Biochemistry W2-2
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Questions and Answers

Which technique uses a solid phase to selectively bind proteins based on their affinity?

  • Affinity Chromatography (correct)
  • Gel Filtration Chromatography
  • Size Exclusion Chromatography
  • Ion Exchange Chromatography

    • In gel filtration chromatography, which characteristic of proteins is primarily utilized for separation?

  • Polarity
  • Charge
  • Hydrophobicity
  • Size (correct)

    • What is the primary purpose of protein purification techniques?

  • To degrade unwanted proteins
  • To enhance protein folding
  • To isolate proteins from complex mixtures (correct)
  • To analyze protein structure

    • Which of the following correctly describes the structure of a peptide?

    <p>A sequence of amino acids connected by peptide bonds</p> Signup and view all the answers

    Which amino acids typically have side chains that can influence their pKa values?

    <p>Amino acids with ionizable side chains</p> Signup and view all the answers

    What is a common characteristic of amino acids with three pKa values?

    <p>They possess more than one ionizable group</p> Signup and view all the answers

    Which of the following methods can be used for protein sequencing?

    <p>Edman Degradation</p> Signup and view all the answers

    MALDI MS is primarily used for which of the following purposes?

    <p>Analyzing large biomolecules</p> Signup and view all the answers

    What is the main principle behind tandem mass spectrometry (MS/MS)?

    <p>Fragmentation of ions for structural analysis</p> Signup and view all the answers

    What factor significantly influences the isoelectric point (pI) of an amino acid?

    <p>The ionizable side chain groups</p> Signup and view all the answers

    What principle does affinity chromatography rely on to separate proteins?

    <p>Specific binding affinity</p> Signup and view all the answers

    In denaturing gel electrophoresis, which factor does NOT influence the migration of proteins through the gel?

    <p>Buffer pH</p> Signup and view all the answers

    Which method is most appropriate for separating proteins with similar isoelectric points under the same buffer solution?

    <p>Ion-exchange chromatography</p> Signup and view all the answers

    Which technique is least suitable for separating proteins based on their molecular mass?

    <p>Affinity chromatography</p> Signup and view all the answers

    Which amino acid's R group has a pKa value that indicates it can act as both an acid and a base within physiological pH?

    <p>Cysteine</p> Signup and view all the answers

    Which factor does NOT affect the isoelectric point of an amino acid?

    <p>Solvent polarity</p> Signup and view all the answers

    What is the primary mechanism of action for affinity chromatography?

    <p>Binding target proteins to a specific ligand</p> Signup and view all the answers

    Which statement about mass spectrometry is incorrect?

    <p>It can analyze solid-phase samples directly.</p> Signup and view all the answers

    Which statement about the sequence of amino acids in peptides is accurate?

    <p>Peptides have a defined amino acid sequence starting from the N-terminus.</p> Signup and view all the answers

    When considering protease treatment in mass spectrometry, which statement is most accurate?

    <p>It simplifies the analysis of complex mixtures.</p> Signup and view all the answers

    Which of the following amino acids is likely to have the highest pKa value due to its side chain?

    <p>Lysine</p> Signup and view all the answers

    In size-exclusion chromatography, which protein would elute last from the column?

    <p>Protein B, with Mr = 58,400</p> Signup and view all the answers

    The four levels of protein structure include all EXCEPT:

    <p>Quasi structure</p> Signup and view all the answers

    What is the main characteristic that distinguishes peptides from proteins?

    <p>Proteins contain one or more polypeptide chains.</p> Signup and view all the answers

    Which amino acid would least likely be found in the interior of a globular protein?

    <p>Lysine</p> Signup and view all the answers

    What differentiates orthologs from paralogs in protein nomenclature?

    <p>Paralogs are homologs found in different species.</p> Signup and view all the answers

    Which of the following statements accurately describes peptide bond formation?

    <p>Peptide bonds form through dehydration reactions.</p> Signup and view all the answers

    What is the primary method of distinguishing proteins using gel filtration chromatography?

    <p>Size exclusion based on molecular weight</p> Signup and view all the answers

    What is the correct order of pKa values for cysteine's functional groups?

    <p>pK1: 1.96, pK2: 8.18, pK3: 10.28</p> Signup and view all the answers

    How does denaturing gel electrophoresis primarily separate proteins?

