Protein Structure and Sequencing Methods
26 Questions
0 Views

Choose a study mode

Play Quiz
Study Flashcards
Spaced Repetition
Chat to lesson

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

What is the significance of studying the 3D shape of proteins?

  • It helps to understand their molecular mechanisms of action. (correct)
  • It identifies the amino acid sequence.
  • It assesses the temperature stability of proteins.
  • It determines the weight of the protein.

    • The first complete amino acid sequence of a protein was determined using just a few micrograms of protein.

    False

    What reagent is used to sequence protein fragments according to the outlined methodology?

    Edman reagent

    To determine the number of protein chains, one method that can be used is __________.

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

    Match the following methods with their appropriate descriptions:

    <p>SDS-PAGE = Technique used to determine the number of protein chains End group analysis = Method for identifying the number of N- and C-terminal ends Cleave disulfide bridges = Step for separating polypeptide chains Sequencing fragments = Using Edman reagent to identify amino acids</p> Signup and view all the answers

    What is the primary purpose of using endopeptidases in the fragmentation of polypeptide chains?

    <p>To cleave internal peptide bonds based on side chain requirements</p> Signup and view all the answers

    Direct sequencing can be used effectively for polypeptides longer than 60 amino acids.

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

    What reagents are commonly used to perform the cleavage of peptide bonds in acidic conditions?

    <p>HCl or 70% formic acid</p> Signup and view all the answers

    The complete sequence obtained by combining peptide fragments is: Phe-Trp-Met-Gly-Ala-Lys- _____ -Met-Asp-Gly-Arg-Cys-Ala-Gln.

    <p>Leu-Pro</p> Signup and view all the answers

    Match the following methods with their uses in peptide sequencing:

    <p>Edman's degradation = Identify S-S linked fragments Reverse phase HPLC = Separate peptide fragments Limited proteolysis = Optimize fragmentation conditions Direct sequencing = Applicable to 40-60 amino acids</p> Signup and view all the answers

    Which method is specifically limited to identifying di-tripeptides?

    <p>Sanger’s method</p> Signup and view all the answers

    Hydrazine is used to identify the N-terminal of amino acids.

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

    What is the advantage of using the Edman reagent for N-terminal residue identification?

    <p>It breaks only the first peptide bond, allowing the remaining peptide to be used for further identification.</p> Signup and view all the answers

    The method using dansyl chloride is used for the identification of the N-terminal amino acid by ___________________.

    <p>colorimetric detection at a specific wavelength</p> Signup and view all the answers

    Match the following identification methods with their corresponding end (N-terminal or C-terminal):

    <p>Dansyl chloride = N-terminal Exopeptidase = C-terminal Hydrazine = C-terminal Phenylisothiocyanate (PITC) = N-terminal</p> Signup and view all the answers

    What is a feature of the process that uses alkaline conditions for amino group identification?

    <p>The amino group is uncharged.</p> Signup and view all the answers

    Insulin has a phenylalanine (phe) N-terminal.

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

    What happens to the peptide after the removal of the N-terminal residue?

    <p>It can be used again to detect the next amino acid.</p> Signup and view all the answers

    What is the primary role of carboxypeptidase in protein analysis?

    <p>It cleaves C-terminal amino acids.</p> Signup and view all the answers

    Hydrazine treatment is effective for identifying C-terminal amino acids without any side reactions.

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

    What is the purpose of alkylation in protein analysis?

    <p>To prevent the reformation of disulfide bonds.</p> Signup and view all the answers

    The cleavage of disulfide bonds in proteins often requires reducing agents such as __________ or __________.

    <p>2-mercaptoethanol, dithiothreitol</p> Signup and view all the answers

    Match the following terms with their descriptions:

    <p>Exopeptidases = Cleave terminal residues of peptides Hydrazine treatment = Used to identify C-terminal residues 2-mercaptoethanol = Reducing agent to break disulfide bonds Denaturation = Process to unfold proteins</p> Signup and view all the answers

    Which of the following methods can be used to separate polypeptide chains based on size?

    <p>Sec-HPLC</p> Signup and view all the answers

    Denaturing conditions can include low temperature and high salt concentration.

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

    What is the purpose of exposing proteins to heat during denaturation?

    <p>To expose the thiol groups (SH) of cysteine residues.</p> Signup and view all the answers

    Study Notes

    Protein Sequencing

    • Protein sequencing is the process of determining the order of amino acids in a protein.
    • Covalent structures of proteins and nucleic acids are important for protein sequencing.
    • Proteins have various functions: enzymes (active sites for substrate binding), structural components (like creatinine, hemoglobin, myoglobin), and hormones (like peptide hormones and insulin).
    • Proteins have four levels of structure.

