Biology Chapter 5: Proteins Primary Structure
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Questions and Answers

What is the primary structure of a protein?

  • The combination of multiple polypeptide chains
  • The folding pattern of the polypeptide chain
  • The three-dimensional shape of the protein
  • The sequence of amino acids in the polypeptide chain (correct)

    • Which of the following accurately describes polypeptide chain diversity?

  • Polypeptides must have a maximum of 100 residues
  • A protein with n residues has 20^n possible sequences (correct)
  • Polypeptides contain only 40 residues or fewer
  • There are only 20 possible sequences for polypeptides

    • What role do proteins play in biological processes?

  • They are the primary source of energy
  • They perform functions unrelated to cellular metabolism
  • They mediate nearly all molecular transformations (correct)
  • They act solely as structural components

    • What is a characteristic of multisubunit proteins?

    <p>They are made up of several polypeptide chains</p> Signup and view all the answers

    Which of the following amino acids is considered the rarest in proteins?

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

    What is a common application of cloning genetically engineered organisms?

    <p>To produce recombinant proteins</p> Signup and view all the answers

    Which protein has the largest known polypeptide chain?

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

    How many residues are typically required for a polypeptide chain to fold into a functional shape?

    <p>At least 40 residues</p> Signup and view all the answers

    What is the primary purpose of buffers in the protein purification process?

    <p>To provide a stable pH range</p> Signup and view all the answers

    At what temperature is protein purification typically carried out to avoid damage?

    <p>Near 0°C</p> Signup and view all the answers

    Which of the following is NOT a recommended practice when storing proteins?

    <p>Storing at room temperature</p> Signup and view all the answers

    Which type of assay uses radioactive labeled substances?

    <p>Radioimmunoassay (RIA)</p> Signup and view all the answers

    What is the role of the second protein-specific antibody in the ELISA method?

    <p>To bind to the protein-antibody complex and allow enzyme measurement</p> Signup and view all the answers

    What do coupled enzymatic reactions accomplish in assays?

    <p>They convert a product to an easily quantified substance</p> Signup and view all the answers

    Which of the following practices can help minimize protein damage during purification?

    <p>Using buffers with stabilizing pH</p> Signup and view all the answers

    What is a characteristic feature of immunoassays?

    <p>They specifically bind proteins using antibodies</p> Signup and view all the answers

    What characterizes anion exchangers in chromatography?

    <p>They use diethylaminoethyl (DEAE) groups.</p> Signup and view all the answers

    Which of the following statements about cation exchangers is correct?

    <p>Cations bind to anionic groups on the exchanges.</p> Signup and view all the answers

    What is the primary reason proteins separate into discrete bands during elution?

    <p>They have different affinities for the exchanger.</p> Signup and view all the answers

    What does gel filtration chromatography separate molecules based on?

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

    Why do larger molecules elute from a gel filtration column faster than smaller molecules?

    <p>They have a lower affinity for the gel matrix.</p> Signup and view all the answers

    What technique is used to monitor the presence of proteins in the column effluent?

    <p>Measuring absorbance at 280 nm</p> Signup and view all the answers

    Which characteristic of proteins is utilized in affinity chromatography?

    <p>Their ability to bind specific molecules tightly</p> Signup and view all the answers

    What factor influences the elution of proteins in an ion exchange chromatography setup?

    <p>The salt concentration in the eluant</p> Signup and view all the answers

    What type of enzyme is trypsin classified as?

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

    Which amino acid residues does trypsin cleave next to?

    <p>Arg and Lys</p> Signup and view all the answers

    During Edman degradation, what reaction does Edman's reagent (PITC) undergo?

    <p>It forms a Phenylthiocarbamyl (PTC) adduct.</p> Signup and view all the answers

    Which technique has become dominant for characterizing and sequencing proteins?

    <p>Mass Spectrometry</p> Signup and view all the answers

    What does electrospray ionization mass spectrometry (ESI) primarily measure?

    <p>The mass to charge (m/z) ratio for ions</p> Signup and view all the answers

    What is the purpose of dry nitrogen (N2) in ESI mass spectrometry?

    <p>To promote solvent evaporation from charged droplets</p> Signup and view all the answers

    What does the mass spectrum consist of?

    <p>A series of peaks corresponding to ions differing by ionic charge</p> Signup and view all the answers

    What result does protonation of basic side chains like Arg and Lys produce in mass spectrometry?

    <p>Ionic charges in the range +0.5 to +2 per kilodalton</p> Signup and view all the answers

    What is the purpose of the ligand in affinity chromatography?

    <p>To specifically bind to the protein of interest</p> Signup and view all the answers

    What property determines the migration of proteins during electrophoresis?

