:
What caused an increase in resolution? (to atomic level) 4
Why is cyroEM better than X-ray?
Why can it be worse?
- Select the 2D images of particles
- Classify the images of same orientation & average them (higher signal to noise)- many cycles
- Use Fourier transform & signal to noise image to form 3D image/proteosome
Better detectors (high signal to noise) Advances in image processing (correct signal to noise) Brighter light sources More powerful electron guns Higher vacuum
Don’t need crystallise. Provides structural info at atomic resolution
More demanding- requires expertise, doesn’t provide info on dynamic regions
What are the advantages of NMR?
Disadvantages?
Which isotopes are most found in proteins/NAs?
E = h y Bo - what is the significance of this?
What happens when a radiowave irradiates the population of nuclei?
The strength of the NMR signal is based on the difference in population, what does this mean about NMR’s sensitivity?
Info on structure, motions (dynamics), interactions including transient, chemistry. Provides info other systems don’t as it’s solution based
Size limited, needs lots of sample
1H, 13C, 15N, 31P
In the presence of an external magnetic field, nuclei exists in 2 spin states- energy difference of the 2 states depends on field strength & efficiency factor (y)
Moves to non-equilibrium state- then returns to equilibrium state inducing a current in receiver coil
Low- not a big difference
What is essential for the magnet to be superconducting in NMR?
Where is the NMR sample tube?
How is a current recorded?
Very cold ~4K (helium & nitrogen)
In a probe (moveable)
Probe contains circuitry to pump in energy & record signal- transformed into NMR spectra
What is the 1D NMR spectra?
What does the frequency of a peak depend on?
Why does a smaller molecule give a sharper peak? What does this mean about NMR?
1H
Microenvironment/nuclear shielding of the proton
Tumbles quicker so nuclei relax slower = sharper signal. Size limited 35kDa
How do magnetic nuclei interact with eachother?
How can you obtain 15N proteins?
What does a 1H-15N 2D NMR topographic map show you?
What are each individual peak? What are they sensitive to?
What is this spectrum used for?
Where are the peaks most likely to shift?
Space (dipole-dipole coupling) or bonding (scalar coupling)
Insert a recombinant protein gene in bacteria in 15N medium- proteins produced with 15N
Correlation between proton & covalently bound nitrogen- so can find NH backbone groups to distinguish the amino acids
Amino acid. Microenvironment of amino acid
Monitor folding/unfolding
Peaks corresponding to amino acids on the surface of the protein
What is NOE caused by?
What can it be used to measure?
What 3rd dimension could be added to the 2D 1H-1H NOESY spectra? What could this tell you?
What does a large peak in this 3D spectra indicate?
What are the steps in accumulating a final image of structures from NMR?
Interaction dipole-dipole between 2 proton nuclei
Distance between them (structure)- NOE is inversely proportion to r^6- observed 5Å away
1H-15H correlation- connect an amide to a proton up to 5Å away
Smaller distance between proton and amide group
- Record NMR spectra, assign the resonance
- Calculate inter-proton/constraints distances
- Restrained molecular dynamics & minimisation protocol- applying random velocities & optimise force fields to guide final fold to satisfy NOE & 1H-15H distances
- Evaluate result with fitting of experimental data (NMR & chemistry) & quality- iterative optimisation to go and repeat cycle
What information do intra-residues provide?
Inter residues?
Dihedral angle restraints? How are these obtained?
How are distance restrains obtained?
Orientation of NH vectors?
Local info conformation side chains
Short range = secondary structures
Long range = NOEs between secondary structures
Conformational restraints (local). Scalar couplings between protons/bonding.
Mainly NOE
Residual dipolar couplings from chemical shift & frequency of atoms
What regions of a protein in NMR have low resolution? How does data quality also affect resolution?
Is unfolding reversible? What causes it?
What are the visual differences in spectra between folded & unfolded species?
Dynamic regions. High = high
Yes- phosphorylation/negative charges.
Unfolded = many more identical frequencies (due to similar chemical microenvironments)- appear closer together
What information does CD provide?
What are its main advantages?
Disadvantages?
What are the requirements?
Why is it not that sensitive of a technique?
Secondary & tertiary structure, folding & stability, macromolecular interactions if structural change happens
(mainly structural change information)
Fast, no labelling
Not sensitive, no atomic resolution info
Chromophore needs to be chiral or in an asymmetric environment
Represents only small percentage of differential absorption
What region is the Far-UV region? What does the signal contribute to?
Why is Far-UV better than Near-UV?
What is Far-UV used for? (4)
180-250nm- backbone of peptide- conformational changes of backbone (secondary structure)
DIRECTLY reports on secondary structure, 50 times more intense
- Look at secondary structures (see if its folded)
- Look at stability & changes (mutations)
- Folding & unfolding
- Macromolecular interactions
What is near-UV CD used to measure?
What is the region wavelength?
Where is the best place to measure changes?
