Lecture 11 Protein Folding

Question 1

History

Answer 1

Bridgman 1914 - high pressure changes protein structure

H. Wu 1924-40 pioneering protein folding

Chris Anfinsan purifying proteins

Cyrus Leviathal analysing DNA sequences

Question 2

Non covalent interactions

Answer 2

Comparitively weak non-cov bonds but more can form than cov bonds in any macromolecule. Proteins are not very stable an H bond is 10kj/mol - easily broken.

So cov bonds give framework but overall shape is determined by non-cov bonds

These allow specific reversible interactions

Question 3

Folding

Answer 3

Decreases entropy - but often offset by increase in entropy of surrounding solvent.

When unfolded hydrophobic side chains force order of water molecules which is not entropically favourable

Folding removes interactions between hydrophobic regions of protein and solvent.

Hydrophobic core remains

Question 4

Forces holding 3D structures together are comparitively weak

Answer 4

Folded form is more stable than unfolded form

It is a balance between 2 competing energies delta H and T delta A

Stability (delta G) is a summation of a large no. of weak delta H interactions balanced against a decrease in entropy delta S in protein caused by folding and an increase in delta S of solvent (hydrophobic effect)

Question 5

Disulphide bonds can stabilise 3D structures

Answer 5

But do not determine them.
S-S bonds make a protein more stable at higher temperatures

Question 6

Summary: protein folding is a balance of:

Answer 6

Energy of non-cov interactions within the protein/within the solvent

Entropy effects of folding protein/ in the solvent

Combining to give free energy for folding that determines whether it it thermodynamically favoured or not.

If folding is not thermodynamically favoured the protein unfolds (denatures)

Protein folding easily affected by a range of factors affecting balance e.g. temp. pH and chemicals

Question 7

Is protein folding reversible - Anfinsen refolding experiment

Answer 7

Active folding protein chemically unfolded then refolded to active form

Disulphide bonds not essential for formation of correct structures but act to stabilise correct structures once formed (incorrect disulphide bonding prevents correct structure forming)

Anfinsen experiment doesn’t work with all proteins.

Many do not recover function on refolding once denatured - protein misfolding

So chaperone proteins aid folding in vivo

Question 8

Protein folding not a simple problem - Levinthal paradox - protein folding is not a random process

Answer 8

A small protein of 100 amino residues each of which can assume 3 confirmations

Total no. of structures would be 3¹⁰⁰ or 5x10⁴⁷

If it takes 10-¹³ S for a cov bond vibration to convert one structure to another then total search time to cover all possible confirmations would be 5x10⁴⁷ x 10-¹³S or 1.6x10²⁷ years
greater by ~17 orders of magnitude than the age of the universe
It would take too long for even a small protein to fold properly by randomly trying all confirmations

Protein folding is not a random process

Question 9

Progressive protein folding

Answer 9

Progressive model exploits local folding in secondary structures

Unfolded polypep (U) undergoes folding by a pathway of partially folded intermediates
Involves local folding of a small no. Of residues so relatively fast
“Centres” of local folding accumulate to a point where most of the molecule is folded (I1- In)
Final folding steps bring regions of local folding together to form final folded structure (F)
Protein domains often fold independently of the rest

Question 10

Protein folding funnel analogy

Answer 10

Similar to rolling a coin down a funnel

Confirmational entropy is referring to the protein and ignoring entropy of solvent

Denatured state: high confirmational entropy and high free energy

Native state: low confirmational entropy and low fee energy

Question 11

Protein folding in vivo

Answer 11

Does not involve a completely unfolded polypep because the polypep starts to fold as soon as it is synthesised - because of translation process. Protein folding in vitro starts with a complete unfolded polypep.
Folding funnel and progressive model applies to both.

Question 12

Protein unfolding

Answer 12

Denaturation by heat force or chemical

Question 13

Protein unfolding: heat

Answer 13

Increases movement in molecule, breaks weak interactions like H-bonds

causing loss of 3D structure
>loss of function
> Insoluble aggregates form

E.g. egg white

Raw: folded soluble aqueous 10% globular protein solution

Poaching: unfolding denatured by heat

Poached: insoluble egg white protein aggregates formed as result of heat denaturation

Question 14

Denaturation at hydrophobic surfaces

Answer 14

E.g. whisked eggs form meringue - loss of tertiary structure due to removal of surrounding “shell” of water molecules from proteins at air interface tipping balance towards unfolding

Bubbles of air coated with precipitated denatured protein
Similarly protein + organic solvent leads to denaturation and precipitation

To prevent this - treat surface to avoid interactions with proteins - a process used in medicine and chemical plants

Question 15

Chemicals that chase protein unfolding (denaturants)

Answer 15

Denaturing agents disrupt non-cov interactions causing denaturation

e.g. disruption of salt bridges and other electrostatic interactions involving ions: by extreme pH causing loss of charge or introduction of new charge causing them to repel oneanother

. v. high salt conc. can disrupt electrostatic interactions in org solvents e.g. acetone interferes with hydrophobic interactions leading to protein aggregation and insolubility

Question 16

Chemical denaturants

Answer 16

Disruption of hydrophilic interactions by non polar compounds in solution such as alcohols or detergents

Detergents contain hydrophobic groups linked to a charge that makes them soluble in water overall e.g. dodecyl sulphate ions

Sodium dodecyl sulphate (SDS) both denatures and solubilises proteins this is why detergents are effective for cleaning fabrics and other materials coated with protein deposits.