    Levels of Protein Structure

    • Primary structure: The linear sequence of amino acids in a polypeptide chain.
    • Secondary structure: Local folding of the polypeptide chain, stabilized by hydrogen bonds (e.g., alpha-helices, beta-pleated sheets).
    • Tertiary structure: Three-dimensional folding of a protein due to side chain interactions (e.g., hydrogen bonds, ionic interactions, van der Waals forces, disulfide bonds).
    • Quaternary structure: Protein consisting of more than one amino acid chain that interact. Insulin is an example.

    Primary Structure

    • Is the amino acid sequence
    • Amino acids are connected by peptide bonds.
    • Each amino acid unit is called a residue.

    Determination of Primary Structure

    • The primary structure is crucial for understanding a protein's 3D shape, function, and evolutionary relationships.
    • Mutations in amino acid sequences can lead to diseases, providing insights for developing therapies.
    • First complete amino acid sequence of a protein (insulin) was determined in 1953 using 100 grams. Now, it can be done using micrograms.

    Sanger's Strategy

    • For protein sequencing:
      • Preparation: Determine the number of subunits, cleave disulfide bridges, separate/purify polypeptide chains.
      • Fragmentation: Cleave chains into fragments less than 50 amino acids, then separate and purify the fragments.
      • Sequencing: Sequence the fragments using the Edman reagent.
      • Organization: Compare overlaps of fragments to determine the order and position of the disulfide bonds.

    Protein Sequencing Example

    • Purify a protein with 7 amino acids
    • Expose to different proteolytic agents to obtain fragments.
    • Fragments are used to determine the sequence based on overlaps.

    Sequencing of Proteins

    • Once the amino acid compositions are known, determine the specific sequence.
    • Edman Degradation: A technique to determine the first amino acid.
    • Exposure to phenyl isothiocyanate labels the first amino acid.

    Cleaving Peptide Bonds

    • Mildly acidic conditions cleave the first peptide bond, releasing the labeled amino acid.
    • Repeated procedures determine the entire sequence.

    Step 1: Preparation for Sequencing

    • Identify the number of chains using SDS-PAGE.
    • Identify N-terminal and C-terminal amino acids.
    • Use methods like Sanger's method (DNFB), Dansyl chloride, PITC/Edman reagent, aminopeptidase (identifying N-terminus), and exopeptidases (identifying C-terminus).

    Step 2: Fragmentation

    • Use enzymes or chemicals (e.g., CNBr) to fragment polypeptide chains.
    • Endopeptidases cleave peptide bonds within a protein's internal regions.

    Specificity of Peptide Cleavage

    • Enzymes like trypsin, chymotrypsin, elastase, and thermolysin have specificities for cleaving peptide bonds.
    • Knowing their specificities gives insights into the sequence of the peptide.
    • Reaction rates vary depending on the residue. This can impact the order of the amino acids.

    Separation and Purification of Polypeptides

    • Reduction of disulfide bonds is necessary to separate chains.
    • Denaturation (heat) exposes SH groups to cleave the disulfide bonds.

    Protection of Sulfhydryl Groups

    • Alkylation: Preventing disulfide reformation by attaching a molecule (like iodoacetate) to the sulfur atom in the SH group.

    Separation and Purification and Characterization of Polypeptide Chain

    • Denaturation: Acidic-basic- low salt- high temperature- denaturant (urea-guanidin hypochloride- SDS)
    • Separate chains by charge (IEC) or size (SEC-HPLC).
    • Estimate the number of residues from the molecular weight (MW) using SEC-SDS-PAGE, and MS.

    Step 2: Fragmentation of Polypeptide Chains

    • Use enzymes (e.g., endopeptidases like trypsin) or chemicals (e.g., CNBr) to fragment the polypeptide chain.
    • Endoproteases cleave internal peptide bonds.
    • Some enzymes (e.g., trypsin, chymotrypsin) have specificities for cleaving peptide bonds after certain amino acid residues.

    Ordering the Peptide Fragments

    • Overlapping fragments from different cleavage points are compared.
    • This provides information to establish the correct order of amino acids.

    Identifying the Position of Disulfide Bonds

    • This requires maintaining disulfide bonds during the cleavage process.
    • Repeat fragmentation followed by separation (reverse phase HPLC) to identify disulfide bonds.

    Determining Amino Acid Composition

    • Employ hydrolysis (e.g., 6M HCl at high temperature) to break peptide bonds.
    • Separate the amino acids via ion-exchange chromatography.
    • Analyze the quantity of each amino acid using ninhydrin.

    Example: Sequencing a Heptapeptide

    • Provides illustration of the steps to determine the sequence of a small peptide using Sanger reagent, different enzyme treatments, and cleavage reactions (like by CNBr).

    Studying That Suits You

    Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

    Quiz Team

    Related Documents

    Protein Sequencing - Chapter 2 (PDF)

    Description

    Explore the essential concepts related to protein structure and sequencing methodologies through this quiz. Test your knowledge on the significance of 3D protein shapes, methods of protein fragmentation, and various reagents used in the sequencing process. This quiz is ideal for those studying biochemistry or molecular biology.

    More Like This

    Use Quizgecko on...
    Browser
    Open
    Browser
    Browser