    <p>The net charge of the proteins</p> Signup and view all the answers

    How can proteins be visualized after electrophoresis?

    <p>By soaking the gel in a stain that binds tightly to protein</p> Signup and view all the answers

    What technique is employed to detect a specific protein on a gel after electrophoresis?

    <p>Immunoblotting or western blotting</p> Signup and view all the answers

    What does SDS-PAGE stand for?

    <p>Sodium dodecyl sulfate - polyacrylamide gel electrophoresis</p> Signup and view all the answers

    What kind of relationship exists between the molecular mass of a protein and its electrophoretic mobility in SDS-PAGE?

    <p>Logarithmic relationship</p> Signup and view all the answers

    What component is essential for the separation of proteins in polyacrylamide gel electrophoresis?

    <p>The electric field applied</p> Signup and view all the answers

    What is the typical molecular range that can be separated and detected by gel electrophoresis?

    <p>Less than a nanogram of protein</p> Signup and view all the answers

    What minimum number of residues is required for a polypeptide chain to fold into a functional shape?

    <p>40 residues</p> Signup and view all the answers

    Which of the following amino acids is considered most abundant in proteins?

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

    What is the largest known polypeptide chain composed of, in terms of residues?

    <p>35213 residues</p> Signup and view all the answers

    Which factor can influence the functional composition of a protein's primary structure?

    <p>Frequency of amino acids</p> Signup and view all the answers

    How does the number of possible unique polypeptide chains change with the length of the chain?

    <p>$20^n$, where n is the number of residues</p> Signup and view all the answers

    In what manner do recombinant proteins arise from genetically engineered organisms?

    <p>By integrating genes that direct protein expression</p> Signup and view all the answers

    What characteristic defines multisubunit proteins?

    <p>They consist of several identical or nonidentical chains</p> Signup and view all the answers

    Which type of amino acid is more prevalent in the polypeptide sequences of proteins?

    <p>Standard amino acids</p> Signup and view all the answers

    What is the significance of determining the amino acid sequence in proteins?

    <p>It helps in understanding the protein's 3D structure.</p> Signup and view all the answers

    Which method was used by Sanger to label the N-terminal amino acid in proteins?

    <p>Formation of a DNP derivative with DNFB</p> Signup and view all the answers

    What is the first step in the protein sequencing process described?

    <p>Separation of polypeptide chains by reducing disulfide bonds.</p> Signup and view all the answers

    Which of the following accurately describes endopeptidases in protein sequencing?

    <p>They are used to cleave polypeptides into smaller peptides.</p> Signup and view all the answers

    What can be inferred about the importance of overlapping peptide sequences in protein sequencing?

    <p>They are critical for reconstructing the sequence of the intact protein.</p> Signup and view all the answers

    What is the role of the ligand in affinity chromatography?

    <p>To covalently bind to the matrix and specifically bind the target protein</p> Signup and view all the answers

    In polyacrylamide gel electrophoresis, what factors influence molecular separation?

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

    What is the purpose of using SDS in SDS-PAGE?

    <p>To provide a uniform charge-to-mass ratio among proteins</p> Signup and view all the answers

    How are protein bands visualized after electrophoresis?

    <p>By staining the gel with a dye that binds specifically to proteins</p> Signup and view all the answers

    What information can be obtained from an autoradiograph following electrophoresis?

    <p>The positions of radioactive proteins on the film</p> Signup and view all the answers

    What determines the net charge of proteins during electrophoresis in a high pH gel?

    <p>The pH of the gel, making nearly all proteins negatively charged</p> Signup and view all the answers

    What is a characteristic of the log relationship in SDS-PAGE?

    <p>It shows a direct linear correlation with the molecular mass</p> Signup and view all the answers

    How can immunoblotting be described in the context of protein detection?

    <p>A method employing specific antibodies to visualize proteins in a gel</p> Signup and view all the answers

    What is the significance of using a high pH gel in protein analysis?

    <p>To achieve consistent negative charges on proteins for accurate tracking</p> Signup and view all the answers

    What outcome can be derived from running a protein sample through SDS-PAGE?

    <p>Estimation of molecular masses of protein subunits</p> Signup and view all the answers

    What is the primary function of diethylaminoethyl (DEAE) groups in chromatography?

    <p>To bind anions in a selective manner</p> Signup and view all the answers

    How do small molecules behave differently from large molecules in gel filtration chromatography?

    <p>They are eluted first due to their smaller size</p> Signup and view all the answers

    Why is absorbance at 280 nm used to monitor protein presence in a chromatography column?

    <p>Proteins absorb UV light at this wavelength</p> Signup and view all the answers

    What is a primary characteristic of affinity chromatography?