    <p>Based on their size and shape.</p> Signup and view all the answers

    What is a key characteristic of mass spectrometry that distinguishes it from other techniques?

    <p>It provides high precision in determining mass-to-charge ratios.</p> Signup and view all the answers

    Which of the following statements about protein structure is incorrect?

    <p>Orthologs are homologs found in different species.</p> Signup and view all the answers

    In affinity chromatography, how can a specific protein be separated from another similar protein?

    <p>Binding the protein to a specific ligand attached to resin.</p> Signup and view all the answers

    What type of analysis is performed by mass spectrometry in regard to cellular proteins?

    <p>Monitoring changes in the proteome related to metabolic states.</p> Signup and view all the answers

    Which factor does NOT influence the migration of proteins during electrophoresis?

    <p>The solvent used in the gel.</p> Signup and view all the answers

    Which amino acid would you expect to have a non-polar side chain?

    <p>Alanine</p> Signup and view all the answers

    What characteristic of an amino acid's side chain primarily influences its pKa values?

    <p>Functional group structure</p> Signup and view all the answers

    In which scenario would an amino acid with a charged side chain likely exist?

    <p>At low pH environments</p> Signup and view all the answers

    Which of the following is a key feature of SDS-PAGE?

    <p>It utilizes proteins' size for separation after denaturation.</p> Signup and view all the answers

    Which gel electrophoresis technique is best suited for determining the molecular weight of proteins?

    <p>SDS-PAGE</p> Signup and view all the answers

    What is a common outcome when carrying out Edman degradation?

    <p>Sequencing proteins by detecting the N-terminal amino acid</p> Signup and view all the answers

    Which of the following mass spectrometry (MS) techniques involves the use of laser energy?

    <p>MALDI MS</p> Signup and view all the answers

    What is the primary advantage of using mass spectrometry in protein analysis?

    <p>It determines the mass-to-charge ratio for identification.</p> Signup and view all the answers

    Which amino acid is highly hydrophilic and typically found on the surface of proteins?

    <p>Arginine</p> Signup and view all the answers

    Which amino acid has the lowest pKa value and is likely to have a carboxyl group?

    <p>Cysteine (Cys)</p> Signup and view all the answers

    Which statement correctly describes alanine's characteristics regarding its amino acid sequence?

    <p>Alanine has a hydrophobic R group.</p> Signup and view all the answers

    Which amino acid has a side chain that can influence the isoelectric point (pI) significantly?

    <p>Lysine (Lys)</p> Signup and view all the answers

    In size-exclusion chromatography, which protein would elute last from the column?

    <p>Protein B with Mr = 58,400</p> Signup and view all the answers

    What is a characteristic feature of peptides compared to proteins?

    <p>Peptides consist of fewer than 50 amino acids.</p> Signup and view all the answers

    Which method is ideal for separating proteins based on differing affinities to ligands?

    <p>Affinity chromatography</p> Signup and view all the answers

    Which amino acid's R group indicates it can donate a proton at physiological pH?

    <p>Histidine</p> Signup and view all the answers

    Which method would provide the best separation for proteins with significantly different molecular weights?

    <p>Size-exclusion chromatography</p> Signup and view all the answers

    Which factor primarily determines the order of elution in gel filtration chromatography?

    <p>Molecular weight of the proteins</p> Signup and view all the answers

    Which component is least critical in the successful purification of a protein?

    <p>Gene sequence of the protein</p> Signup and view all the answers

    What is the primary basis for the separation of proteins in denaturing gel electrophoresis?

    <p>Size and shape of the proteins</p> Signup and view all the answers

    Which statement about mass spectrometry in protein analysis is accurate?

    <p>Mass spectrometry measures the mass-to-charge ratio with high precision.</p> Signup and view all the answers

    Which statement incorrectly defines orthologs in the context of protein evolution?

    <p>Orthologs are homologs found in different species.</p> Signup and view all the answers

    What characteristic is NOT typically used to define the four levels of protein structure?

    <p>Tertiary structure involves interactions among multiple polypeptide chains.</p> Signup and view all the answers

    What principle is utilized by affinity chromatography for protein separation?

