AA, peptides, proteins

Question 1

What are the 5 major classifications for amino acids?1

Answer 1

nonpolar/alphatic, aromatic, polar uncharged, polar and positively charged, polar and negatively charged

Question 2

Do you remember the 6 nonpolar, aliphatic amino acids?

Answer 2

glycine, alanine, valine, leucine, methionine, isoleucine (listed in increasing hydrophobicity)

Question 3

Do you remember the three aromatic amino acids?

Answer 3

Tyrosine, tryptophan, phenylalanine (listed in increasing hydrophobilicty

Question 4

Do you remember the 6 polar uncharged amino acids?

Answer 4

Proline, Glutamine, Asparagine, cysteine, serine, threonine.

Question 5

Do you remember the 3 polar positively charged amino acids?

Answer 5

Histidine, Lysine, Argenine.

Question 6

Do you remember the 2 polar negatively charged amino acids?

Answer 6

Aspartate, Glutamate

Question 7

What is the function of disulphide bonds within a protein?

Answer 7

Under oxidizing conditions disulphide bonds can be made. Disulphide bonds a an important contributor to tertiary protein structure. Examples, RNAase, and insulin molecules.

Question 8

What are the main post translational modifications that are done to proteins?

Answer 8

(6)

1. hydroxyproline,
2. gamma carboxy glutamate,
3. glycosylation of Ser or Thr or asn
4. acetylation or methylation (modification of side chains),
5. phosphorylation of dephos. of Ser, Thr, Tyr
6. Ubiquitination

Question 9

What is the medical significance of hydroxyproline modification?

Answer 9

Is very abundant in collogen. Vitamin C is required for the enzyme that hydroxyalates the proline. This is why Vitamin C deficiency causes scruvy

Question 10

What is the medical significance of the gamma carboxyglutamate

Answer 10

present in proteins involved with blood clotting pathway. The enzyme that does this carboxylation requires Vitamin K so Vitamin K deficiency can lead to bleeding disorders. The drug Warfarin, or coumadin, inhibits this carboxylation, which is why it is considered a blood thinner

Question 11

What is the medical significance of glycosylation

Answer 11

O-linked added to Ser or Thr, N-linked added to asn
Many secreted or cell surface proteins are glycosylated.
1. This can protect protein stability, (in plasma for example)
2. make it more soluble (it is a charged addition)
3. can also be important for recognition of other factors.
4. Congenital disorder of glycosylation (defect in unglycosylation pathway). (symptoms can include psychomotor retardation, seizures)

Question 12

What is the medical significance of acetylation and methylation

Answer 12

Acet of lysine, methyl of lysine and argenine.
These AAs are very abundant in histones. Acetylation and methylation of these two AAs, affect txn regulation. When it is deregulated by drugs, you
can cause cells, like cancerous cells to do apoptosis.

Question 13

What is the medical significance of phosphorylation

Answer 13

of Ser, Thr, Tyr, A major way to transduce signals in cells. For example, Gleevec, a bcr-abl tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML)

Question 14

What is the medical significance of Ubiquitination

Answer 14

adding of ubiquitin (a small protien) added to lysines by ubiquitin ligases. Polyubiquitination of a protein targets this protein for degredation by proteosomes. Bortezomib, used to treat multiple melanoma. inhibits proteosome by targeting this ubiquitin pathway.

Question 15

What are the three bonds that make up the backbone of a polypeptide chain?

Answer 15

1. Bond between AlphaC of residue1 to Carbonyl carbon of residue1
2. peptide bond between carbonyl carbon of residue1 and amide nitrogen of residue2
3. bond between amide nitrogen of residue2 and alpha carbon on residue2

Question 16

Of the three bonds that make up a polypeptide chain backbone, which ones can and cannot rotate?

Answer 16

the peptide bond has partial double bond characteristics and cannot rotate. the other two bonds can rotate.

Question 17

What determines the function of a protein?

Answer 17

the amino acid sequence, because that determines the structure of the protein

Question 18

What are some examples of proteases and the specific breaking of peptide bonds that have important functions.

Answer 18

Some proteases chew all proteins (trypsin, chymotrypsin, pepsin)

Question 19

Understand that the amino acid sequence determines the function; mutations in amino acid sequence can cause genetic disease.

Answer 19

sickle cell anemia, and E6V mutation causes the sickle shape, leading to severe anemia. However, there are many proteins that are polymorphic, meaning you can have slightly different AA sequences and still have a perfectly functional protein (if a hydrophobic residue which out any special function is replaced by another hydrophobic residue of approximately the same size, it probably would not affect protein structure, thus its function.

Question 20

Describe hydrogen bonds and their role in secondary structure formation.

Answer 20

Hydrogen bonds are interactions between hydrogen donors and acceptors (usually N and O in proteins). 1. H bonds between backbones are important in formation of beta sheets.

2. Beta turns are also stablized by carbonyl O of 1st residue, and Amide proton of 4th residue
3. Alpha helices are stablized by H bonds from CO of residue n and NH of residue n+4.

Question 21

Describe two major types of protein secondary structures.

Answer 21

1. Alpha helices: Right handed helical structure, H bonds between n CO and n+4 NH. Alanine and leucine have higher tendancy to form A.helices, Proline and glycine cannot form them. Example: hemaglobin
2. Beta sheets: parallel or antiparallel sheets, H bonds between backbone. Example, immunoglobulins

Side note: circular dichroism is a good way to measure secondary structure of a protein

Question 22

. What is the difference between a tertiary and Quaternary structure?

