Proteins: Protein Folding

Question 1

How do proteins have to fold?

Answer 1

With high fidelity, remaining dynamic, binding tightly and specifically to ligands, with control and degradation pathways, with a method to unfold.

Question 2

On a folding funnel what does the diameter of the opening (top) represent?

Answer 2

Entropy

Question 3

On a folding funnel what does the length represent?

Answer 3

Energy

Question 4

In its native state what properties does the protein have?

Answer 4

Low enthalpy and low entropy

Question 5

What is a molten globule?

Answer 5

When proteins populate partially folded states under mildly denaturing conditions (such as acidic pH, low urea concentration or when a cofactor/metal is removed).

Question 6

What is key to many cellular processes?

Answer 6

Constant folding and unfolding.

Question 7

What is protein folding important for?

Answer 7

Structure prediction, biotechnology, de novo protein design, protein folding in vivo, medicine etc.

Question 8

What did Anfinson do?

Answer 8

Found that the primary sequence determines the structure, and that it was an example of spontaneous self-assembly. Also came to the realisation that mutations caused diseases of protein misfolding.

He won the Nobel prize in 1972

Question 9

What dud Levinthol do?

Answer 9

Found that proteins fold along defined pathways on funnel shaped landscapes and stated protein folding was not random.

Question 10

What did Ranganathan find?

Answer 10

The idea of convolution and conservation and that only a few amino acids are required to define the protein folding pathway to native state and therefore protein folding is a lot less complex than initially thought.

Question 10

What do you need to define protein folding?

Answer 10

To describe the structured intermediate partially folded state, to describe the energetics of the process, to understand chain collapse, structural properties of intermediates, when tertiary structure forms, if non-native structure forms, how proteins misfold and when the reaction is complete.

Question 11

What is hen lysozyme?

Answer 11

A glycosidase enzyme which breaks down bacterial cell walls.

Question 12

What is the structure of hen lysozyme?

Answer 12

Small, soluble, globular 129 amino acid protein with a mixed alpha and beta fold and 4 disulphide bonds.

Question 13

Why are methods of initiating and monitoring folding combined?

Answer 13

To give a detail picture of the folding and unfolding process.

Question 14

What does hydrogen exchange labelling and NMR provide?

Answer 14

Information on formation of persistent hydrogen bonds burial from solvent at specific sites.

Question 15

What does hydrogen exchange labelling and ES-MS provide?

Answer 15

Information on folding populations.

Question 16

What does far UV CD provide?

Answer 16

Secondary structure information.

Question 17

What does near UV CD provide?

Answer 17

Tertiary structure information.

Question 18

What does intrinsic fluorescence provide?

Answer 18

The environment of tyrosine and tryptophan residues.

Question 19

What does ANS binding provide?

Answer 19

Information on exposure of hydrophobic surface area.

Question 20

What does inhibitor binding provide?

Answer 20

Information of the formation of active site.

Question 21

What method/s provide information on the environment of aromatic residues?

Answer 21

Absorbance and real-time NMR.

Question 22

How does stopped flow tryptophan fluorescence tell you about the tryptophans environment?

Answer 22

When exposed tryptophan express more and when exposed the protein is likely to be in its nascent state.

Question 23

What environments are hen lysozyme tryptophans in?

Answer 23

Six in total, 2 are highly exposed in native state despite their hydrophobicity as they are involved in substrate binding.

Question 24

What can be used to obtain residue specific information?

Answer 24

Hydrogen exchange and NMR, or mutational analysis.

Question 25

How does hydrogen exchange and NMR give you important information?

Answer 25

The amide on the backbones can rapidly exchange with solvent, the rate is critically dependent on pH and in native protein the rate is very slow. All -NH can be assigned to individual residues using NMR.

Question 26

How does lysozyme fold?

Answer 26

By domains and multiple rates, the transition state simplifies the protein folding problem.

Question 27

How do small proteins fold?

Answer 27

On relatively smooth landscapes, with unpopulated intermediates and only a few contacts required to define the native fold the remaining structure consolidates around the nucleus.

Question 28

How do large proteins fold?