    <p>Proteins bind to specific ligands, allowing separation based on affinity.</p> Signup and view all the answers

    What is the significance of attaching calcium to beads in an affinity chromatography setup?

    <p>It creates a matrix that selectively binds proteins that interact with calcium.</p> Signup and view all the answers

    What role do side chains play in determining the pKa values of amino acids?

    <p>They can confer charged properties influencing pKa values.</p> Signup and view all the answers

    Which of the following amino acids is most likely to exist in a zwitterionic form at physiological pH?

    <p>Alanine</p> Signup and view all the answers

    In the context of SDS-PAGE, what is primarily measured to determine protein size?

    <p>The molecular mass-to-charge ratio (m/z).</p> Signup and view all the answers

    Which amino acid is least likely to be found in the interior of a globular protein due to its polar side chain?

    <p>Lysine</p> Signup and view all the answers

    What is the pKa value of the α-carboxyl group of cysteine?

    <p>1.96</p> Signup and view all the answers

    Which method effectively separates proteins based on their interactions with specific ligands?

    <p>Affinity chromatography</p> Signup and view all the answers

    What technique is primarily used to evaluate the secondary structure of proteins?

    <p>Nuclear magnetic resonance (NMR)</p> Signup and view all the answers

    Which method would be least effective in separating proteins with very similar isoelectric points?

    <p>Size-exclusion chromatography</p> Signup and view all the answers

    What is the unique feature of tandem mass spectrometry compared to simple mass spectrometry?

    <p>It allows for sequencing of proteins.</p> Signup and view all the answers

    What is the function of peptide bonds in proteins?

    <p>To link amino acids together</p> Signup and view all the answers

    Which protein is expected to elute last from a size-exclusion chromatography column?

    <p>Protein C, a homodimer with protomer Mr = 11,300</p> Signup and view all the answers

    What characteristic differentiates charged and non-charged amino acids?

    <p>Type of side chain functional groups.</p> Signup and view all the answers

    Which amino acid is most likely to have a side chain influencing its pKa significantly?

    <p>Cysteine</p> Signup and view all the answers

    Which statement about the amino acid sequencing of peptides is accurate?

    <p>Peptides have their sequences written from the N-terminus</p> Signup and view all the answers

    What is the primary operation during the protein purification process using ion exchange chromatography?

    <p>Ion binding followed by elution.</p> Signup and view all the answers

    In what scenario would an amino acid with a side chain pKa of 8.18 primarily exist in its deprotonated form?

    <p>In basic pH conditions</p> Signup and view all the answers

    Which property of peptides makes them potentially biologically active despite being short chains of amino acids?

    <p>The formation of specific secondary structures</p> Signup and view all the answers

    What technique is crucial for differentiating two proteins with similar molecular weights but different affinities to ligands?

    <p>Affinity chromatography</p> Signup and view all the answers

    Study Notes

    Amino Acid Structure and Properties

    • Amino acids are the building blocks of proteins.
    • Amino acids have a central carbon atom (α carbon) with four different groups attached:
      • Amino group (-NH2)
      • Carboxyl group (-COOH)
      • Hydrogen atom (H)
      • Side chain or R group
    • R group determines the properties of an amino acid.
    • Hydrophobic R groups tend to cluster in the interior of proteins.
    • Hydrophilic R groups tend to be on the surface of proteins.

    Types of Amino Acids

    • There are 20 standard amino acids.
    • Amino acids can be categorized based on their R groups:
      • Aromatic Ring: Phenylalanine, Tyrosine, Tryptophan
      • Positively charged: Lysine, Arginine, Histidine
      • Negatively charged: Aspartic acid, Glutamic acid
      • Nonpolar: Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Methionine
      • Polar, uncharged: Serine, Threonine, Asparagine, Glutamine, Cysteine

    Ionizable Side Chains

    • Some amino acids have ionizable side chains that can gain or lose protons, affecting their charge and the overall charge of the protein.
    • Titration curves can be used to determine the pKa values of ionizable groups in amino acids.

    Protein Structure and Function

    • The primary structure of a protein is the linear sequence of amino acids.
    • The secondary structure of a protein refers to local folding patterns, such as α-helices and β-sheets.
    • The tertiary structure of a protein is the overall three-dimensional shape of a single polypeptide chain.
    • The quaternary structure of a protein refers to the arrangement of multiple polypeptide chains (subunits).
    • The function of a protein is determined by its three-dimensional structure.
    • Mutations in the amino acid sequence can disrupt protein structure and function.

