3. Protein Stability Folding Diseases

Question 1

What are the various covalent and non-covalent interactions that stabilize the protein structure?

Answer 1

• Covalent
  
  • Disulfide bonds (Cys-Cys)
• Non-covalent
  
  • Hydrogen bonds
  • Hydrophobic interactions
  • Electrostatics
  • Ion pairs
  • van der Waals

Question 2

What are the three classifications of disease-causing mutations?

Answer 2

1. Missense mutations.
2. Premature STOP codons or nonsense mutations.
3. Frameshift mutations.

Question 3

In general, the mutations that trigger diseases are non-conservative mutations.

Explain.

Answer 3

99% of diseases are caused when there is an amino acid substition that causes the change on an amino acid with different properties, thereby destabilizing the protein.

Question 4

Vitamin A deficiency:

Glycine → Aspartate

Answer 4

• Substitutes most flexible, non-polar residue
• Replaces with a negatively-charged amino acid

Question 5

Sjorgen-Larsson Syndrome:

Leucine → Arginine

Answer 5

• Substitutes a non-polar, aliphatic, short sidechain residue
• Replaces with a positively charged, long sidechain residue

Question 6

Galactosemia:

Leucine → Proline

Answer 6

• Mutation L226P occurs in galactose-1-phosphate uridylyltransferase
• Mutant introduces a proline (helix breaker) into an α-helix, causing
  
  • The loss of a main-chain hydrogen bond
  • The loss of hydrophobic interactions of the side chain

Question 7

Isovaleric acidemia:

Arginine → Leucine

Answer 7

• Mutation R382L occurs in isovaleryl coenzyme A dehydrogenase
• R382 usually forms a salt-bridge (charge-charge interaction)
  
  • Mutation causes the loss of a salt-bridge

Question 8

Aspartylglycosaminuria:

Glycine → Aspartate

Answer 8

• Mutation G60D occurs in aspartylglucosaminidase
• Substitutes uncharged residue
• Replaces wth a charged group into the interior of the protein (buried charge)
  
  • Causes helices to push apart
  • Causes overpacking

Question 9

Gaucher’s disease:

Phenylalanine → Isoleucine

Answer 9

• Mutation F411I occurs in glucocerebrosidase
  
  • Substitutes a large, buried, non-polar side-chain
  • Replaces with a smaller sidechain
    
    • Creates an internal cavity (cavity formation)
    • Loss of hydrophobic interaction

Question 10

Night blindness:

Glycine → Aspartate (neighboring glutamic acid)

Answer 10

• The mutation G38D occurs in guanine nucleotide binding protein
• Introduction of an aspartic acid side-chain
  
  • Electrostatic repulsion with neighboring glutamic acid
  • Limited over-packing

Question 11

Aspartylglycosaminuria:

Cysteine → Serine

Answer 11

• Mutation C163S occurs in aspartylglucosaminidase
• Substitutes cysteine with serine
  
  • Breaks disulfide bond (replaces one component of a disulfide bond)

Question 12

What is the largest factor that can contribute to genetic mutation and therefore trigger a disease?

Answer 12

A large number of genetic mutations trigger diseases by decreasing protein stability, which decreases the net functional protein concentration.​

Question 13

What are the two protein states?

What is the relationship between the two protein states?

Answer 13

Proteins exist in two states:

• functional native state (N)
• non-functional unfolded state (U)

The two protein states are in equilibrium. The unfolding equilibrium constant (KU) is the ratio of unfolded to native state concentration.

Note: Decreased stability increases the non-functional unfolded population.​

Question 14

What is the equation that determines the stability of a protein?

Answer 14

The stability (ΔG) of a protein can be calculated from:

• K_U - the ratio of unfolded to native state concentration
• R - universal gas constant R
• T - absolute temperature (T) in Kelvins.​

Many factors contribute to the stability of proteins. Protein structures are often stable over very narrow experimental conditions such as temperature, pH etc.

Question 15

denaturants

Answer 15

Small molecules which unfold proteins are often referred to as denaturants, e.g., guanidinium salts and urea.

Question 16

Protein stability can be measured by the addition of ________.

Answer 16

denaturants

Question 17

Explain what is occuring at C_m.

Answer 17

At the midpoint denaturant concentration C_m, equal concentrations of unfolded state and native states are present.​

Assuming [N] + [U] = 1, [N] = [U] = 0.5 at Cm.​

Question 18

Protein stability can be measured by _______ of the structure with temperature.

Answer 18

melting

Question 19

Explain what is occuring at T_m.

Answer 19

At the midpoint temperature T_m, percent of unfolded state population is the same as that of the native state.

Question 20

Based on the graph below, which protein is more stable?

Answer 20

Apomyoglobin, because it requires higher temperatures to unfold (higher T_m) compared to the other protein ribonuclease A.

