lecture 2 biochemistry (week 2)

Question 1

describe protein structures

Answer 1

every protein has one main native structure –> active and correct functional structure

determined by amino acid sequence

Question 2

what determines whether a protein is active?

Answer 2

whether it has been folded into the correct structure

other active conformations can be achieved by binding and post translational modifications

Question 3

how do proteins fold?

Answer 3

spontaneously under physiological conditions to their native structure

Question 4

where is the information for folding stored?

Answer 4

inside the protein

Question 5

compare the unfolded and folded forms

Answer 5

the folded form is more thermodynamically stable

Question 6

what does folding depend on?

Answer 6

multiple often weak interactions existing simultaneously

folding starts slowly and then accelerates (same for unfolding)

Question 7

what are the forces that drive protein folding?

Answer 7

hyrdrophobic forces are the most important - stron effect that dictates the location of amino acid residues

close packing of the core of the protein is important

h bonds and salt bridges within the protein also play a part

Question 8

describe the flexibility in the amino acid chain

Answer 8

rotation is permitted about the N-C bond (the phi bond) and abouy the carbonyl bond (the psi bond)

allows protein to fold in different ways

Question 9

what makes protein folding possible?

Answer 9

the restrictions by the rigidity of the peptide bond

restricted set of allowed phi and psi angles (steric hindrance)

Question 10

what are the kinetics of protein folding?

Answer 10

proteins fold spontaneously in 10ms - 1 second

Question 11

describe the pathway of protein folding

Answer 11

unfolded chain –> short stretches a-helix/b strand –> extended a-helix/ b strand –> secondary structures associate –> molten globule –> folded domains –> native tertiary structures –> quaternary structure

approach to the correct structure is associated with sharply increasing conformational stability

Question 12

what can happen when protein folding goes wrong?

Answer 12

proteins get into an energetic cul-de-sac when misfolded.

aggregates cause protein plaques and disease

Question 13

what proteins/enxymes assist protein folding?

Answer 13

folding accessory proteins - enzymes that reorganise certain bonds in the nascent proteins
- protein disulfide isomerases, peptidyl cis-trans isomerases

molecular chaperones - these bind to proteins as they are synthesised on the ribosomes and prevent them from aggregating

chaperonins - these are multi subunit complexes that help misfolded proteins to achieve the correct conformation

Question 14

describe protein disulfide isomerase

Answer 14

shuffles the S-S bonds until they form between the correct Cys residues

enzyme has a groove for binding the substrate and an exposed disulfide on the surface

Question 15

describe peptidyl prolyl cis-trans isomerase

Answer 15

polypeptides are synthesised with all pro-X bonds in the trans form

this enzyme converts some trans bonds into cis bonds - allowing proline to fit into turns

Question 16

describe molecular chaperones

Answer 16

prevent newly synthesised young proteins from forming undesirable interactions with other proteins

ie hydrophobic patches sticking together - they bind these exposed patches and release the nascent protein allowing it to fold correctly

Question 17

how is the molecular chaperone process driven?

Answer 17

ATP is used

chaperones come in different sizes/degrees of complexity, but often involve complexes of several associated proteins

Question 18

describe the Hsp70 (and Hsp40) system

Answer 18

Hsp70 has ATPase activity

ATP binding, hydrolysis and release all conformational changes that helps to fold and compact the protein substrate

DnaJ (Hsp40) assists in the process by the unfolded protein

Question 19

describe the Hsp70 system

Answer 19

unfolded protein –> phosphate release (rate-limiting step) –> ADP release –> ATP binding –> native protein released

Question 20

describe chaperonins (GroEL-GroES)

Answer 20

this is an E.coli chaperonin

TCiP is found in eukaryotes

Cpn60 is in chloroplasts

chaperonins are a specific class of molecular chaperones that have a typical 14 subunit structure

individual polypeptides are heat shock proteins

only required for folding of some particularly difficult proteins and are very large multi subunit complexes

barrel shaped and the protein to be folded enters the barrel where it folds to the native state.

sometimes several cycles are required

release of the protein requires hydrolysis of 14 ATPs and removal of GroES which forms a cap over the barrel

Question 21

describe post transational modifications

Answer 21

phosphorylation - on hydroxyl groups of Ser, Thr, Tyr - used to regulate the activity of enzymes in metabolism or signalling

glycosylation - addition of a sugar moiety on Asn (N-glycosylation) Thr or Tyr (O-glycosylation), most commonly on extracellular proteins, protects the proteins and may be involved in recognition

n-terminal acetylation - occurs in ~80% of proteins and prevents rapid degradation

other modifications include hydroxylation of proline(essential in collagen), farnesylation (may facilitate attachment to cell membranes), ubiquitinylation (degradation), and carboxylation of glutamate (in blood clotting factors).

Question 22

describe protein lifetime and degradation

Answer 22

all proteins have a finite lifetime - may vary from minutes to years

eventually senescent proteins are degraded and the amino acids are reused

non-specific degradation occurs in lysosomes – membrane-bound acidic compartments containing proteases

specific degradation occurs in proteasomes – multi-subunit proteins similar to chaperonins. Proteins to be degraded are tagged with the small (8.5 kDa) protein ubiquitin.

Question 23

describe the nobel prize in chemistry for 2004

Answer 23

“for the discovery of ubiquitin-mediated protein degradation”

jointly to

Aaron Ciechanover Technion - Israel Institute of Technology, Haifa, Israel

Avram Hershko Technion - Israel Institute of Technology, Haifa, Israel

Irwin RoseUniversity of California, Irvine, USA

Question 24

describe the ubiquitin-activating enzyme

Answer 24

in the first step, ubiquitin is linked through its C-terminal glycine to a cysteine residue of an E1 class enzyme via thioester bond

in the second step, the activated ubiquitin is transferred to a cysteine residue of an E2 class enzyme

in the third step E3 class enzymes catalyse the amide linkage between the C-terminal glycine of ubiquitin with the sigma-amino group of a lysine residue within the target protein. the transfer takes place either directly or through an E3-ubiquitin intermediate

Question 25

where is ubiquitin attached?

Answer 25

to proteins at a lysine residue ubiquitinylation is affected by the N-terminal residue in the target protein - N-terminal Arg, Lys, Phe, Tyr, Leu cause rapid degradation (3 mins). Ala, Val, Ser, Thr, Gly, Cys, Met are stabilising and protect against proteolysis (30 hrs). ubiquitinoylation is like a kiss of death labelled proteins are fed into the cells 'waste disposers' - the proteasomes ubiquitin is a signal that the protein is on the way for disassembly ubiquitin is disconnected for reuse prior to degradation in the proteasome

Question 26

what can protein misfolding cause?

Answer 26

can cause a loss of function may target the protein for destruction or sometimes prevent proteasomal degradation alzheimers disease is caused by accumulation of fragments of amyloid precursor protein and the microtubule-binding protein Tau. These associate into stable filaments > form tangles > amyloid plaques in the brain. they are resistant to degradation BSE and vCJD are caused by prions - abnormally folded and degraded brain protein. They have different structures and the conformational change is autocatalytic. The abnormal PrP is partially resistant to proteolysis, and accumulates in the brain > aggregation > plaques.

Question 27

what is the structure of a prion?

Answer 27

PrPC has 3% b-sheet and 42% a-helix (normal structure),alpha-helix rich prion protein (PrPC) PrPSc has 54% b-sheet and 21% a-helix (abnormal structure)