Disruption of H bonds by compounds which form H bonds themselves and compete with internal H bonds e.g. urea and guanidine do not make proteins insoluble and proteins remain as single unfolded polypeptides

Question 17

Effects of denaturing agents are conc. Dependent

Answer 17

Diff proteins show diff degrees of resistance to denaturation due to diff degrees of stabilisation in their 3D structure.
Any proteins unstable outside a limited pH range

A high conc. Of H bonds disruptors like urea is required for denaturation typically >4 moles

Detergents are effective in protein denaturation and solubilisation at low conc. <1%

Question 18

Effect of denaturant is conc dependent so stability of protein can be analysed with 2 state model

Answer 18

NATIVE <-> DENATURED

Equilibrium constants for this process can be calculated at each denaturant conc. Then extrapolation to 0 denaturant performed.

Gives equilibrium constant and free energy of stability (delta G) of protein in norm. Solution

Question 19

Unfolding

Answer 19

Often leads to formation of insoluble aggregates of protein and an irreversible loss of protein function unless specific chemicals such as detergents are present

Aggregation clearly a problem to living organisms

Question 20

Protein misfolding countered by chaperones and chaperonins

Answer 20

There are protein chaperones and chaperonins. They require energy input by ATP to force confirmational change in their structure affecting bound partially folded protein aubstrate

e.g hsp70 chaperonin prevents protein folding prematurely by binding to patches on nascent polypep

E.g. hsp60 chaperonin complex unfolds and refolds misfolded proteins

E.g. GroEL-GroES bacterial chaperonin complex acts as a refolding bin providing an environment in which partially folded proteins can be remodelled and folded correctly

Question 21

Aggregates and amyloid deposits: both insoluble but diff

Answer 21

Aggregates contain polypeps that have a random structure, may have residual secondary structure but no regularity

Amyloid fibrils have a secondary structure high in beta pleated sheets organised in repeating arrays. Amyloid forming process can be self-catalysing - if a protein is partially unfolded and misfolds to form an amyloid precursor structure further protein molecules can add by partial unfolding and refolding to amyloid structures.
Amyloid formation is sequence dependent - mutations in protein sequence can alter tendency to form amyloid deposits.
Certain proteins are prone to misfolding and amyloid formation e.g amyloid beta protein that causes Alzheimer’s disease

Question 22

Prion proteins : degenerative CJD

Answer 22

Form amyloid deposits when misfolded and can act as infective agents

Degenerative conditions like Creutzfeldt Jakob disease (CJD) result from self-catalysing conversion of a pre-existing protein called a prion from soluble to insoluble form.

This can occur as the result of a mutation or by exposure to insoluble prion protein in this case the protein can act as an infective agent. Accumulation of insoluble protein deposits in amyloid fibrils and plaques leads to irreparable cell damage to neuronal tissue

Change in secondary and Tertiary structure between soluble prion and insoluble plaque forming state - disease causing prion converts normal prions causing a chain reaction.

Question 23

Degenerative amyloid conditions

Answer 23

Alzheimer’s, spongiform encephalitis and type ll diabetes also result from conversion of soluble proteins to insoluble fibres

E.g. lewy body structures develop grey filamentous structures in Parkinson’s disease

Question 24

Protein folding and denaturation summary

Answer 24

Protein folding is the process that generates secondary and tertiary structure responsible for 3D shape - maintained by weak non cov bonds

Denaturation of proteins is loss of correct folding state usually accompanied by loss of function usually loss of secondary and tertiary structure

Denaturation can be caused by physical conditions: temp., hydrophobic interfaces, solvents or chemicals that disrupt non-cov interactions

H bond breaking chemicals and detergents can keep denatured proteins in solution as soluble polypeptides, most other denaturing conditions cause insolubility and aggregation

Some denatured proteins can spontaneously regain 3D structure and functional activity - shows primary structure dictates all levels of folding structure

Folding occurs by localised folding foci in progressive manner

Folding in vivo is associated with chaperones and chaperonins many proteins need these to fold correctly

Protein misfolding can cause formation of insoluble deposits and is responsible for some degenerative diseases