Answer 28

On rough energy landscapes, following multiple pathways into domains, with populated intermediates.

Question 29

Describe hydrophobic collapse?

Answer 29

The exclusion of water and the formation of short-range interactions in a molten globule state.

Question 30

Describe consolidation?

Answer 30

Formation of long range interactions and finding the lowest energy conformation.

Question 31

What can folded states lead to?

Answer 31

Damage, unfolding, misfolding or aggregation.

Question 32

What can all states lead to?

Answer 32

Aggregation.

Question 33

What are proteins half lives?

Answer 33

Short

Question 34

How do you favour folding over aggregation?

Answer 34

Reduce the concentration of unfolded proteins to reduce folding and thre gore possible aggregation. Everything that binds to the unfolded reduces the concentration of free unfolded and will therefore favour folding.

Question 35

Define molecular chaperones?

Answer 35

A protein that is involved in the correct folding, transport, assembly or degradation of a polypeptide chain without becoming part of the final functional complex.

Question 36

Describe molecular chaperones?

Answer 36

A diverse family of gene products whose expression is generally heat induced (HSP). They are often classified into families according to their molecular weight. They are not foldases as they do not catalyse (they don’t stabilise the transition state). They are promiscuous and bind to almost any non-native protein. They are particularly held in large storage assemblies and protect polypeptides from cradle-to-grave.

Question 37

What do small HSP do?

Answer 37

Act as a buffer of protein aggregation, they reversible bind the misfolding protein to prevent aggregation under heat shock or stress. Under no stress folding is favoured and allowed.

Question 38

What HSP bind the nascent chains?

Answer 38

HSP 70/40 (DNA K/J in E.coli)

Question 39

What binds to the polypeptide as it emerges from the ribosome?

Answer 39

Trigger factor and a peptidyl-prolyl isomerase NAC

Question 40

What does HSP90 do?

Answer 40

Least well understood, binds ATP and other proteins acting on protein conformation, involved in Raf-signalling, steroid hormone receptor activation and it’s at the hub of many processes in eukaryotes.

Question 41

Which HSP disaggregated proteins?

Answer 41

HSP 100, performs am ATP driven conformational change.

Question 42

What aids in aggregate binding and feeding into a protease?

Answer 42

HSIUV, ClpAP and ClpXP

Question 43

Describe HSP 70?

Answer 43

The most ubiquitous heat shock protein, useful for cells activity in heat shock, cold shock, removing the clathrin coat, chaperoning nascent chains, import into the mitochondria and important in the ER.

Question 44

Describe the structure of HSP70?

Answer 44

Monomeric, 70 kDa. Has a weak ATpase activity and a strong affinity for non-native polypeptides.

Question 45

what works with HSP 70?

Answer 45

Nucleotide exchange factor GrpE in the ATPase cycle and co-protein HSP 40 (DNAJ on E.coli)

Question 46

What is HSP 90 used for?

Answer 46

Steroid hormone loading receptor activation and modulating protein conformation.

Question 47

Where is the GroE chaperonin complex from?

Answer 47

E.coli

Question 48

What is GroEL?

Answer 48

Homologous to rubisco binding protein in chloroplasts and HSP60 in mitochondria. Required for cell viability at all temperatures and conditions. It has a weak ATPase activity and a strong affinity for non-native proteins. GroES binds in the presence of ATP or ADP.

Question 49

Describe the structure of GroEL?

Answer 49

57.4 kDa monomer, 14 subunits (7-fold symmetry) and a molecular weight of 800 kDa.

Question 50

What is the GroE effect?

Answer 50

Increases the efficiency of folding of a wide range of E.coli proteins by producing a complex with a large cavity which is used as a cage for the proteins to fold in.

Question 51

How are specimens prepared for cryo-EM?

Answer 51

The protein is embedded in a layer of heavy metal salt to allow for contrast between water and protein atoms. The sample is then dried onto a carbon film and negatively stained or cooled by plunging the sample with a weak buffer into cold solvent with high thermal conductivity which causes vitrification.

Question 52

How does basic EM work?

Answer 52

High energy electrons focused with magnetic lenses to form a magnified image.