Podcast
Questions and Answers
What is the primary reason for proteins to adopt a specific secondary structure?
What is the primary reason for proteins to adopt a specific secondary structure?
- To minimize the steric strain between atoms in the peptide backbone. (correct)
- To facilitate cis-conformation which reduces steric hindrance.
- To increase the flexibility of the peptide bonds for efficient enzymatic activity.
- To maximize the steric allowances between the phi and psi angles.
In protein sequencing, why is it necessary to reduce disulfide bonds before proceeding with techniques like Edman degradation?
In protein sequencing, why is it necessary to reduce disulfide bonds before proceeding with techniques like Edman degradation?
- To separate polypeptide chains linked by disulfide bonds, allowing individual sequencing of each chain. (correct)
- To increase the solubility of hydrophobic proteins in aqueous sequencing buffers.
- To prevent the protein from aggregating during the sequencing process.
- To ensure that the protein is fully denatured and all amino acids are accessible for modification.
What is the significance of the 'fold purification' parameter calculated during protein purification?
What is the significance of the 'fold purification' parameter calculated during protein purification?
- It indicates the total amount of protein recovered after each purification step.
- It measures the increase in purity of the protein relative to the starting material. (correct)
- It determines the percentage of initial enzymatic activity retained throughout the process.
- It reflects the efficiency of removing non-target proteins in each purification step.
When using endopeptidases in protein sequencing, what factor determines whether cleavage will occur at a specific site?
When using endopeptidases in protein sequencing, what factor determines whether cleavage will occur at a specific site?
How does a Ramachandran plot aid in assessing the quality of a theoretically derived protein structure?
How does a Ramachandran plot aid in assessing the quality of a theoretically derived protein structure?
When using cyanogen bromide (CNBr) to cleave a protein, which amino acid residue is targeted?
When using cyanogen bromide (CNBr) to cleave a protein, which amino acid residue is targeted?
Why is it essential to know the specific activity of an enzyme during a purification protocol?
Why is it essential to know the specific activity of an enzyme during a purification protocol?
What distinguishes hydrophobic interaction chromatography from ion exchange chromatography?
What distinguishes hydrophobic interaction chromatography from ion exchange chromatography?
Why is it important to use multiple enzymes or chemical methods to fragment a protein during sequencing?
Why is it important to use multiple enzymes or chemical methods to fragment a protein during sequencing?
In mass spectrometry, what limits the maximum size of peptides that can be effectively sequenced?
In mass spectrometry, what limits the maximum size of peptides that can be effectively sequenced?
Given that the Edman degradation procedure cleaves amino acids sequentially from the N-terminus of a peptide, what modification prevents its direct application to proteins with a blocked N-terminus?
Given that the Edman degradation procedure cleaves amino acids sequentially from the N-terminus of a peptide, what modification prevents its direct application to proteins with a blocked N-terminus?
How do stabilizing sucrose gradients facilitate the separation of macromolecules during ultracentrifugation?
How do stabilizing sucrose gradients facilitate the separation of macromolecules during ultracentrifugation?
Which of the following modifications to a protein would MOST likely cause a significant change in its migration pattern during SDS-PAGE?
Which of the following modifications to a protein would MOST likely cause a significant change in its migration pattern during SDS-PAGE?
In the context of protein purification, what does the term 'salting out' refer to?
In the context of protein purification, what does the term 'salting out' refer to?
When comparing protein sequences across species to understand evolutionary relationships, why are highly conserved regions particularly significant?
When comparing protein sequences across species to understand evolutionary relationships, why are highly conserved regions particularly significant?
What aspect of peptide bond structure contributes most to the limited flexibility and predominantly trans configuration observed in proteins?
What aspect of peptide bond structure contributes most to the limited flexibility and predominantly trans configuration observed in proteins?
How does the principle of isoelectric focusing enable the separation of proteins?
How does the principle of isoelectric focusing enable the separation of proteins?
A researcher is using ultracentrifugation to separate cellular organelles. Which strategy would BEST improve the resolution of separation?
A researcher is using ultracentrifugation to separate cellular organelles. Which strategy would BEST improve the resolution of separation?
Why is it crucial to include a 'wash' step in affinity chromatography before eluting the target protein?
Why is it crucial to include a 'wash' step in affinity chromatography before eluting the target protein?
Which biochemical characteristic makes 2D gel electrophoresis particularly useful for proteomics studies comparing healthy and diseased cells?