Question 21

Describe how the Gibbs-Hemholtz equation is related to protein denaturation.

Answer 21

Proteins can be denatured either by increasing the temperature or by decreasing the temperature. As the temperature is increased or decreased from the temperature at which the protein is most stable, ΔG_U decreases, leading to hot or cold denaturation.

Note: Protein drugs aggregate upon freeze-thaw because they pass through cold denaturation.

Question 22

Explain the similarities and differences of Duchenne and Becker muscular dystrophy.

Answer 22

Both DMD/BMD affects and progressively weakens all types of muscles, leading to an array of disease manifestations, including cardiomyopathy and congestive heart failure (decreased heart muscles).

• Duchenne muscular dystrophy (DMD)
  
  • More common than BMD
    
    • 1 in 3,500 live male births
  • Most severe MD
    
    • Patients’ life spans rarely exceed teenage years
• Becker muscular dystrophy (BMD)
  
  • Symptoms develop with a slower rate of progression
  • Patients’ life spans are typically ~40 years

Question 23

Loss of dystrophin triggers DMD/BMD.

Explain why this protein is integral to muscle contraction.

Answer 23

• Dystrophin stabilizes the cell membrane of muscle cells against the mechanical forces associated with muscle contraction and stretch. ​
• Acts as a link between the sarcolemma transmembrane glycoprotein complex and actin filaments.​
• Protects actin filaments from depolymerization.​

Question 24

Which dystrophin mutation triggers Duchenne muscular dystrophy?

Answer 24

L54R places a positively charged residue in the hydrophobic core formed by A80, V83, and L84.

Question 25

Which dystrophin mutation triggers Becker muscular dystrophy?

Answer 25

A168D

• Places a negatively charged residue in the hydrophic cluster formed by V154, V156, and Trp 164
• A168 is part of an α-helix. Ala has the highest helix propensity compared to any other amino acid, and hence mutating it will decrease the helix stability
• After mutation, Asp at position 168 is three residues from another Asp at position 165 in the same helix, which will result in electrostatic repulsion leading to helix destabilization

A171P

• Part of an α-helix. Mutation replaces the strongest helix promoter with the strongest helix breaker.​

Y231N

• Places a polar residue in the hydrophobic cluster formed by F200, L213, and L234.

Question 26

Besides denaturation, diseases can also get triggered when mutations make the protein more _______.​

Explain.

Answer 26

active

Mutations in disordered regions can make the protein structured and bind more effectively.

Question 27

Describe how a gain-of-function mutation results in familial hypertrophic cardiomyopathy.

Answer 27

• Gain-of-function R403Q mutation in myosin
• Heart walls become much thicker (hypertrophied). The thickened heart muscle makes it harder for the heart to pump blood. ​
• 80% of the muscle mass corresponds to myosin and actin. ​
• R403Q mutation in myosin enhances its ATPase activity by twice, leading to faster sliding on actin filaments.​

Question 28

Describe what mutation causes cystic fibrosis?

Answer 28

• Cystic fibrosis disease is caused by the deletion of a single residue Phe at position 508 in Cystic Fibrosis Transmembrane Regulator (CFTR) protein (70% patients).
• CFTR is a chloride channel, and the mutation results in ineffective chloride ion pumping out of the cell. ​

The symptoms of cystic fibrosis include difficulty in breathing, thick mucus production, and lung infections. The thick mucus becomes more susceptible to trapping bacteria.

Question 29

Why is a sweat test used to diagnose cystic fibrosis?

Answer 29

• Sweat ducts in patients differ in the ability to reabsorb chloride before the emergence of sweat.
• A major pathway for Cl- absorption is through CFTR.
• Diminished chloride reabsorption results in a decrease of the total sodium chloride flux, leading to increased salt content.

Question 30

T/F: The genetic mutation that causes cystic fibrosis ultimately affects the function of the folded CFTR protein.

Answer 30

False.

Deletion of F508 residue slows down the folding of CFTR, and hence more protein is degraded by the proteasome resulting in a decrease in the functional protein concentration and hence decrease in net function. But the mutation does not affect the function of folded CFTR protein.

Question 31

What are the two steps of protein synthesis?

Answer 31

1. Transcription where genetic information from DNA is transcribed to messenger RNA (mRNA)
2. Translation where mRNA is translated into amino acid sequence by transfer RNAs and ribosome machinery.

Question 32

What are thre three possible pathways for polypeptides?

Answer 32

A newly synthesized polypeptide has three choices:

1. Folding to a functional protein
2. Resulting in protein aggregation because of the exposed hydrophobic residues which hate water
3. If the protein is not folded well, it is chewed up by proteases and the amino acids are recycled to synthesize new polypeptide chains.

Question 33

How do proteins fold into their functional forms?