    <p>It uses specific binding interactions without covalent bonds</p> Signup and view all the answers

    In anion and cation exchange chromatography, what primarily affects the binding of proteins to the matrix?

    <p>The pH of the buffer solution</p> Signup and view all the answers

    What is the main limitation of using size exclusion (gel filtration) chromatography?

    <p>It does not provide any information about protein binding affinity</p> Signup and view all the answers

    Which of the following statements about gel beads in gel filtration chromatography is correct?

    <p>They create a sieve-like barrier for molecule separation</p> Signup and view all the answers

    During the elution process in ion exchange chromatography, how is the salt concentration manipulated?

    <p>It is increased to compete with protein binding sites</p> Signup and view all the answers

    Which chromatography technique separates molecules primarily by size rather than charge or binding affinity?

    <p>Gel filtration chromatography</p> Signup and view all the answers

    Which protein characteristic is primarily utilized in ion exchange chromatography?

    <p>The net charge of the protein at a given pH</p> Signup and view all the answers

    Which of the following factors is critical to avoid irreversible damage during the protein purification process?

    <p>Using a stable buffer with a pH range</p> Signup and view all the answers

    What is a primary characteristic of inclusion bodies in genetically engineered organisms?

    <p>They are aggregates of foreign proteins</p> Signup and view all the answers

    Which technique is NOT associated with measuring protein concentration in biological samples?

    <p>Polymerase chain reaction (PCR)</p> Signup and view all the answers

    In the ELISA technique, what is the role of the enzyme attached to the second antibody?

    <p>To catalyze a reaction that produces a measurable signal</p> Signup and view all the answers

    What is the main purpose of storing proteins under nitrogen or argon gas?

    <p>To eliminate exposure to oxygen that may cause degradation</p> Signup and view all the answers

    Which of the following describes a coupled enzymatic reaction in protein assays?

    <p>It uses the product of one enzyme to be a substrate for another enzyme.</p> Signup and view all the answers

    Which statement about the function of proteases during protein purification is true?

    <p>They can irreversibly damage proteins if not inactivated.</p> Signup and view all the answers

    What role do antibodies play in immunoassays?

    <p>They specifically bind to the protein antigen.</p> Signup and view all the answers

    For which method is determining the binding competition with a radioactive standard a key feature?

    <p>Radioimmunoassay (RIA)</p> Signup and view all the answers

    Which of the following is NOT a reason to keep proteins concentrated during purification?

    <p>To reduce shipping costs</p> Signup and view all the answers

    Study Notes

    Proteins and Their Functions

    • Proteins are essential in biological processes, acting as catalysts, regulators, structural components, and in transport and immunity.
    • Techniques like mass spectrometry allow paleobiologists to analyze proteins in ancient bones, aiding species identification and insights into early human agriculture.

    Primary Structure of Proteins

    • The primary structure consists of the amino acid sequence in polypeptide chains; proteins with multiple chains have a combination of polypeptides.
    • The total possible sequences for polypeptides with n residues are 20^n, highlighting extreme diversity; e.g., a 100-residue protein has approximately 1.27x10^130 unique sequences.
    • Proteins typically have a minimum of 40 residues to achieve a functional conformation; smaller chains are termed peptides.
    • Titin, the largest known protein, comprises 35,213 residues and plays a vital role in muscle fiber structure.

    Diversity in Protein Composition

    • Polypeptides generally range from 100 to 1000 residues, with the average size varying across different proteins.
    • Composition of proteins is not uniform; the most frequent amino acids in proteins include Leu, Ala, Gly, Val, Glu, and Ser, while Trp, Cys, Met, and His are among the rarest.

    Genetic Engineering and Protein Production

    • Genetic engineering manipulates genes in host cells to produce recombinant proteins for research and medicine.
    • Proteins in engineered organisms may form aggregates called inclusion bodies, which require careful purification to prevent damage.

    Protein Purification Techniques

    • Proteins should be dissolved in buffered solutions to maintain stable pH, typically purified around 0°C to slow enzymatic degradation.
    • Storage under inert gases or at very low temperatures (e.g., -80°C) maintains protein integrity.
    • Several assays are used to quantify proteins: enzyme assays, immunoassays, and techniques like RIA and ELISA which utilize antibodies for detection.

    Chromatography Methods

    • Ion exchange chromatography separates proteins based on charge via anion and cation exchangers (DEAE for anions and CM for cations).
    • Gel filtration chromatography, or size exclusion chromatography, separates proteins based on size, with larger proteins eluting first.
    • Affinity chromatography utilizes specific binding properties of proteins to purify them based on affinity to immobilized ligands.