Which biochemical characteristic makes 2D gel electrophoresis particularly useful for proteomics studies comparing healthy and diseased cells?
After conducting Edman degradation on a purified peptide, a researcher observes that the PTH-amino acid derivative obtained in each cycle is consistently low in yield or undetectable. What is the MOST likely explanation for this?
After conducting Edman degradation on a purified peptide, a researcher observes that the PTH-amino acid derivative obtained in each cycle is consistently low in yield or undetectable. What is the MOST likely explanation for this?
Which of the following mutations would MOST likely disrupt a protein’s overall structure, as predicted by a Ramachandran plot?
Which of the following mutations would MOST likely disrupt a protein’s overall structure, as predicted by a Ramachandran plot?
Which statement best describes the chemical basis of protein separation by ion exchange chromatography?
Which statement best describes the chemical basis of protein separation by ion exchange chromatography?
A researcher aims to purify an enzyme with a known affinity for a specific substrate analog. Which chromatography method would be MOST appropriate?
A researcher aims to purify an enzyme with a known affinity for a specific substrate analog. Which chromatography method would be MOST appropriate?
A protein has a high percentage of hydrophobic amino acids. Which chromatography would be most effective for purification?
A protein has a high percentage of hydrophobic amino acids. Which chromatography would be most effective for purification?
How does the structure of a peptide bond affect the conformation of a protein?
How does the structure of a peptide bond affect the conformation of a protein?
Which technique would be most appropriate for separating proteins that differ significantly in size?
Which technique would be most appropriate for separating proteins that differ significantly in size?
Cyanogen bromide cleaves peptide bonds specifically after methionine residues. If a peptide has the sequence 'Ala-Lys-Met-Ser-Met-Gly', how many fragments would result from cyanogen bromide cleavage?
Cyanogen bromide cleaves peptide bonds specifically after methionine residues. If a peptide has the sequence 'Ala-Lys-Met-Ser-Met-Gly', how many fragments would result from cyanogen bromide cleavage?
A protein is subjected to Edman degradation. After three cycles, the following PTH-amino acids are identified: Cycle 1: Alanine (Ala), Cycle 2: Proline (Pro), Cycle 3: Valine (Val). What is the N-terminal sequence?
A protein is subjected to Edman degradation. After three cycles, the following PTH-amino acids are identified: Cycle 1: Alanine (Ala), Cycle 2: Proline (Pro), Cycle 3: Valine (Val). What is the N-terminal sequence?
Which statement accurately describes the role of a Ramachandran plot in protein structure validation?
Which statement accurately describes the role of a Ramachandran plot in protein structure validation?
A researcher wants to purify a protein based on its ability to bind a specific DNA sequence. Which approach would be MOST appropriate?
A researcher wants to purify a protein based on its ability to bind a specific DNA sequence. Which approach would be MOST appropriate?
Which chromatographic technique separates proteins based on differences in their net electric charge at a particular pH?
Which chromatographic technique separates proteins based on differences in their net electric charge at a particular pH?
In protein purification, what is the purpose of performing a 'wash' step after binding the target protein to a chromatography column but before eluting it?
In protein purification, what is the purpose of performing a 'wash' step after binding the target protein to a chromatography column but before eluting it?
Which property of proteins is exploited in two-dimensional (2D) gel electrophoresis to achieve high-resolution separation?
Which property of proteins is exploited in two-dimensional (2D) gel electrophoresis to achieve high-resolution separation?
Flashcards
Protein Purification Process
Protein Purification Process
A procedure to purify and enrich a protein of interest.
Quantifying Protein
Quantifying Protein
Measuring a protein's unique feature to determine its quantity.
Ultracentrifugation
Ultracentrifugation
Separates molecules by mass using high-speed rotation.
Purification Table
Purification Table
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Total Protein
Total Protein
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Total Activity
Total Activity
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Specific Activity
Specific Activity
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Initial Homogenate
Initial Homogenate
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Yield
Yield
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Purification Level
Purification Level
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Protein Sequencing
Protein Sequencing
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Endopeptidases
Endopeptidases
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Cyanogen Bromide
Cyanogen Bromide
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Dansyl Chloride Method
Dansyl Chloride Method
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Edman Degradation
Edman Degradation
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Mass Spectrometry
Mass Spectrometry
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Primary structure
Primary structure
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Secondary Structure
Secondary Structure
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Tertiary structure
Tertiary structure
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Quaternary Structure
Quaternary Structure
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Electronegativity
Electronegativity
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Resonance Structure
Resonance Structure
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Phi and Psi angles
Phi and Psi angles
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Ramachandran plot
Ramachandran plot
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Study Notes
- Problem set #1 is due on 02/12/2025 at 11:59 p.m., with a signed academic integrity page.
