Exam 1 Steele Lecture 2

Question 1

Whare are four ways that proteins can be sorted?

Answer 1

• Size
• Shape
• Charge
• Locaton in the cell

Question 2

What are polypeptide or peptide?

Answer 2

• Protins that are 50 amino acids

Question 3

What are the two shapes for proteins?

Answer 3

• Globular proteins: dominant types (enzymes are globular)
• Structural proteins: elongated (fibrous), e.g. collagen, elastin, keratin

Question 4

What are the four sources of charge for proteins?

Answer 4

• alphaNH2 + alphaCOOH (at ends of polypeptides)
• side chains of charged amino acids = D,E,H,K,R
• covalent modifications (e.g. phosphorylation, often transitory)
• metal ions

Question 5

What kind of charge does phosphorylate add to a protein?

Answer 5

• Negative

Question 6

What is a zwitterion?

Answer 6

• A zwitterion is a molecule that carries two distinct ionizable groups. In the case of a free amino acid, these are the amino and carboxyl groups.
• Shows the ionic state of these two groups as a function of the pH of the solution in which the amino acid is located.
• AA are zwitterions

Question 7

Describe what pKa is.

Answer 7

• A measure of how strongly a proton is held by the group that contains it. The higher the pKa, the more strongly the proton is held, and the less likely it is to be removed in an aqueous solution.
• As the pH of the solution changes relative to the pKa, the ratio of the two forms of the amino acid (protonated or unprotonated) changes.

Question 8

Describe buffering as it pertains to pH change.

Answer 8

• Buffering refers to the phenomenon in which a molecule resists changes in pH of a solution at 1 pH unit above and below the pKa for the molecule.
• Amount of base/acid usually not linear. Takes more of acid/base to change pH in the buffer zone.
• Buffer zone below is between 4 and 5.5

Question 9

Are proteins good buffers?

Answer 9

• pKa values of most amino acid side chains are not useful for physiological buffering
• Only histidine and α-amino groups have pKa values in the right range, but they occur too sparsely in serum proteins to be of significance - even in albumin, by far the most abundant human serum protein
• Blood buffering is accomplished by carbonic acid and bicarbonate

Question 10

How is amino acid distributed in proteins?

Answer 10

• Charge in proteins is distributed according to the environment surrounding the amino acids. Hydrophobic amino acids are buried in the interior of the protein, away from water. The surface of the protein, which interacts with water, is enriched in charged amino acids.

Question 11

Describe the two types of membrane proteins.

Answer 11

• Those associated with the membrane surface but not penetrating it (peripheral membrane proteins, in green in 7 and 8 below)
• Those that are embedded in the membrane either directly or indirectly via a lipid anchor (integral membrane proteins, 1-6 below)

Question 12

Describe transmembrane proteins.

Answer 12

• Single and multiple membrane pass structures
• External domain: glycosylation and disulfide bonds
• Transmembrane segment: highly hydrophobic alpha-helix
• Internal domain: reduced sulfhydryl groups, phosphorylation (transient), no complex carbohydrate on the internal part
• Note that the SH groups of cysteines are reduced on the cytoplasmic side, but oxidized (i.e. disulfide bonded) on the extracellular side.

Question 13

What are the two major types of protein glycosylation?

Answer 13

• N-linked - the sugar is linked to the side chain N of asparagine
• O-linked - the sugar is linked to the OH group of serine or threonine

Question 14

What kind of modifications are made to proteins that are secreted extracellular environment?

Answer 14

• Proteins have added carbohydrate chains.

Question 15

What are the 5 destinations of proteins in the cell?

Answer 15

1. endoplasmic reticulum and its continuum
2. nucleus (reversible)
3. mitochondrion
4. cytoplasm (“default mode”)
5. peroxisome

Question 16

How do proteins know where to go in a cell?

Answer 16

• Proteins have targeting seqeunces.
• These are essentially molecular mailing addresses for proteins. There are two types of nuclear localization signals, a monopartite one and a bipartite one.
  *

Question 17

What are the four protein isolation methods?

Answer 17

• Ion-exchange chromatography (based on charge)
• Gel filtration chromatography (based on size)
• Absorption chromatography (based on hydrophobicity)
• Affinity chromatography (based on interaction with other molecules)

Question 18

Describe ion exhange chromatography.

Answer 18

• Based on the net charge of the protein

Question 19

Describe gel filtration chromatography.

Answer 19

• Based on the size of the protein

Question 20

Describe affinity chromatography.

Answer 20

• Affinity chromatography takes advantage of the specificity of protein interactions. In some cases such interactions are known ahead of time and are used to guide the purification. In other cases, interactions are not known ahead of time.
• Ligands/substrates/products, binding proteins, antibodies against the protein

Question 21

What are orthologs versus paralogs?

Answer 21

• Orthologs are proteins that have the same function in different species.
• Paralogs are proteins in the same specicies, that have the same structure/seqeunce but have different functions

Question 22

Describe sickle cell anemia.

Answer 22

Hemoglobin is a tetramer composed of two alpha chains and two beta chains

In individuals with sickle cell disease, the E at position 6 in the beta chain is changed to a V. The hemoglobin molecules incorporating this mutant  chain are called HbS (the normal major Hb in adults is HbA).

This change leads to aggregation of hemoglobin molecules due to hydrophobic interactions (V is hydrophobic, E is charged).

Affected red blood cells “sickle” as a result of precipitated HbS.

Question 23

Describe electrophoresis

Answer 23

• Used as a tool to analyze proteins for research and in the clinic
• Can be used to separate proteins on the basis of size, charge, or both
• Abnormal hemoglobins were first detected by electrophoresis

Question 24

What is an allozyme?

Answer 24

forms of a protein encoded by different alleles of a gene (e.g. maternal and paternal alleles)

Question 25

What are isozymes?

Answer 25

forms of a protein encoded by different genes, but catalyzing the same reaction (however, often with different kinetics)

Question 26

Why is creatine kinase clinically relevant?

Answer 26

• 30% of the creatine kinase in heart is the MB isozyme (CK2) and 70% is MM isozyme (CK3); skeletal muscle contains mostly the MM isozyme (CK3). When heart muscle is damaged by infarction, creatine kinase is released into the blood stream. Release of the MB (CK2) isozyme is diagnostic of damaged heart muscle. When skeletal muscle is damaged, the MM isozyme is released but not the MB isozyme.
• CK2 isozyme (MB), heart
• CK3 isozyme (MM), skeletal muscle
• The creatine kinase isozyme profile can be used to diagnose myofcardial infarction. Useful when the electrocardiogram is hard to interpret.

Question 27

Why is cardiac troponin clinically relevant?

Answer 27

• Cardiac troponin is an even better marker than CK-MB because of its greater specificity and the fact that it remains elevated longer following the infarction than does CK-MB.