Protein Structure, Folding, Proteostasis (L2,3)

Question 1

How are IgG monoclonal antibodies made?

Answer 1

An antigen binds to one immature B cell among a pool of 1E8 immature B cells (each presenting UNIQUE antigen binding sites). Then the B cell matures and proliferates secreting IgG which can only bind to that single epitope.

Question 2

Describe the 3D structure of IgG

Answer 2

It is a tetramer with 2 identical light chains and 2 identical heavy chains held together by disulfide bonds.

Question 2

The antibody-binding surface is ____ and recognizes antigens via what interactions?

Answer 2

small;

1.Complimentary H bonding, 2.Electrostatic & VDW interactions

Question 3

What is an epitope?

Answer 3

It is the site on the antigen to which an antibody binds to.

Question 4

What do chaperonins do?

What are the 3 main types and which is most common?

Answer 4

They can help the particularly stubborn proteins (eg ACTIN and TUBULIN) attain a native fold.

1. Group I (most common)
2. HSP60 (eukaryotic cells)
3. GroEL-GroES (bacteria)

Question 4

Describe the problem that can arise with Islet Amyloid Polypeptide (IAPP)

Answer 4

IAPP, AKA Amylin, is an intrinsically disoredered peptide hormon that is co-secreted from pancreatic Islet cells w/ insulin at a 100:1 ratio (along w/ its precursor, proamylin).

Type II diabetes increases insulin demand which causes high levels of amylin and proamylin that form fibrillar amyloid plaques (by adopting beta-strand secondary structure) that lead to islet cell death from apoptosis.

Question 5

Why is Protein Disulfide Isomerase (PDI) formed in the ER?

Answer 5

B/c lots of extracellular membrane proteins have disulfide bonds, and they are formed in the ER, so that is where the chaperone protein PDI needs to be to make sure everything goes smoothly.

Question 6

What do Protein Disulfide Isomerases do?

Answer 6

They are a type of chaperone that reduce disulfide bonds so proper bonds can form

Question 6

What do Peptidyl Prolyl Isomerases do?

Give an example

Answer 6

They are a type of chaperone that will change stereo isoforms from trans to cis or vice-versa.

15% of X-Prolyl peptide bonds in native proteins have cis conformations (85% have trans). But conversion from trans to cis is very slow so the PPIs will help the kinetically trapped (of the 15%) go from trans to cis.

Question 7

What is glycosylation?

What is the purpose?

What are the 2 main types of glycosylation to amino acids?

Answer 7

The addition of sugar residues (oligosaccharaides) to proteins.

Purpose: alter solubility, promote protein-protein or cell-cell interactions, or PROTECT AGAINST PROTEOLYSIS.

1) O-linked glycosylation: occurs on OH groups (eg side chains of Ser and Thr)
2) N-linked glycosylation: occurs on amide groups (eg side chains of Asn and Gln)

Question 7

Describe the HAT selection process

Answer 7

Use immortal myeloma cells that have mutations in enzymes required for the sythensis of Deoxyribonucleotides via the “salvage pathway” (Thus cells must sythesize them de novo). These myeloma cells will die in Hypoxanthine-aminopterin-thymadine (HAT) media b/c AMINOPTERIN INHIBITS DE NOVO SYNTHESIS.

After fusing mortal spleenic B-cells w/ immortal HAT-sensitive myeloma cells, select for them in HAT media b/c only the hybrids will be able synthesize DNA due to restored kinase function and they will have immortality from myeloma cells.

Question 7

What are the 3 main causes of proteins to form amyloid filaments?

Answer 7

1) Amyloid diseases are inherited due to mutations in the gene that encode proteins that ultimately form fibrils
2) Some mutations inster poly glutamine (which is polar) into non-polar parts of proteins (eg huntingtin and ataxin proteins)
3) Some proteolytic fragments of the amyloid protein forms (eg aggregation of tau protein)

Question 8

What do chaperone proteins do?

Name the 4 main types of chaperone proteins.

Answer 8

They rescue folded intermediates from kinetic traps (ie improperly folded states that have a lower free energy than the native state)

1. Protein Disulfide Isomerase (PDIs)
2. Peptidyl Prolyl Isomerase (PPIs)
3. Non-ribosome Heat Shock Protein 70 (HSP70)
4. Chaperonins

Question 10

1) What is an Intrisically Disordered Protein (IDP)?
2) How do regulatory IDPs enhance their range of activity?
3) Why do many regulatory IDPs have “reversible regulation”?

Answer 10

1) An IDP is a protein with NO defined structure.
2) They adapt to diff binding partners b/c regulatory proteins exhibit varying degrees of disorder or unstructured regions.
3) Regulatory IDPs bind to targets relatively weakly allowing reversible regulation

Question 10

What do Non-ribosome Heat Shock Protein 70s do?

How do they do this?

Answer 10

They help partially folded, stable intermediates that have more hydrophobic side chains on their surface than the native fold does go back to normal.