Answer 33

Amino acid sequence contains all the information required to fold the polypeptide chain into its native, three-dimensional structure.

• Bury hydrophobics inside, hydrophilics outside
• In the interior, satisfy all polar and charged groups (both mainchain & sidechain)

Question 34

What happens when a nonpolar solute is dissolved in water?

Answer 34

When a nonpolar solute is dissolved in water, more water molecules get structured (increased order) due to Van der Waal’s interactions.

Question 35

What did Anfinsen’s experiments find about protein folding?

Answer 35

Anfinsen’s ‘Nobel’ Experiments:

Ribonuclease contains four disulfide bonds. Upon adding reducing agent which breaks disulfide bonds and urea which denatures proteins, the protein unfolds. Upon removing the urea and the reducing agent, the protein folds to its native, functional state.

Question 36

Do reactions proceed towards or away from disorder?

Answer 36

Reactions always proceed in the direction in which the disorder (Entropy) of the universe increases.

(Second law of thermodynamics)

Question 37

What is the major contribution of hydrogen bonds and electrostatic interactions to protein structure?

Answer 37

The MAJOR contribution of hydrogen bonds and electrostatic interactions to protein structure is reducing the energetic cost of having unpaired polar or charged groups in the hydrophobic interior of the protein.

Hydrogen bonds and electrostatic interactions are considered favorable free energy.

Question 38

What is the major contribution of hydrophobic interactions in protein structure?

Answer 38

The MAJOR contribution of hydrophobic interactions in protein structure is increasing the entropy of the solvent.

Question 39

What three bonds result in higher order of protein structures?

Explain.

Answer 39

• The MAJOR contribution of hydrogen bonds and electrostatic interactions to protein structure is reducing the energetic cost of having unpaired polar or charged groups in the hydrophobic interior of the protein.
• The MAJOR contribution of hydrophobic interactions in protein structure is increasing the entropy of the solvent.
• Results in higher order of protein structures

Question 40

Is a peptide bond hydrophobic or hydrophilic?

Answer 40

Hydrophilic; it cannot be buried in the protein interior; neutralizes charges by H-bonds or electrostatics.

Question 41

Where can hydrophobic sidechains be located on a protein?

Answer 41

Hydrophobic sidechains are located in the protein interior.

Question 42

What are the four nucleotides that make up DNA?

Answer 42

1. Adenine
2. Thymine
3. Guanine
4. Cysteine

Question 43

What are the three components of a nucleotide?

Answer 43

• Nucleobase
• Sugar
• Phosphate

Question 44

What is the START codon?

Answer 44

ATG

Question 45

What are the STOP codons?

Answer 45

1. TAA
2. TAG
3. TGA

Question 46

What occurs when there is a premature STOP codon?

Answer 46

When nonsense mutations occur, STOP codon occurs prematurely, resulting in an incomplete synthesis of polypeptide chains.

Question 47

What are three diseases caused by a premature STOP codon?

Answer 47

1. 5% CF patients are with premature STOP codons in the gene encoding CFTR.
2. 15% of Duchenne muscular dystrophy patients are with premature STOP codons in the gene encoding dystrophin, which stabilizes muscle cell membrane.
3. Beta-thalassemias, a genetic blood disorder, is due to a nonsense mutation in the gene encoding ß chains of hemoglobin, resulting in severe anemia (incomplete synthesis of the ß chain).

Question 48

frameshift mutations

Answer 48

• Extra base pairs may be added to or deleted from the DNA of a gene.
• The number of bases can range from a few to thousands.
• Insertions and deletions of one or two bases or multiples of one or two cause frameshifts (shift the reading frame).

Question 49

Where on the DNA can frameshifts have particularly devastating effects?

Explain.

Answer 49

Frameshifts can have devastating effects if they occur in exons, because the mRNA is translated in new groups of three nucleotides and the protein being produced may be useless.

Question 50

Tay-Sachs disease is caused by a frameshift mutation.

Describe the effect of how the frameshift mutation causes Tay-Sachs disease.

Answer 50

Tay-Sachs disease (GM2 gangliosidosis):

• This disease is caused by insufficient activity of the enzyme hexosaminidase A, a vital enzyme that breaks down phospholipids.
• Hexosaminidase A specifically breaks down fatty acid derivatives called gangliosides; these are made and biodegraded rapidly in early life as the brain develops.
• When hexosaminidase A is no longer functioning properly, the lipids accumulate in the brain and interfere with normal biological processes.

Question 51

Cystic fibrosis is caused by a frameshift mutation.

Describe the effect of how the frameshift mutation causes cystic fibrosis.

Answer 51

Two frameshift mutations, CF1213delT and CF1154-insTC. Both of these mutations commonly occur in tandem with at least one other mutation. They both lead to a small decrease in the function of the lungs and occur in about 1% of patients tested.