Lecture 12 - 16

Question 1

Advantages of NMR over X-ray Crystallography

Answer 1

Protein can be in solution - natural state
Can study protein dynamics
Can see interactions that protein makes

Question 2

Atoms used

Answer 2

1H, 13C, 15N - spin of 1/2

Question 3

Halpha chemical shift in secondary structure

Answer 3

Lower for alpha helix than beta sheet

Question 4

How to determine alpha helix/beta sheet from chemical shift index

Answer 4

4 or more consecutive -1/+1

Question 5

J

Answer 5

spin-spin coupling constant - when nearby nuclei split a signal

Question 6

Relaxation

Answer 6

Time (T) taken for NMR signal to disappear - shorter T means there are other nuclei nearby

Question 7

Nuclear Overhauser Effect (NOE)

Answer 7

Depends on relaxation between spins - if you saturate one nuclei, the nuclei nearby will have a stronger signal

Question 8

R

Answer 8

Distance between 2 relaxing nuclei

Question 9

NOE signal + distance between nuclei

Answer 9

Strong - 1.8 - 2.7A
Medium - 2.7 - 3.5A
Weak - 3.5 - 5A

Question 10

Amide protection

Answer 10

When amide protons are involved in hydrogen bonds in a folded protein, they are protected from exchange with solvent and exchange more slowly

Question 11

Protonation factor

Answer 11

Rate of expected change (unfolded)/ Rate of actual exchange

Question 12

Problems with large molecule NMR

Answer 12

Issues with unresolved signals
Overlapping signals
Complex multiplets
Low sensitivity - use high B0
Large quantity of info - use computational methods

Question 13

Ligand

Answer 13

Non-macromolecules that interact with proteins - water not normally counted as ligand e.g. peptides nucleotides, ions

Question 14

Surface characteristics of protein

Answer 14

Size, shape, charge, hydrophobicity, dynamics e.g. flexibility

Question 15

Most favourable protein shape

Answer 15

Globular/spherical

Question 16

Protein crystallisation steps

Answer 16

Purify protein
Crystallise protein
Collect data
Evaluate data
Model building
3D structure

Question 17

Why is it hard to crystallise proteins?

Answer 17

Proteins not evolved to crystallise, may be dynamic, every protein requires different parameters, can be unstable

Question 18

Structural proteins

Answer 18

Keratin, collagen, fibroin

Question 19

Motor proteins

Answer 19

Actin/myosin, kinesin, ATP synthase

Question 20

Fibroin

Answer 20

Antiparallel beta sheets with ala and gly side chains

Question 21

To get pure spider silk

Answer 21

Ligate consensus sequence into expression vector, transform into e.coli and express and purify

Question 22

MotA/MotB

Answer 22

convert proton gradient into energy for movement

Question 23

FliG, FliM and FliN

Answer 23

Interact with MotA/B to cause a direction change

Question 24

FliF

Answer 24

self-assembled, forms template for rest of structure to assemble from

Question 25

Amino acid in outlier range in ramachandran plot

Answer 25

Glycine - so small that doesn't really have many steric clashes

Question 26

Protein homeostasis maintained by...

Answer 26

protein folding/unfolding

Question 27

Folding is exothermic/endothermic

Answer 27

exothermic

Question 28

Half unfolding point

Answer 28

melting point - Tm. sharp change between folded and unfolded state due to cooperative binding

Question 29

Excreted misfolded proteins

Answer 29

Aggregate, which can form amyloids - result in disease

Question 30

Promiscuous activity

Answer 30

duplicated gene has a small degree of secondary activity , this can become the main activity if beneficial through mutation.

Question 31

Glycosyl hydrolases involved in..

Answer 31

Degredation of poly/oligosaccherides

Question 32

Saccherification

Answer 32

hydrolysis into soluble sugar

Question 33

Hydrophobic cluster analysis

Answer 33

shows folding similarities through hydrophobic/hydrophillic residues - can determine structural families

Question 34

Digestive protease

Answer 34

Low specificity, works well in acidic environment

Question 35

General proteases

Answer 35

high specificity, only cleave at one specific peptide bond in one specific molecule. adapted to specific environment

Question 36

How do mutations make drug ineffective?

Answer 36

Change in sequence, change in structure, change in binding site = ligand/drug can no longer bind