    Protein Analysis Techniques

    • SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separates proteins based on their size.
    • Mass Spectrometry (MS) determines the mass-to-charge ratio (m/z) of molecules.
    • Edman degradation is a method for determining the amino acid sequence of a protein.
    • Chromatography techniques are used to separate proteins:
      • Ion-exchange chromatography: separates proteins based on their charge.
      • Size-exclusion chromatography: separates proteins based on their size.
      • Affinity chromatography: separates proteins based on their specific binding to a ligand.

    Protein Sequencing

    • MALDI MS (Matrix-assisted laser desorption/ionization mass spectrometry) is a method for determining the mass of biomolecules.
    • Tandem MS (MS/MS) involves multiple stages of mass analysis to identify and sequence peptides.

    Protein Purification

    • Protein purification is the process of separating a protein from other molecules in a mixture.
    • Affinity chromatography is a commonly used technique for protein purification.
    • Isoelectric focusing separates proteins based on their isoelectric point (pI).

    Questions

    • The amino acid with three pKa values is cysteine.
    • Alanine, with a nonpolar side chain, is typically found in the interior of a globular protein.
    • Peptides are named from the N-terminus.
    • A means of detecting the protein is absolutely necessary for protein purification.
    • Protein B with the highest molecular weight will elute first from a gel filtration column.
    • Affinity chromatography is the most suitable method to separate a new protein from myosin based on its calcium binding property.
    • Denaturing gel electrophoresis separates proteins based on size and shape.
    • Mass spectrometry can be used to monitor changes in the cellular proteome as a function of metabolic state.
    • Orthologs are homologs found in different species.

    Amino Acids

    • Alanine and Valine can be represented using "Ball and Sticks"
    • Aromatic ring amino acids: Trytophan, Phenylalanine, Tyrosine
    • Positively charged amino acids: Arginine, Lysine, Histidine
    • Negatively charged amino acids: Aspartic acid, Glutamic acid
    • Glycine and Alanine are non-polar amino acids
    • Ionizable side chains are indicated by red circles
    • Amino acids can be titrated, they have pKa values
    • Non-ionizable side chains have simpler titration curves
    • Ionizable side chains affect the titration curves of the amino acid
    • Lysine is a positively charged amino acid

    Proteins

    • Protein structure can be studied using SDS-PAGE
    • SDS-PAGE denatures proteins, separating them by size
    • Ion exchange chromatography separates proteins based on charge
    • Size exclusion chromatography separates proteins based on size
    • Affinity chromatography separates proteins based on their affinity for a specific molecule
    • Edman degradation and Mass Spectrometry (MS) can be used to sequence proteins
    • Edman degradation sequentially cleaves amino acids from the N-terminus
    • MS determines the mass-to-charge ratio (m/z) of ions generated from proteins
    • MALDI MS is a type of MS that uses a laser to ionize proteins
    • Tandem MS sequentially fragments ions to obtain structural information
    • A peptide is a short chain of amino acids
    • Cysteine has three pKa values due to its ionizable thiol side chain
    • Alanine is a hydrophobic amino acid, often found in the interior of globular proteins
    • Peptides are named from the N-terminus to the C-terminus
    • A means of detecting the protein is essential for successful protein purification
    • Larger proteins elute first from a gel filtration column
    • Affinity chromatography is useful for separating proteins based on their affinity for a specific molecule
    • Denaturing gel electrophoresis separates proteins based on size and shape
    • Mass spectrometry can be used to analyze proteins in the liquid phase
    • Mass spectrometry can determine the sequence of multiple polypeptide segments
    • Mass spectrometry can monitor changes in the proteome over time
    • Orthologs are homologous proteins found in different species
    • A protein's function is determined by its amino acid sequence
    • It is possible to change a protein's amino acid sequence without affecting its function