- Mini Exam 1 will be in-person on Friday, February 14, 2025, at 10:05 a.m.
- Weekly Homework #2 (Achieve) is due on Sunday 02/16/2025 at 11:59 p.m.
Protein Separation and Purification Strategies
- Protein characteristics and purification procedures:
- Solubility: salting out
- Ionic Charge: ion exchange chromatography, electrophoresis, isoelectric focusing
- Polarity: hydrophobic interaction chromatography
- Size: gel filtration chromatography, SDS-PAGE
- Binding Specificity: affinity chromatography
Proteomics - 2D Gel Electrophoresis Comparative Analyses
- Two sets of cells (healthy vs cancerous) are considered.
- Orange proteins are present in healthy cells but not in cancerous cells.
- Blue proteins are found in cancerous cells but not in healthy cells.
- Black proteins are equally abundant in both healthy and cancerous cells.
Ultracentrifugation
- Ultracentrifugation separates macromolecules by mass
- Ultracentrifuges can spin at speeds of up to 150,000 revolutions per minute (RPM).
- Ultracentrifuges are capable of generating 1,000,000 x g.
Protein Purification Process
- Purification enriches for the protein of interest.
- Quantifying the protein of interest involves measuring its unique character.
- Enzyme purification involves measuring its activity via assay.
Protein Purification Table for a Hypothetical Enzyme
- A hypothetical purification table outlines the purification process for an enzyme.
- Crude cellular extract: fraction volume 1,400 ml, total protein 10,000 mg, activity 100,000 units, specific activity 10 units/mg
- Precipitation with ammonium sulfate: fraction volume 280 ml, total protein 3,000 mg, activity 96,000 units, specific activity 32 units/mg
- Ion-exchange chromatography: fraction volume 90 ml, total protein 400 mg, activity 80,000 units, specific activity 200 units/mg
- Size-exclusion chromatography: fraction volume 80 ml, total protein 100 mg, activity 60,000 units, specific activity 600 units/mg
- Affinity chromatography: fraction volume 6 ml, total protein 3 mg, activity 45,000 units, specific activity 15,000 units/mg
- After the affinity chromatography step, the enzyme is purified by a factor of 1,500.
- The yield of the enzyme is 45%
Total Protein
- Quantity of total protein in fraction, measured in mg or g.
Total Activity
- The quantity of the target protein measured by its activity or function.
- An assay is needed
Specific Activity
- Total activity divided by the amount of protein, measured in units of activity per mg of protein.
Yield
- Measure of total activity retained after each purification step.
- Expressed as a percentage of the total activity in the initial homogenate.
Purification Level
- Measures the increase in purity.
- Calculated by dividing the specific activity by the specific activity of the initial homogenate.
Pepsinogen C example of purification
- A table shows the purification of pepsinogen C
- Crude Extract: 130 mg protein, 11,500 units activity, 88.5 units/mg specific activity.
- Salting out: 23 mg protein, 7,980 units activity, 347 units/mg specific activity,.
- Ion exchange: 11.4 mg protein, 5,130 units activity, 450 units/mg specific activity.
- Gel filtration: 1.6 mg protein, 750 units activity, 457 units/mg specific activity.
- Yield calculation: total activity retained after each step/ total activity in initial homogenate X 100
- Yield (%) for Pepsinogen C at the gel filtration step: 6.5%.
- Fold purification is specific activity at purification stage/ specific activity of initial homogenate
- Fold purification at gel filtration step: is 5.16-fold.
- Specific activity = activity divided by total amount of protein. Increases as purity increases.
Protein Sequencing
- Determining the amino acid sequence of a protein involves several steps:
- Obtain a purified protein sample.
- Convert the protein into a single chain, especially if disulfide bonds are present.
- Cut the protein into short fragments using more than 2 enzymes (endo peptidases) or chemicals.
- Run chemical reactions to determine the order of amino acids:
- Dansyl Chloride targets the N-terminal amino acid only.
- Edman degradation sequences from the N-terminal.
- Assemble the fragments into the correct order.
Disulfide Bonds
- Disulfide bonds are cut in protein sequencing
- Oxidized structures will be reduced using reducing agents
Amino-Terminal Analysis with Dansyl Chloride
- Dansyl chloride is used in amino-terminal analysis
- Memorizing the structures for dansyl chloride or dansyl-amino acids is not necessary.
