Lecture 6 - Protein structure and protein folding

Question 1

Supersecondary structure

Answer 1

Super-secondary structure contains 3D secondary elements ie helices and strands that are connected by turns or regions of less ordered structure (loops or coils)
Some examples are helix-turn-helix, beta hairpin, Greek key and strand-helix-strand

Question 2

Common motifs of super secondary structures

Answer 2

Helix-turn-helix, beta hairpin, Greek key and strand-helix-strand

Question 3

Helix-turn-helix examples

Answer 3

DNA binding proteins - dimers and have 2 helices each, they dimerise across the helix and then two outer helices fit inside the DNA major groove , calcium binding proteins

Question 4

What is a beta hairpin and what are the key features?

1. What shape is it is most like?
2. How many residues?
3. Most common AA?
4. What % of residues in turn?
5. What bonds are common?

Answer 4

1. Hairpin like
2. involve usually 3 or 4 residues,
3. High Gly, Pro content
4. Almost 30% of residues involved in turns
5. Hydrogen bonds across the turn are common

Also common, antiparallel, length varies, short stretches of beta strands that are antiparallel to each other, beta stand-turn-beta strand

Question 5

Greek key

Answer 5

N to C
Connected so that the first strand is in the middle connects to 2 and foes to 3 and then goes to 4
4 stranded beta sheet, composed of 4 individual antiparallel strands

Question 6

Strand helix strand

Answer 6

Helix is depicted as a cylinder

The 2 strands are next to one another and they hydrogen bond to one another and helix is out of the plane

Question 7

Protein domains and motifs

Answer 7

Super-secondary structure elements combine to form regions with a specific function

Typically, a protein domain has a hydrophobic core and the hydrophilic parts of the protein are arranged on the surface in contact or near solvent.
Small proteins contain usually one domain, larger proteins may have multiple domains.

Question 8

Three examples of protein families based off tertiary structure

Answer 8

Alpha domain family (alpha helicies dominant)
alpha/beta family
Antiparallel beta family

Question 9

Alpha domain family

Answer 9

4 helix bundle - connected from N to C, tilted about 20 degrees to each other which has a purpose
Helix - turn - helix - turn - helix - turn - helix - turn
Folds together because it can create its own hydrophobic core, hydrophobic residues are in the core which helps to stabilise the protein

Globin fold - 8 helices that bind around a heme group and there are 2 histodines there that interact with the heme group

Question 10

alpha/beta family

Answer 10

Mixture of alpha and beta structure - alternating alpha helices and beta strand

alpha/beta barrel - strand-helix-strand-helix (8 strands), all of the strands interact, hydrophobic inside of barrel - next layer of side chains point out (hydrophilic outside the barrel)

alpha/beta open twisted sheet

Alpha/beta horseshoe fold

Question 11

Antiparallel beta family

Answer 11

No alpha
Mostly antiparallel beta structure … beta strand - turn - beta strand - turn - beta strand
8 strand generally and it forms a barrel with a hydrophobic interior

Retinal binding protein - every atom in retinal is hydrophobic with exception of the terminal hydroxide. Binds retinal and transports it

Hydrophobic inside of barrel

Mostly carrier proteins or binding proteins

Question 12

Protein folding

Answer 12

Proteins are synthesized as linear polymers that have to fold into a 3-dimensional functional structure 

Protein are made at the ribosome, and then generally they fold into their active shape spontaneously 

The only “instructions” needed are embedded in the aa sequence 

Essentially the sequence contains the instructions! 

This was proven by a famous biochemist, Christian Afinsen, in a series of experiments that led to a Nobel Prize

Question 13

The Afinsen Experiment

Answer 13

Anfinsen treated a ribonuclease enzyme with excess β-mercaptoethanol and 8M urea. This broke all the hydrogen and disulphide bonds and the protein denatured. When the agents were removed, the enzyme eventually reformed its original tertiary structure

Question 14

Folding pathways

Answer 14

Protein folding is directed largely by its internal hydrophobic residues, which form an internal core, while hydrophilic residues are solvent exposed. Not a random process
A likely sequence of events is:
(i) Formation of short secondary structure segments 

(ii) Nuclei come together, growing cooperatively to form a domain 

(iii) Domains come together (but tertiary structure still partly disordered) 

(iv) Small conformational adjustments to give compact native structure

Question 15

Stabilisation of protein folding

Answer 15

Non-covalent interactions, while individually weak in proteins, collectively make a significant contribution to protein conformational stability 

In some proteins additional covalent bonds (eg. disulfide bonds) may be present that contribute to conformation stability 

The hydrophobic core is likely the most important noncovalent contributor to protein stability in aqueous solution.

Metal ion coordination, hydrophobic interactions, electrostatic attraction, side chain hydrogen bonding (non covalent), disulphide bond (covalent)

Question 16

Some protein folding is assisted by….

Answer 16

Chaperones - prevent aggregation before the final folding can occur

(a) ‘chaperone’-independent 

(b) Chaperone-dependent eg Hsp70 

(c) Chaperonin-dependent eg GroEL-GroES - these are larger molecules that require ATP to function, hydrophobic core that interacts with the hydrophobic part of the amino acid chain. Prevents incorrect folding from taking place. Quite often gets partially folded by chaperones first.

Question 17

What is a chaperone and what role do they play?

Answer 17

Chaperones are independent peptides that assist in the correct folding or unfolding of other proteins.
Some chaperones eg Hsp70 are thought to crowd and stabilise the unfolded molecule until it folds properly.

No ATP required for these

Question 18

What is a chaperoning and what role does it play?

Answer 18

Chaperonins have the same function as chaperones (independent peptides that assist in the correct folding or unfolding of other proteins). but are barrel-shaped and the protein folding happens protected from the cell environment inside the structure.

Question 19

Unfolding of proteins

Answer 19

Weakening of non-covalent interactions can lead to unfolding and loss of biological function (denaturation)
May result from eg:
- change in pH (lab 1) - detergents - urea
- heating (lab 1) - organic solvents - guanidium HCL

Question 20

Misfolding of proteins

Answer 20

Proteins in living organisms that are folded normally can sometimes change their shape and become misfolded 

Some misfolded proteins can cause other proteins to change their shape as well, sometimes with disastrous consequences 

Can be associated with disease in living organisms

Question 21

Misfolding of proteins in disease

Answer 21

In the brain three conditions have been identified as being due to a protein, PrP, that changes its shape and then forms aggregates that cause brain damage: bovine spongiform encephalopathy (BSE), Creutzfeld-Jacob Disease (CSD) and Kuru 

The proteins that cause the problem are called prions for “proteins infectious agent” 

It is thought that the abnormal form of a prion protein, PrP, induces the normal form of this protein to become misfolded 

alpha → beta transformation 

No treatment available, always fatal.

Other diseases in which protein misfolding or aggregation is thought to contribute:
– Alzheimer’s Disease 

– Type 2 Diabetes 

Prions are not involved in these ailments 

Abnormally folded protein called amyloid is thought to contribute