They do this through the hydrolysis of ATP

Question 11

Describe the process to make immortal monoclonal antibodies generated from hybridomal cells.

Answer 11

1) Eject mice with the antigen, then isolate the spleen cells that will produce the antibodies.
2) Fuse the spleen cells with cell-culture myeloma immortal cells in POLYETHYLENE GLYCOL
3) Select and grow hybrid cells
4) Select cells making antibody of desired specificity
5) Grow in mass culture or inject new mice which will induce tumors and produce the antibody.

Question 11

What is the amount of components found relative to eachother b/w chaperones, ubiquitin/proteosome system, autophagy?

Answer 11

Ubiquitin/proteosome system (600 components)>Chaperones(180)>Autophagy (30)

Question 12

What is characteristic of most proteins associated with amyloid fibers?

Answer 12

They are Intrisically Disordered Proteins, have significant regions of intrisic order, or/and have a high beta-sheet content.

Question 14

What are 3 main factors that aid proteins in folding such that it is not entirely a random process?

Answer 14

1) For water soluble proteins, the hydrophobic residues quickly form the core
2) Secondary structures (esp helixes) form fast and then rearrange
3) Smaller domains can fold independently and then assemble into the final structure

Question 15

Which domains in IgG are the variable domains?

Answer 15

The N-terminal domains of each light and heavy chain are the variable domains (4 in total). The others are Constant domains (8 in total).

Question 17

How many AA’s/turn in an alpha helix?

What is the rise distance/turn?

Answer 17

3. 6 AA/turn
4. 4 Angstroms rise/turn

Question 19

In an antiparallel beta pleated sheet, how are side chain residues arranged relative to each other?

Answer 19

i and i+1 side chains are on opposite surfaces of the s sheet

Question 20

What is the most common regulatory modification?

Answer 20

Phosphorylation of OH group on Ser, Thr, and Tyr

Question 20

Explain why a high intercellular protein concentration presents enormous problems (talk about invitro vs intracellular). List 3 problems.

Answer 20

Invitro protein folding exps use low micromolar concentrations of protein. Intracellular protein concentrations are millimolar so they are much more.

1) proteins can lead to amorphous aggregates w/ diff types of polypeptides
2) You can have formation of oligomers that wouldn’t normally exist in dilute solutions
3) You can have formation of very stable amyloid fibrils

Question 22

Amino Acids IN PROTEINS are exclusively ___ isomers

Answer 22

L-isomers

Question 23

What 2 common methods of regulatory modifciation play a key role in gene activity and chromosme structure?

Answer 23

ACETYLATION (eg of Lys residues in histones to modify gene activity) and METHYLATION (eg of histones also to modify gene activty)

Question 25

What was Anfinsen’s experiment and what was fundamental dogma derived from it?

Why are some proteins an exception?

Answer 25

He denatured native ribonuclease (using beta-mercaptoethanol) and then gradually removed the denatured ribonuclase dialysis.

The dogma: The information necessary for protein folding is ENCODED BY THE PRIMARY SEQUENCE OF THE PROTEIN and does not require cellular compenents.

Some proteins are an exception b/c they are too big and refold very slowly or not at all in vitro.

Question 26

Intrisically Disordered Proteins (IDPs) have a ___ content of polar and charged AAs and a ___ content of hydrophobic residues, which ______.

Answer 26

High; Low; Prevents formation of a hydrophobic core.

Question 27

What are IgG polyclonal antibodies?

Answer 27

Pools of IgGs where each pool of IgGs recognize different epitopes on the SAME antigen.

Question 28

How are the tertiary and quaternary structures of IgG molecules stabilized?

Answer 28

Through disulfide bonds.

Question 29

What disease are prions, which are proteins normally found in the bran, related to?

Answer 29

Mad-Cow Disease,

also Creutzfeldt-Jakob disease

Question 31

Which form of transfiguration (trans or cis) is strongly favored for peptide bonds?

How is proline diff?

Answer 31

Trans b/c of steric clashes that occur in cis form.

Proline can be found in cis transfiguration

Question 33

Why will the native fold of a protein accumulate given enough time?

Answer 33

B/c the native fold has the lowest free energy.

Question 34

What is fatty acylation?

What is the purpose?

What are the most common sites of fatty acylation?

Answer 34

The addition of fatty acid groups via acyl bonds.

Purpose: ALLOWS PROTEIN TO ASSOCIATE W/ MEMBRANES, or can provide regulatory function

Common sites: side chains of Cys residues, and amino group of N-terminal Gly residues.

Question 35

What is the problem with amyloid plaques?

Answer 35

They are aggregates of proteins that can bind w/ other proteins and halt function of those proteins. An accumulation can also lead to apoptosis of the region. It is the cause of many neurological diseases.

Question 36

What are the Complimentary Determining Regions (CDR)?

Answer 36

They are the hypervariable loops in the V_L and V_H domains (there are 3 CDRs in each one so 6 in total).

They form the antigen bidning surface on the antibody.

Question 37

All amino acids except _____ are optically active

Answer 37

Glycine