Proteins/Protein Folding (Symes)

Question 1

Which mutation of the CFTR gene is the most common amongst different groups in the US? What defect does this encode?

Answer 1

F508 deletion (68.6% population); the CFTR protein fails to reach the cell membrane, decreasing Cl- conductance across epithelial cell linings

Question 2

In what order do proteins fold?

Answer 2

in the configuration with the lowest energy and highest amount of hydrogen bonds

Question 3

Ways in which proteins fold?

Answer 3

• on their own directly to the native state
• help of chaperone proteins

Question 4

primary structure

Answer 4

sequence of amino acids; includes all covalent (peptide) bonds
- written N-terminus to C-terminus

Question 5

Secondary Structure

Answer 5

every 4 amino acids per alpha helix

Question 6

R groups of amino acids

Answer 6

what differentiates amino acids, giving them their properties that contribute to protein stability

Question 7

Would a protein of just glycine molecules be stable? Why?

Answer 7

No. Glycine has no R group to contribute to angle rotation of the growing polypeptide, making it incredibly flexible but very unstable since no R group to stabilize folding

Question 8

Which amino acids favor alpha helix formation?

Answer 8

Met, Ala, Leu

Question 9

Which amino acid disrupts alpha helical formation and why?

Answer 9

Proline- bulky= steric hindrance
charged amino acids= electrostatic repulsion/interactions in LARGE NUMBERS would hinder helix formation

Question 10

Motif

Answer 10

STRUCTURAL element of protein folding that occurs in tertiary and quaternary structures and some secondary structures
i.e. transcription factors contain variety of motifs (helix-turn helix, helix-loop helix)
i.e Zinc finger motif, where different parts of the motif coordinate the Zn ion

Question 11

Domains

Answer 11

FUNCTIONAL element of protein folding; these regions are part of polypeptide chains that can fold stably and
independently with respect to the entire protein; proteins can have multiple domains and the domains can have different functions
- i.e Dishevelled

Question 12

Fibrous Proteins

Answer 12

function: provide STRUCTURAL support
shape: organized into strands or sheets (one form of secondary structure), and simple tertiary structure
solubility: insoluble in water d/t high concentration of hydrophobic amino acids (i.e collagen)

Question 13

Globular Proteins

Answer 13

MOST PROTEINS ARE GLOBULAR!
function: VARIES; more diverse in function and structure (contains enzymes, immunoglobulins, transport proteins etc)
shape: polypeptide folded into globular shape; combo of secondary and complex tertiary structures

Question 14

Disordered Proteins/Protein Regions

Answer 14

RARE!!
some proteins that lack specific structure or function; flexible structure that allows them to interact with other proteins (hard to predict what structure/function they will take; depends on interacting protein)
- include scavenger proteins, structural proteins, components of protein
interaction networks, and versatile inhibitors
- i.e. the tumor suppressor
p53 contains a disordered region at the C-terminus that can bind to at least 4 diff proteins, all requiring a different conformation

Question 15

Aims of Protein Therapy

Answer 15

• replace a protein that is deficient or abnormal
  – augment an existing pathway (i.e CF therapies)
  – provide a novel function or activity (to bypass regular pathway by doing something else to compensate)
  – inhibit/interfere with a molecule or organism
  – deliver another compound/ drug
  these proteins are grouped based on their molecular types i.e antibodies, enzymes, etc

Question 16

Current protein therapies

Answer 16

• insulin (lowers blood glucose levels for type 2 diabetics)
• Dornase alfa (breaks down the extracellular DNA of WBCs left behind from autoimmune response in the lungs to improve respiratory symptoms without targeting intracellular DNA bc cannot penetrate the cell)
• Ivacaftor= CFTR potentiator–>G551D mutation; improves the transport of chloride through the ion channel by binding to the channels DIRECTLY to induce a new gating mechanism
  which INCREASES the probability that the channel is open allowing more cl to exit the cell and more sodium and more water to restore fluidity of the cell surface
  does NOT fix the channel, just helps by doing something else
• Orkambi= HOMOZYGOUS DF508 mutation;

Question 17

Buffering capability of proteins

Answer 17

amino acids can act as weak acids or bases due to the amino,
carboxyl groups, and ionizable R groups
- some diseases alter pH of the cell, so proteins can be a buffer and restore pH since they can accept and donate protons

Question 18

pKa

Answer 18

-log(Ka) Ka= [H+][A-]/[HA]
- small pKa= STRONG acid
- can be determined experimentally by titration = it is the pH when
the molecule is 50% in the acid form and 50% in the base form

Question 19

Henderson-Hasselbalch equation

Answer 19

pH = pKa + log ([conjugate base]/[conjugate acid]

Question 20

What is the pI for Aspartic acid
(pKas: 2.1, 3.9, 9.8)?

Answer 20

3

Question 20

Histidine as a buffer

Answer 20

GREAT buffer bc its pka is very close to physiological pH of 7 (6)
- it has more sources of Hydrogens than other amino acids, so they will be donated BEFORE the amino group

Question 21

Key proteins in diagnostic testing

Answer 21

• ALT (alanine aminotransferase) Healthy range: 8 to 37 IU/L; liver enzyme where high levels are indicative of liver damage
• Albumin Healthy range: 3.9 to 5.0 g/dL; liver protein where high levels indicate liver or kidney damage
• Alkaline phosphatase Healthy range: 44 to 147 IU/L; enzyme is involved in
  both liver and bone, so elevations may indicate problems with the liver or bone-related disease
• Bilirubin Healthy range: 0.1 to 1.9 mg/dL; critical for liver and kidney functions; indicative of problems in bile ducts, anemia

Question 22

Antibiotic resistance

Answer 22

• quinolones work to bind to particular region of DNA gyrase (a topoisomerase) which inhibits it and leads to DNA fragmentation of bacteria
• antibiotic resistance is due to a difference in the way the quinolone interacts with that particular topoisomerase that is ineffective in facilitating the binding of the two molecules
• sites critical to topoisomerase-quinolone binding through the water-metal ion
  bridge are affected
  (n Bacillus anthracis, quinolone resistant
  mutants include S81F and S81Y)