    Amino Acid Structure

    • Amino acids are the building blocks of proteins.
    • Alanine and valine are amino acids.
    • Amino acids have a central carbon atom attached to an amino group (NH2), a carboxyl group (COOH), a hydrogen atom (H), and a side chain (R group).
    • The side chain (R group) is what distinguishes amino acids from each other.
    • The structure of amino acids can be represented using ball and stick models.
    • There are 10 amino acids to learn:
      • 3 with aromatic rings: Phenylalanine, Tyrosine, Tryptophan
      • 3 positively charged: Lysine, Arginine, Histidine
      • 2 negatively charged: Aspartic acid, Glutamic acid
      • Glycine, Alanine

    Amino Acid Properties

    • Amino acids can be classified as hydrophobic (nonpolar) or hydrophilic (polar).
    • Hydrophobic amino acids tend to be found in the interior of a protein, away from water.
    • Hydrophilic amino acids tend to be found on the surface of a protein, interacting with water.
    • The ionization of amino acids depends on their side chain (R group) and creates a positive charge or negative charge.
    • Titration: The pH at which an amino acid has no net charge is called its isoelectric point (pI).
    • Identifying Amino Acids: The properties of amino acids, their ionization states, and their reaction with specific chemical solutions can be used to identify and classify amino acids.

    Proteins

    • Proteins are formed from long chains of amino acids called polypeptides.
    • Peptide bonds: The amino acids in a polypeptide chain are joined together by peptide bonds.
    • The polypeptide chain can be written from the N-terminus to the C-terminus based on the placement of the amino and carboxyl groups.
    • Protein structure: The three-dimensional structure of a protein is essential for its function.
    • Levels of Structure: There are four levels of protein structure:
      • Primary structure: The linear sequence of amino acids.
      • Secondary structure: Local folding of the polypeptide chain, including alpha helices and beta sheets.
      • Tertiary structure: The overall three-dimensional shape of a single polypeptide chain.
      • Quaternary structure: The arrangement of multiple polypeptide chains (subunits) in a protein complex.
    • Protein function: The function of a protein is determined by its three-dimensional structure.
    • Protein folding: The process by which a protein folds into its correct three-dimensional structure is a complex process.
    • Chaperone proteins: Proteins that help other proteins to fold correctly.

    Studying Proteins

    • Studying Proteins Scientists study proteins using various methods:
      • SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE): This technique separates proteins based on their size.
      • Chromatography: This technique separates proteins based on their properties, such as size, charge, or binding affinity.
        • Ion exchange chromatography: Separates proteins based on their charge.
        • Size exclusion chromatography (gel filtration): Separates proteins based on their size.
        • Affinity chromatography: Separates proteins based on their binding affinity to a specific ligand.
      • Mass Spectrometry (MS): This technique measures the mass-to-charge ratio (m/z) of ions.
        • MALDI MS: This technique is used to analyze proteins and peptides. It involves ionizing the sample in the form of a gas phase.
        • Tandem MS: This technique is used to sequence peptides by breaking them down into smaller fragments and then analyzing their fragment ions.
      • Edman degradation: A method for determining the amino acid sequence of a protein.

    Protein Sequencing

    • Edman Degradation: This method involves sequentially removing amino acids from the N-terminus of a polypeptide chain. The cleaved amino acids are then identified to determine the sequence.
    • Mass Spectrometry: This technique measures the mass-to-charge ratio of peptides by ionizing them. The resulting spectrum provides information about the peptide's mass and sequence.

    Important concepts

    • Homologs: Proteins that share a common ancestor and have similar amino acid sequences.
    • Orthologs: Homologs from different species.
    • Paralogs: Homologs within the same species.

    Additional Notes

    • Cysteine: The only amino acid with three pKa values because the thiol group in its side chain can ionize.
    • Gel filtration chromatography: Large proteins elute first because they cannot fit through the pores in the column matrix.
    • Affinity chromatography: Specific ligands are attached to the column to bind the protein of interest.
    • Denaturing gel electrophoresis: The SDS (sodium dodecyl sulfate) detergent disrupts the protein's structure and provides a uniform negative charge per unit of length. This allows separation based on molecular mass.
    • Mass Spectrometry: Can be used to analyze proteins in the liquid, solid, or gaseous phases.
    • Protein Function and Structure: The function of a protein is determined by its amino acid sequence and its three-dimensional structure. Changes to the sequence of amino acids can affect the protein's structure and function.

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