Answer 22

Tertiary structure is the overall spacial arrangement of a single polypeptide chain. Quaternary structure is a complex of multiple polypeptide chains, and their interactions and overall spacial arrangement

Question 23

Explain the role of loops in protein structure and function.

Answer 23

This is how protein backbone changes direction! Glycine and proline are often found in loops.
They also have functional functions. Loops in antibody proteins at the tip of the variable domain, can bind antigens.

Question 24

Explain how to use Kd to represent binding strength.

Answer 24

For proteins, such as globular proteins, that have to interact with or bind other molecules, Kd, the dissociation constant, is a measure of binding strength. The binding specificity and strength is achieved through the lock and key complementary model or the induced fit model.

Examples: Hemoglobin binds a heme which allows it to bind oxygen. but It has a higher Kd for carbon monoxide, so it preferentially binds to it. different structures of hemoglobin also have different Kds for oxygen. This allows it to transport O2, as opposed to myoglobin which can only store O2, because it lacks the different conformations with different Kds

Question 25

Explain how binding specificity can be achieved.

Answer 25

The binding specificity is achieved through the lock and key complementary model or the induced fit model. Factors include size, shape, charge, hydrophobicity or hydrophilicity,

There may be changes in structure upon binding, in both protein and ligand to increase affinity. This is called induced fit

Question 26

Explain how heme enables myoglobin to bind oxygen.

Answer 26

the protein itself lacks affinity to O2, but a reduced Iron in heme can bind O2, so myoglobin binds heme and encloses it within itself to protect it from oxidizing agents. This heme can then bind O2. bound O2 does not oxidize the heme

Question 27

Explain the molecular basis of carbon monoxide poisoning.

Answer 27

CO has a similar size and shape as O2, and it has a much higher affinity for heme. The fact that the heme is in a protein pocket reduces its affinity for CO but it is still many times higher than for O2.

Question 28

Explain why hemoglobin is a good oxygen transporter.

Answer 28

Being a tetramer, Hemoglobin has multiple binding sites that interact with each other, are positive allosteric affectors of each other (sigmoidal binding curve) , and this protein has two conformations.

T(tense) state has low O2 affinity. O2 binding triggers change to R(relaxed) state that has a higher O2 affinity.

Affinity for O2 is also affected by pH, Metabolic tissue blood has lower pH which reduces O2 affinity. (as compared to lungs) Bohr effect

Question 29

Identify the factors that cause protein denaturation

Answer 29

Heat, pH, chemicals (such as urea, organic solvent, detergents, guanidinium)

Question 30

What do the ribonuclease folding experiments show?

Answer 30

1. All the information needed to fold the protein correctly is embedded in the primary sequence.
2. The environment provided by the inside of the cell is not always required.

Question 31

What is the general function of chaperones, and what are the two main classes?

Answer 31

The two major classes are Hsp70/40 and Chaperonin GroEL/GroES complex.

Their function is to help proteins fold into their native states.

Hsp’s are activated by increased temp. They bind to hydrophobic regions to prevent aggregation. Can also transport proteins across membranes in their unfolded states. They can provide a “safe compartment” for a protein to fold

Chaperonins also provide an enclosed space for proteins to fold.

Question 32

Explain why sometimes protein disulfide isomerase is required for protein folding.

Answer 32

Proteins with a lot of disulfide bonds can sometimes make the wrong bond. Protein disulfide isomerase can reduce the improper disulfide bonds, and give the protein another chance to make the correct ones.

Question 33

Explain why sometimes protein prolyl isomerases is required for protein folding.

Answer 33

In some proteins, some structures, proline needs to take a cis conformation, rather than the typical trans conformation, such as in beta turns or some specific active sites. Protein prolyl isomerases helps these important prolines to convert to a cis conformation.

Cyclophilin, a PPI, is important for activation of calcineurin for example, which dephosphorylates and activates NFAT which starts and immune response. Side note: cyclosporin can bind and inhibit cyclophilin, acting as an immune suppressant.

Question 34

What are some diseases that are caused by protein misfolding?

Answer 34

Cystic fibrosis, Prion diseases, alzheimer’s, parkinsons, amyloidosis (misfolded proteins other than in the brain)

Question 35

Identify the secondary structure changes and the infectious agent in prion disease.

Answer 35

The infectious agent is an insoluble prion protein, which has a lot of beta sheets. (normal form has a lot of alpha helices) This is resistant to heat and proteases. It causes normal form of prp (prion protein) to assume the insoluble form.

Question 36

Describe the major approaches for purifying a protein.

Answer 36

1. gel filtration chromatography which separates proteins based on their size differences;
2. ion-exchange chromatography which separates proteins based on their charge differences;
3. affinity chromatography which utilizes the ability of proteins to bind specific ligands.

The mass of the protein can be accurately determined by mass spectrometry. The N-terminal sequence of the protein can be determined by Edman degradation. Proteins can be identified on a Western blot using protein-specific antibodies.

Question 37

How can you monitor protein purities?

Answer 37

gel eletrophoresis on an SDS polyacylamide gel.

Question 38

How can the mass of a protein be determined?

Answer 38

mass spectrometry

Question 39

What is a degredation method to determine the N terminal sequence

Answer 39

Edman degredation. you attach a chemical compound to the N terminus of a protein. Then you lower the pH to hydrolyze peptide bond between first amino acid and the rest of the peptide to release the labeled N terminus, and you can identify it by chromatography. you can the do the same thing over again to identify the second amino acid. usually 5-10 AA is enough to ID the protein.

Question 40

What can you do with a western blot?

Answer 40

you can identify specific proteins by using protein specific antibodies to bind them.