Lecture 8 - 11 Flashcards
Examples of protein-DNA interactions
Histones recognising DNA for packaging
RNA polymerases recognising correct DNA sequence for transcription
Topoisomerases recognise DNA to unwind it
Why is histidine not often used in binding site
Has a pKa of 7 - so in biological conditions exists in 50:50 charged: uncharged - too unpredictable to be involved in binding
Zinc Finger
Zinc found in 2+ form
Zinc finger binds to DNA at 3 points - not sufficient for selectivity - require more than 1 zinc finger
Found in steroid receptors e.g. oestrogen receptor
Each finger = 2 beta sheets attached to 1 alpha helix
Helix-turn-Helix
2 alpha-helices -each binds to major groove in DNA
Results in DNA bending - can bring domains together to result in dimerisation
Example - lac repressor
Leucine zipper
2 alpha helices that form coiled coil around each other
Basic - basic area involved in binding to the major groove of DNA
Leucine every 7 aa
Reasons for having membrane in cells
Allows selective permeability
Allows generation of electrical/chemical gradient - can be used to generate energy
Creates a scaffold for cytoskeleton
Protects from environment
Sterols
Alter membrane fluidity
Peripheral protein removal
Via mild treatment e.g. pH
Integral protein removal
Via strong detergent
Type of residues in membrane areas
non-polar
Difficulties in obtaining structural info about membrane proteins
Difficult to find host, can be toxic to cell
MP are unstable outside of membrane
Difficult to crystalise - every protein requires different detergent concentration
Use antibodies to prevent aggregation
Amino acids in membrane spanning regions
hydrophobic residues at surface
Tyr/Trp at interface
Charged residues act as snorkels
Hydropathy index
Used to predict if segment could be membrane spaning - free energy of transfer to water
Sec pathway
Adds alpha-helix transmembrane protein
Driving factors for integration of alpha-helix transmembrane protein
Positive-inside rule (intracellular residues more +ve)
Charge difference
TMH hydrophobicity
Transport channels
Can be active or passive
Carrier proteins
Can be saturated - shuttle specific molecule. Gates not open both ways simultaneously e.g. glucose
Channel
Has no limit to capacity - hydrophillic. Open and close due to stimuli
k+
Voltage gated - requires action potential
Where is ATP synthetase found?
Chloroplast thylakoid membrane, cell membrane (prokaryotes), inner mitochondiral membrane (eukaryotes)
Bragg’s Law
Will see diffraction if waves scatter in phase
What does diffraction pattern show?
Distance between the atoms
Crystallisation procedure
- Purify protein (>95%)
- Crystalliation
- Diffraction pattern
- Electron density map
- Atomic model
- Validation
- PDB deposition
Crystallisation variables
Protein (purity, conc)
Precipitant ( conc, type)
Temperature
Buffer (pH, conc)
Unit cell
Smallest volume containing all structural and symmetry information required to build the proten structure via translation
Assymetric unit
Smallest unique volume contains all structural info - through applying symmetry can get unit cell
Resolution required to see individual atoms
1.2A
Refinement and R factor
Refinement = fitting side chains to correct errors. R-factor compares model to experimental data
B-factor
Shows thermal movement. The higher the number, the greater the movement = less valid result
Occupancy
The fraction of time an atom spends in a specific position, 1= always, 0 = never
cryo-EM resonance
proportional to wavelength
proteins and nucleic acids have a low amplitude contrast compared with…
ice
Cryo-EM: defocus contrast…
changes phase constrast to amplitude contrast
Vitreous Ice
Want ice to free very quickly so that sample doesn’t form crystals - use liquid ethane
Why do not want proteins to stick to ice/air boundary?
That would result in all proteins being in same conformation - want random conformations
Cryo-EM - how to correct for thermal drift/vibration?
Take many rings
What do the Thon rings show?
The resolution of the sample
Single particle reconstruction method (Cryo-EM)
- Motion correction
- CTF estimation
- Particle picking
- 2D classification
- 3D classification and refinement
- Postprocessing
Fourier shell correlation (Cryo-EM)
compare 2 independent models and see at what resolution they correlate
How to carry out Cryo-EM in situ?
Use electron cryo-tomography
What size can carry cryo-EM out on?
Samples <100kD
Limits of Cryo-EM?
Not good for small samples
Gives low contrast data - require more information
Not very high resolution
