Protein Dynamics

Question 1

What types of proteins dynamics are there?

Answer 1

Atomic vibrations
Collective movement of domains
Triggered conformational changes of whole subunits
Transient unfolding

Question 2

How fast do individual atoms vibrate?

Answer 2

10^15 to 10^11 per second

Question 3

What distance do atoms vibrate?

Answer 3

0.01 to 1A

Question 4

How fast do domains move?

Answer 4

10^12 to 10^3 per second

Question 5

How far do domains move?

Answer 5

0.01 to 5A

Question 6

How far do subunit conformational changes move?

Answer 6

0.5 to 10A

Question 7

How fast do subunits move?

Answer 7

10^-3 to 10^9 per second

Question 8

Why are dynamics important?

Answer 8

Catalysis
Ligand binding
Formation of complexes
Active site availability

Question 9

What effect do dynamics have on a reaction progression energy plot?

Answer 9

Not smooth as rapid interconversions

Question 10

What effect does temperature have on an energy diagram?

Answer 10

More energy so smoother

Question 11

How can dynamics be measured?

Answer 11

Empirical measurements of ligand release
Quenching of aromatic amino acids
HX NMR

Question 12

What small molecules can be used for quenching?

Answer 12

I-, O2, acrylamide

Question 13

Why is quenching not a good measurement?

Answer 13

Some non-specific binding

Question 14

What molecules are used for HX NMR?

Answer 14

D20 exhanges with amides

Question 15

Why is deuterium used for HX?

Answer 15

Different spin for NMR

different mass for MS

Question 16

What is rate of HX determind by?

Answer 16

Ki exchange,
Ko opening
Kc closing

Question 17

Which residues exchange first?

Answer 17

Exposed, un-bonded ones

Question 18

How is rate of HX dependant on pH?

Answer 18

10x increase for each pH unit above pH3

Question 19

Why is 2D NMR used for large proteins?

Answer 19

To separate overlapping peaks of similar environments

Question 20

What are the types of 2D NMR?

Answer 20

COSY
NOESY
TOCSY
HSQC

Question 21

What spectrum does HSQC produce?

Answer 21

15N-1H spectrum of all amino acids except Proline

Question 22

What spectrum does COSY produce?

Answer 22

Protons coupled by 3 or fewer bonds

Question 23

What spectrum does NOESY produce?

Answer 23

Protons within 5A space

Question 24

What spectrum does TOCSY produce?

Answer 24

protons coupled by bonds and all protons in a spin system

Question 25

What evidence does NMR provide for protein dynamics?

Answer 25

Phe/Tyr ring flipping faster than 10,000s-1 produces an overlapping peak
Dynamic regions have no measurable distance between active nuclei
Modern techniques measure small scale sidechain movements on psec scale

Question 26

Why do His/Trp not show ring flipping?

Answer 26

Too bulky and unsymmetrical so do not have 360’ rotation

Question 27

What evidence does X ray crystallography provide?

Answer 27

Smearing of dynamic regions

More conformations at higher temperatures

Question 28

What is the crystallography B factor?

Answer 28

The temperature factor/chain felixibilty

Question 29

How is B factor affected by temperature?

Answer 29

Increases

Question 30

How is B factor affected by ligand binding?

Answer 30

Reduced and rigidity is imposed

Question 31

Why can crystallography show many conformations?

Answer 31

Snapshots in each crystallisation

Question 32

How can molecular dynamics be used to observe dynamics?

Answer 32

Atoms of known structure are given known velocity and movement can be measured based on new position and acceleration
Many small steps is highly detailed

Question 33

What does molecular dynamics show?

Answer 33

2’ fluidity in core
increased mobility in loops
rigid secondary structure
rates of interconversion

Question 34

What forces exist on atoms?

Answer 34

bond length, angles and torsion angles

Non-bonded interactions

Question 35

How does the B factor affect crystallography?

Answer 35

Smaller B factor has more rigidity so better scattering