Introduction to Proteins III
1. Within an aqueous environment, what are the expected characteristics of a globular
protein’s exterior amino acid composition, and of its interior amino acid composition?
a. As a general rule, a globular protein in an aqueous environment would fold into
compact structures with polar hydrophilic amino acids on the surface and nonpolar hydrophobic amino acids in its interior.
2. Define a protein domain.
a. Protein domains are compact globular protein regions connected by a flexible
segment of the polypeptide chain.
b. Protein domains exist within the same protein
3. Define a subunit of quaternary structure.
a. Proteins with quaternary structure contain two or more subunits, each of which is
an individual protein.
4. What does β-mercaptoethanol and 8M urea do to proteins and how can the effects
become reversed?
a. β-mercaptoethanol breaks disulfide bonds by reducing them to –SH groups while
8M urea denatures the protein; together, they effectively destroy protein function.
b. These effects can be reversed by the removal of 8M urea and β-mercaptoethanol.
i. Removal of only 8M urea will allow the reformation of the protein but
disulfide bonds may reform incorrectly.
ii. The addition of a catalytic amount of β-mercaptoethanol will break
incorrectly reformed disulfide bonds, allowing the reformation of correctly
placed disulfide bonds.
5. List two factors that facilitate protein folding and describe the importance of cooperative
transition.
a. Cooperative transition is the “all or none” folding transition of amino acids where
proteins are either completely denatured or fully folded with half-folded states
existing only transiently.
i. Protein chaperones help prevent the formation of partially folded proteins
ii. Cooperative transition is important because it prevents the accumulation
of partially folded structures that might interfere with cellular processes.
b. Cumulative selection is the process by which partially correct intermediates are
retained, helping the fold to the next partially correct intermediate and leading to
increasing stability as the entire correct conformation is achieved.
i. “If the first fold is correct, it helps the next fold being correct”
6. How do “knowledge-based” methods provide insights into the 3-D conformation of
proteins and their known sequence?
a. The “knowledge-based” method takes the amino acid primary structure and
compares it for sequence compatibility of proteins with known configurations.
b. When a significant match is found, it can be used as an initial model with the
assumption that similar amino acid sequences would yield similarly folded
proteins.
7. Describe the different types of covalent modifications that occur to proteins and
subsequent results.
a. Several different types of covalent modifications occur post-transcriptionally to
proteins and can help to augment or inhibit protein function.
i. Phosphoserine, phosphotyrosine, and phosphothreonine
1. Help modulate cellular functions
ii. Hydroxyl group addition to proline and lysine
1. Stablizes collagen fibers
2. Lack of Vitamin C (ascorbate) results in scurvy as proline and
lysine cannot be hydroxylated and collagen fibers destabilized.
iii. Acetyl group to amino terminal
1. Resists degreatation
iv. Ubiquitin
1. Tags protein for degreation
v. Glychosylation, methylation, sumoyolation, etc.
1. Modifications roles are unknown.
8. Describe the first two essential steps that must be taken when exploring an unknown
cellular protein for function.
a. Purification
i. Separate by differential centrifugation, dialysis, gel filtration,
chromatography, etc. to discriminate my size, charge, molecular mass, and
solubility.
ii. Check for yield and specific activity of the protein
iii. Necessary for determining the amino acid sequence of a specific protein
b. Determine primary amino acid sequence
i. Determine amino acid composition via ion exchange chromatography.
ii. Determine amino acid sequence via Edman degradation.
iii. Necessary for determining tertiary structure, biochemical function,
evolutionary relationships, and genetic sequence.
9. Describe when you would choose affinity chromatography rather than ion exchange
during protein purification.
a. If antibodies are available for affinity chromatography, choose affinity
chromatography because it is the preferred purification method.
b. Affinity chromatography separates and purifies proteins based on specific binding
affinity, allowing high yield and rapid separation.
10. Explain the usefulness of MALDI-TOF.
a. MALDI-TOF uses an electric field to accelerate protein ions to a detector,
measuring the size of fragments and charge through the “time of flight”
b. MALDI-TOF generates mass/charge “fingerprints” of individual proteins that can
be compared to databases to identify proteins of interest.