What are the 2 disadvantages and 1 advantage to CD signals in nucleic acids? Helices have characteristic CD spectra tho
Binding affinities, local tertiary structure & quarternary sometimes, look at non-intrinsic protein chromophores (Fe) (intrinsic e.g = Trp, Tyr Phe give own signal)
250-350nm
Where only 1 signal is contributing
- aromatic bases planar & don’t have intrinsic signals
sugar creates asymmetry so weak signals
+ stacking of bases produce intense signals of oligonucleotides
What is biolayer interferometry BLI used to measure?
What is required? (3)
How is the binding of a protein measured in 3 steps?
How can the material dissociate from the biosensor?
How would you set up protein-RNA binding by BLI?
What does comparing the Kd from CD and BLI do?
Binding affinity & kinetics (measures kd between 10-5 and -9M)
High volume protein solution, optimal conditions to minimise non specific binding, labelling of interacting molecules
- Protein/DNA immobilised on sensor with a tag & interference recorded
- Biosensor dipped into well of diff concentrations of binding partners
- Record the shifts of interference- bound molecules change distance between reference layer & interface (& solvent)
Add buffer
Add RNA target sequence to sensor- then add RNA binding protein (of different concentrations as conc is proportional to binding/affinity)
Removes technique dependent bias
What is fluorescence used to monitor? (4)
Pros?
Cons?
What happens to the energy of So in the presence of a polar solvent?
What happens after a photon is absorbed?
How do polar solvents affect stokes shift?
Protein/NA folding, macromolecular interactions, conformational changes, monitor cellular location of proteins/NAs
Very sensitive, monitors structure, stability and interactions
Labelling, doesn’t provide residue level info
Lowered
Increases energy of S1- so S1 decreases over time with polar solvent rearrangement. Photon is emitted to an So of higher energy level due to rearrangement.
Emission is at a lower energy/longer wavelength than absorption
How does fluorescence differ between Tryptophan in a folded protein & unfolded protein? (think exposure to solvent)
What would happen if you were to bind a protein with a molecule containing a trp residue?
Folded = larger energy difference as not exposed to solvent
Unfolded = exposed to solvent, so energy decreases due to solvent rearrangement & see longer wavelength
Exposed to solvent so wavelength decreases
What conditions are required for FRET?
Why is this significant?
Why do they still need to be separated by a small distance in space?
What does this result in when the donor molecule absorbs light?
On what does the transfer efficiency depend?
What more specifically can FRET be used to measure?
Emission spectrum of donor needs to overlap the absorbance spectra of the acceptor/receiver
So acceptor dipole is induced by donor
For higher probability of energy transfer
Electron moves to higher energy state, & transfers nonradiatively energy to acceptor molecule, so acceptor molecule electron drops to lower energy levels & fluoresces.
The distance between acceptor & donor molecule. Ro is distance at 50% (Förster equation)
Intermolecular interactions, conformational changes on ligand binding, monitor pre-mRNA splicing
What does ITC (isothermal titration calorimetry) study?
What is measured?
Ligand is pushed into the sample cell. How does the energy recorded change with the enthalpy of the reaction?
How does this progress with the reaction of binding?
What is observed when the protein is completely saturated?
Energy associated with interaction, info on stoichiometry & equilibrium constant, binding enthalpy
Energy difference to keep sample (protein) cell & reference cell (buffer) the same temperature in a titration
Exothermic interaction = energy released into cell so less energy used to heat
Endothermic = more energy used to heat cell
More protein-ligand bindings, fewer proteins will bind over time so less energy is needed to heat the cell (as it’s exothermic reaction)
Background heat of dilution
If the area under the curve is equal to the total heat released, how can you plot this to get the binding isotherm?
How do you find the Kd, enthalpy & stoichiometry?
How does signal to noise depend on heat?
What protein concentration is necessary? Why is it not anything else?
Plot integrated heat (kcal/mol ligand) against molar ratio of ligand:protein
Slope is 1/Kd, enthalpy is difference between lowest & highest curve, stoichiometry is kcal/mol at equal ratio of protein:ligand (1.0)
Low heat = low signal:noise
c = 10: see Kd, enthalpy & slope of binding c = 1: get Kd but not H c = 1000: get H but not Kd
What are the strengths of ITC?
Limitations?
What other 2 techniques can be used with ITC for completeness?
No labelling, no specific amino acids/prosthetic groups needed
Direct info on forces driving interaction- relate to structure & interacting partners to see reacting mechanism
Best at protein conc of 1 order of magnitude above Kd
Not sensitive- best with reactions with significant enthalpy
atom force microscopy, magnetic tweezers
What are the strengths of ITC?
Limitations?
What other 2 techniques can be used with ITC for completeness?
How do they work?
No labelling, no specific amino acids/prosthetic groups needed
Direct info on forces driving interaction- relate to structure & interacting partners to see reacting mechanism
Best at protein conc of 1 order of magnitude above Kd
Not sensitive- best with reactions with significant enthalpy
atom force microscopy, magnetic tweezers
AFM: many domain protein attach to surface & to cantilever- force unfolds protein & reveals individual domains
MT: rotating magnet adds torsion to the beads with DNA attached- nickase cleaves DNA strands from molecule