    Electrophoresis for Protein Separation

    • Electrophoresis enables separation of proteins based on charge and size, primarily using Polyacrylamide gel electrophoresis (PAGE).
    • Proteins typically move towards the positive electrode, and their bands can be visualized using stains or radioactive detection methods (autoradiography).
    • SDS-PAGE provides molecular mass data for proteins, facilitating the analysis of protein subunits.

    Proteolytic Enzymes

    • Trypsin, a highly specific endopeptidase, cleaves peptide bonds post Arg and Lys, playing a critical role in protein digestion.
    • Exopeptidases hydrolyze terminal amino acids, and both types are essential in protein processing.

    Determining Amino Acid Sequencing

    • Edman degradation sequencially removes N-terminal residues from peptides using Edman's reagent (PITC), allowing for controlled amino acid analysis.
    • Mass spectrometry, particularly electrospray ionization mass spectrometry (ESI), is the primary technique for protein characterization; it measures ion mass-to-charge ratios to deduce molecular weights.

    Conclusion

    • Understanding proteins' structures, functions, and purification techniques is central to biochemistry and molecular biology, with implications in research, biotechnology, and medicine.

    Protein Functions and Analysis

    • Proteins are essential for biological processes, acting as catalysts, regulators, structural components, and playing roles in transport and immunity.
    • Mass spectrometry helps paleobiologists analyze ancient proteins in bones to identify species and deduce early farming practices.

    Primary Structure of Proteins

    • Primary structure consists of the amino acid sequence of a polypeptide chain, which can be single or multiple chains.
    • The number of possible sequences for a protein of n residues is 20ⁿ; for example, a protein with 100 residues has approximately 1.27 x 10¹³⁰ unique polypeptide chains.
    • A polypeptide must have a minimum of 40 residues to fold into a functional shape; proteins typically range from 100 to 1000 residues.
    • Titin, the largest known polypeptide, has 35,213 residues and is key for muscle fiber structure.
    • Proteins can be single polypeptides or multisubunits, with insulin being an example of a protein synthesized as a single chain and later cleaved.

    Genetic Engineering and Protein Production

    • Cloning involves inserting specific genes into organisms to produce recombinant proteins, crucial for research and medicine.
    • Engineered organisms may sequester foreign proteins in inclusion bodies within cells.

    Protein Purification Methods

    • To avoid irreversible damage during purification, consider:
      • Using buffers that maintain stable pH.
      • Performing purification at near-0°C temperatures.
      • Adjusting pH and temperature to inactivate degradative enzymes.
      • Minimizing foaming and maintaining protein concentration.
      • Storing proteins under nitrogen or frozen at -80°C.

    Protein Quantification Techniques

    • Assays to quantify proteins include:
      • Enzymes that produce detectable products (colored or fluorescent).
      • Coupled enzymatic reactions that yield easily quantified substances.
      • Immunoassays with antibodies binding specifically to proteins.
      • Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antigens.

    Chromatography Techniques

    • Affinity Chromatography: Uses ligands to bind specific proteins, allowing for their purification from a mixture.
    • Ion Exchange Chromatography: Separates proteins based on charge interactions with cation or anion exchangers.
    • Gel Filtration (Size Exclusion) Chromatography: Separates molecules by size, allowing larger proteins to elute faster than smaller ones.

    Electrophoresis

    • Electrophoresis separates molecules based on charge and size, with polyacrylamide gel electrophoresis (PAGE) relying on gel filtration and electric field mobility.
    • Proteins are negatively charged at high pH and migrate toward the positive electrode.
    • Visualization techniques include staining and autoradiography for radioactive proteins; immunoblotting can detect specific proteins.

    Protein Sequencing

    • Protein sequencing is vital for understanding structure, function, evolutionary relationships, and mutations.
    • Frederick Sanger first determined the sequence of bovine insulin, using a method involving dinitrophenol (DNP) labeling for N-terminal amino acid identification.
    • Basic sequencing involves fragmenting the protein, determining peptide sequences, and reconstructing the full sequence from overlapping fragments.

    Key Steps in Protein Sequencing

    • Reduce disulfide bonds to separate polypeptide chains.
    • Use chemical or enzymatic methods to break polypeptides into smaller peptides.
    • Determine sequences of peptide fragments.
    • Reconstruct the full sequence from overlapping data.
    • Eventually, endopeptidases may be employed for cleaving longer peptide chains that cannot be sequenced directly.

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    Description

    Explore the primary structure of proteins and their pivotal roles in biological processes. This quiz will cover topics such as protein function in cellular metabolism, transport, and immunity, as well as techniques used in paleobiology to analyze ancient proteins. Test your knowledge of the importance of proteins in early human farming practices.

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