Lecture 8 - 11

Question 1

Examples of protein-DNA interactions

Answer 1

Histones recognising DNA for packaging
RNA polymerases recognising correct DNA sequence for transcription
Topoisomerases recognise DNA to unwind it

Question 2

Why is histidine not often used in binding site

Answer 2

Has a pKa of 7 - so in biological conditions exists in 50:50 charged: uncharged - too unpredictable to be involved in binding

Question 3

Zinc Finger

Answer 3

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

Question 4

Helix-turn-Helix

Answer 4

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

Question 5

Leucine zipper

Answer 5

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

Question 6

Reasons for having membrane in cells

Answer 6

Allows selective permeability
Allows generation of electrical/chemical gradient - can be used to generate energy
Creates a scaffold for cytoskeleton
Protects from environment

Question 7

Sterols

Answer 7

Alter membrane fluidity

Question 8

Peripheral protein removal

Answer 8

Via mild treatment e.g. pH

Question 9

Integral protein removal

Answer 9

Via strong detergent

Question 10

Type of residues in membrane areas

Answer 10

non-polar

Question 11

Difficulties in obtaining structural info about membrane proteins

Answer 11

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

Question 12

Amino acids in membrane spanning regions

Answer 12

hydrophobic residues at surface
Tyr/Trp at interface
Charged residues act as snorkels

Question 13

Hydropathy index

Answer 13

Used to predict if segment could be membrane spaning - free energy of transfer to water

Question 14

Sec pathway

Answer 14

Adds alpha-helix transmembrane protein

Question 15

Driving factors for integration of alpha-helix transmembrane protein

Answer 15

Positive-inside rule (intracellular residues more +ve)
Charge difference
TMH hydrophobicity

Question 16

Transport channels

Answer 16

Can be active or passive

Question 17

Carrier proteins

Answer 17

Can be saturated - shuttle specific molecule. Gates not open both ways simultaneously e.g. glucose

Question 18

Channel

Answer 18

Has no limit to capacity - hydrophillic. Open and close due to stimuli

Question 19

k+

Answer 19

Voltage gated - requires action potential

Question 20

Where is ATP synthetase found?

Answer 20

Chloroplast thylakoid membrane, cell membrane (prokaryotes), inner mitochondiral membrane (eukaryotes)

Question 21

Bragg’s Law

Answer 21

Will see diffraction if waves scatter in phase

Question 22

What does diffraction pattern show?

Answer 22

Distance between the atoms

Question 23

Crystallisation procedure

Answer 23

1. Purify protein (>95%)
2. Crystalliation
3. Diffraction pattern
4. Electron density map
5. Atomic model
6. Validation
7. PDB deposition

Question 24

Crystallisation variables

Answer 24

Protein (purity, conc)
Precipitant ( conc, type)
Temperature
Buffer (pH, conc)

Question 25

Unit cell

Answer 25

Smallest volume containing all structural and symmetry information required to build the proten structure via translation

Question 26

Assymetric unit

Answer 26

Smallest unique volume contains all structural info - through applying symmetry can get unit cell

Question 27

Resolution required to see individual atoms

Answer 27

1.2A

Question 28

Refinement and R factor

Answer 28

Refinement = fitting side chains to correct errors. R-factor compares model to experimental data

Question 29

B-factor

Answer 29

Shows thermal movement. The higher the number, the greater the movement = less valid result

Question 30

Occupancy

Answer 30

The fraction of time an atom spends in a specific position, 1= always, 0 = never

Question 31

cryo-EM resonance

Answer 31

proportional to wavelength

Question 32

proteins and nucleic acids have a low amplitude contrast compared with…

Answer 32

ice

Question 33

Cryo-EM: defocus contrast…

Answer 33

changes phase constrast to amplitude contrast

Question 34

Vitreous Ice

Answer 34

Want ice to free very quickly so that sample doesn’t form crystals - use liquid ethane

Question 35

Why do not want proteins to stick to ice/air boundary?

Answer 35

That would result in all proteins being in same conformation - want random conformations

Question 36

Cryo-EM - how to correct for thermal drift/vibration?

Answer 36

Take many rings

Question 37

What do the Thon rings show?

Answer 37

The resolution of the sample

Question 38

Single particle reconstruction method (Cryo-EM)

Answer 38

1. Motion correction
2. CTF estimation
3. Particle picking
4. 2D classification
5. 3D classification and refinement
6. Postprocessing

Question 39

Fourier shell correlation (Cryo-EM)

Answer 39

compare 2 independent models and see at what resolution they correlate

Question 40

How to carry out Cryo-EM in situ?

Answer 40

Use electron cryo-tomography

Question 41

What size can carry cryo-EM out on?

Answer 41

Samples <100kD

Question 42

Limits of Cryo-EM?

Answer 42

Not good for small samples
Gives low contrast data - require more information
Not very high resolution