exploaring proteins lecture 2

Question 1

domain

Answer 1

autonomous folding protein unit

Question 2

module

Answer 2

a domain with a contiguous sequence, repeatedly used in diverse proteins

Question 3

repeat

Answer 3

unit that doesn’t fold in isolation, several copies are needed

Question 4

eg of a core domain that is in lots of proteins

Answer 4

IG domain

Question 5

Ig domain facts about it

4 points

Answer 5

about 100 residues
2 layered sandwich of 7 to 9
antiparallel beta sheets
in greek key motif

Question 6

2 main variations of Ig domains

Answer 6

4 strand beta sheet DEBA

3 strand beta sheet EBA

Question 7

what do interdomain linkers also do

Answer 7

they act as hinges

allowing simple shape changes

Question 8

binding motifs are found on

Answer 8

unsaturated protein regions

Question 9

some proteins do not take structure because of the unsaturation why is this

Answer 9

motif recognised by proteins

signalling proteins act by being disordered

Question 10

spectroscopic methods

Answer 10

absorption
concentration
beer lamberts law A=E/c

Question 11

circular dichroism what is it

Answer 11

measures the difference between left and right polarized light as a function of wavelength

Question 12

what is optically active so that circular dichroism can work

Answer 12

peptide bond

which in turn is selective to protein conformation

Question 13

what does CD require

Answer 13

low amounts of protein

Question 14

electron microscopy
what is the resolution
what is needed

Answer 14

image of a single particle is possible -20A

crystalline samples needed

Question 15

what increases contract in EM

however what are the drawbacks

Answer 15

metal staining

the sample could be damaged or changed

Question 16

what does EM provide

Answer 16

the analysis of a whole cell

and distribution of the protein inside the cell

Question 17

EM limitations

Answer 17

radiation damage

transmission EM sample must be very thin

Question 18

Cryo EM
what is the advantage of using this
4points

Answer 18

Flash freeze sample
preserves the structure in aqueous solution
but limits radiation damage
and we don’t need to use metal staining

Question 19

flash freezing what is used to do this

Answer 19

liquid ethane

Question 20

3 ways to enhance signal to noise and resolution in cryo EM

Answer 20

em crystallography (3d/2D possible)
single particle reconstruction
em tomography

Question 21

em crystallography (3d/2D possible) what does it entail

Answer 21

regular lattice
allows averaging over many particles
as well as using periodicity of objects to enhance reso
ie diffraction of electrons leads to diffraction patterns

Question 22

single particle reconstruction what does it entail

Answer 22

accumulates many images of individual particles to improve signal to noise

Question 23

EM tomography what does it entail

Answer 23

rotating object and recording a series of 2D images

reconstructing as a 3D image

Question 24

scattering and molecular shape

what happens

Answer 24

incidence ray is scattered by sample angle is measured

if a domain is know with atomic detail we can rearrange them to give the right shape

Question 25

structure determination via x-ray crystallography
what do you have to do
6 points

Answer 25

make crystal protein
collect diffraction data
convert diffraction data into electron density map
also need additional info not just diffraction spots
because of phase problems
fit molecular model into electron density map

Question 26

step 1 and 2 of x-ray crystalography

Answer 26

making protein crystal

x-ray beam and then diffraction pattern

Question 27

step 3 of crystallography

4 points

Answer 27

make additional crystals with heavy atom derivatives
incorporate seleno-methionine protein using its anomalous diffraction to determine the phase
if similar protein structure is known back calculate the electron map

Question 28

molecules that have a similar structure can be used to work out the electron density map what is this called

Answer 28

homologous replacement

Question 29

step 4 building model of crystallography

Answer 29

anomalous scattering
changing beam intensity
some atoms absorb, we can work out the phase
can substitute Si for sulphur