- Dansyl chloride reagents are florescent and react with primary amines
Edman Degradation
- Cleaves off the first amino acid on N-terminal and derivatizes it.
- The sequence is deduced from N-terminal to the C-terminal.
- Order of appearance of derivatized amino acid residue aids in sequencing.
- What remains of the peptide stays unhydrolyzed for further reactions.
Phenyl Isothiocyanate (PITC)
- PITC is used during Edman Degradation
- Stable phenylthiohydantoin (PTH) products which are identified by mass spec
Mass Spectrometry
- Mass Spectrometry is used to sequence peptides
- It uses HPLC Analysis
Protein Sequencing
- Ultimately determines order within fragment peptides
Endopeptidases in Protein Characterization
- Endopeptidases cleave peptide bonds within the chain.
- Specificity varies, cutting after specific amino acid residues.
- Memorization of the attached table of the slide is not necessary
- Being able to use its information is crucial
Specificities of Various Endopeptidases in Protein Sequencing
- Trypsin cleaves after positively charged residues (Arg, Lys), but not if followed by Proline.
- Chymotrypsin cuts bulky hydrophobic residues (Phe, Trp, Tyr), but not if followed by Proline.
- Elastase, cleaves small neutral residues (Ala, Gly, Ser, Val), but not if followed by Proline.
- Specific 'cut sites' exist for different endopeptidases
Cyanogen Bromide
- Cyanogen bromide (CNBr) cleaves at methionine (Met) residues in chemical cleavage.
Protein Sequencing Example
- An example looks at the 11 residue peptide that gets digested by trypsin and chymotrypsin
- A question of Cyanogen bromide fragmentation is that if you have "EDKQSMERSTWQMSTAG" the fragments are "EDKQSMERSTWQM" "STAG" since cyanogen bromide cleaves after methionine
- Combining fragments from proteins
- CNBr fragments= Asp, Lys-Phe-Met, Tyr-Arg-Gly-Met
- Trypsin fragments= Lys, Gly-Met-Asp, Phe-Met-Tyr-Arg
Protein Sequences and Evolutionary Relationships
- Protein sequences reveal evolutionary relationships among species.
- Cytochrome C amino acid sequences from 38 species are compared
Levels of Protein Structure
- Primary structure: acid sequence
- Secondary structures (α helices and β sheets):
- The driving force of this structure is what facilitates the secondary structure
- Tertiary (3°)structure: 3D folding
- The driving force of this structure?
- How does tertiary structure differ from secondary sequence (of amino acids)
- Quaternary structure: subunits
- Example hemoglobin/RNA polymerase
Electronegativity and Peptide bonds
- Electronegativity is the tendency to attract e-
- H= 2.1, C= 2.5, N= 3.0, O= 3.5
- Differences in electronegativity change electron distribution
Characteristics of Peptide Bonds
- Resonance double bonds stabilize them
- Resonance contributes to rigidity
- There is a planar structure among the atoms
- Trans conformation is more favorable due to Steric hindrance
- Dihedral angles define the arrangements of the secondary structure elements
- Main chain conformations are used to define the secondary structural elements
- Two planes are involved in determining angles for amino acid interactions
- Φ (Phi)= N and Ca (hetero-atoms), Ψ(Psi)= Ca and C (same-atoms)
- Another angle of consideration is Third angle (ω) around N and C (peptide bond)
Ramachandran Plot/Diagram
- This helps to understand distribution of phi (Φ) and psi (Ψ) dihedral angles found on protein
- Its use in protein study includes revealing common and unusual structure of protein
Ramachandran Diagram of Polypeptide
- The Ramachandran Diagram Indicates Allowed Conformations of Polypeptides
- Blue-shaded regions = sterically allowed ϕ and angles for all residues except Gly and Pro.
- Green-shaded regions indicate the more crowded (outer limit) ϕ and angles.
- The yellow circles represent conformational angles of several secondary structures ○α, right-handed α helix; ○ parallel β sheet; ○ antiparallel β sheet; ○ C, collagen helix; ○ α, left-handed α helix.
- Most areas of the Ramachandran diagram (most combinations of ϕ and ) represent forbidden conformations of a polypeptide chain
- 3 regions physically accessible to most residues .
- Observed ϕ and values of accurately determined structures nearly always fall within these allowed regions of the Ramachandran plot
- some notable Ramachandran exceptions
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