Protein Characterisation Techniques
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Questions and Answers

Which spectroscopic technique is primarily used to determine the three-dimensional structure of proteins?

  • Nuclear Magnetic Resonance
  • X-ray crystallography (correct)
  • Fluorescence
  • Circular Dichroism

    • What is the name of the method that combines size-exclusion chromatography and light scattering?

  • Analytical Ultracentrifugation
  • Spectroscopic Techniques
  • SEC-MALS (correct)
  • Light Scattering

    • What is the relationship between energy (E), Planck's constant (h), and frequency (ν)?

  • ν = E/h
  • E = h/ν
  • ν = h/E
  • E = hν (correct)

    • What parameter in light scattering determines the intensity of scattered light?

    <p>All of the above (D)</p> Signup and view all the answers

    Which amino acid has the highest molar extinction coefficient at 280 nm?

    <p>Tryptophan (D)</p> Signup and view all the answers

    What is the Beer-Lambert Law used to determine?

    <p>The concentration of a solution (C)</p> Signup and view all the answers

    In analytical ultracentrifugation, what does the sedimentation coefficient (S) represent?

    <p>The rate of sedimentation of a protein in a centrifugal field. (B)</p> Signup and view all the answers

    What is the principle behind multiangle light scattering (MALS)?

    <p>Light scattering from different parts of a protein is measured at different angles. (C)</p> Signup and view all the answers

    Which type of light is used in Circular Dichroism spectroscopy?

    <p>Circularly polarized light (B)</p> Signup and view all the answers

    What type of scattering occurs in light scattering experiments?

    <p>Rayleigh scattering (C)</p> Signup and view all the answers

    What is the difference between left and right circularly polarized light?

    <p>The direction of the electric field vector (B)</p> Signup and view all the answers

    In analytical ultracentrifugation, what is the relationship between the sedimentation coefficient (S) and the frictional coefficient (f)?

    <p>S is inversely proportional to f. (A)</p> Signup and view all the answers

    What information can be obtained from Circular Dichroism spectra?

    <p>The secondary structure of a protein (A)</p> Signup and view all the answers

    What is the application of spectroscopic techniques in protein studies?

    <p>All of the above. (D)</p> Signup and view all the answers

    What is the molar extinction coefficient of tryptophan at 280 nm?

    <p>5600 M-1cm-1 (C)</p> Signup and view all the answers

    What is the relationship between the intensity of scattered light and protein concentration in light scattering?

    <p>Intensity is directly proportional to concentration. (A)</p> Signup and view all the answers

    What is the relationship between the wavelengths of the absorbed and emitted light in fluorescence?

    <p>The emitted light has a longer wavelength than the absorbed light. (C)</p> Signup and view all the answers

    In the context of the provided content, what does 'eL – eR' likely represent?

    <p>The difference in energy levels between two electronic states within a molecule. (B)</p> Signup and view all the answers

    Why does the fluorescence of tryptophan change upon binding of paracetamol to human serum albumin?

    <p>Binding of paracetamol induces a conformational change in the protein, altering the environment of the tryptophan residue. (B)</p> Signup and view all the answers

    What can fluorescence spectroscopy be used to measure?

    <p>All of the above. (D)</p> Signup and view all the answers

    Which of the following is NOT a characteristic of fluorescence?

    <p>It is a single-step process where the molecule directly transitions from the excited state to the ground state. (B)</p> Signup and view all the answers

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

    <p>To determine the molecular mass of a protein (D)</p> Signup and view all the answers

    Which technique can be used to determine the surface charge of a protein?

    <p>Isoelectric focusing (D)</p> Signup and view all the answers

    What is the significance of using Native PAGE in protein characterization?

    <p>It allows for the determination of protein-protein interactions. (C)</p> Signup and view all the answers

    What is the role of Coomassie Brilliant Blue G-250 in SDS-PAGE?

    <p>It binds to proteins and allows them to be visualized. (A)</p> Signup and view all the answers

    How does analytical ultracentrifugation determine the molecular mass of a protein?

    <p>By measuring the rate at which the protein sediments in a centrifugal field. (A)</p> Signup and view all the answers

    What information can be obtained by combining the results of SDS-PAGE and Native PAGE?

    <p>The number and size of the polypeptide chains in a protein. (A)</p> Signup and view all the answers

    Which of the following techniques is used to determine the mass of a protein in its denatured state?

    <p>Mass spectrometry (C)</p> Signup and view all the answers

    What is the key advantage of using MALDI-TOF mass spectrometry for protein characterization?

    <p>It provides high sensitivity for analyzing protein mixtures. (C)</p> Signup and view all the answers

    Study Notes

    Protein Characterisation Techniques

    • Techniques to determine protein properties include examining polypeptide chains, mass, surface charge, and tertiary/quaternary structure.
    • Determining polypeptide chain composition: Column chromatography separates proteins.

    Characterizing a New Protein

    • Crucial questions in protein characterization include:
      • Whether the protein is composed of one or more polypeptide chains.
      • The mass of each polypeptide chain and the whole protein.
    • Techniques for determining protein mass and charge include:
      • SDS-PAGE and mass spectroscopy (denatured protein)
      • Size-exclusion chromatography, ultracentrifugation, and light scattering (for undenatured protein)
      • Isoelectric focusing (denatured protein)

    SDS-PAGE for Determining Molecular Mass

    • SDS-PAGE is crucial for determining protein molecular mass.
    • A graph of relative mobility versus mass reveals a linear relationship, aiding precise mass determination.

    Native-PAGE

    • In native-PAGE, proteins are not denatured, preserving their tertiary and quaternary structures.
    • Coomassie Brilliant Blue G-250 imparts a negative charge to proteins without unfolding.

    Combining SDS-PAGE and Native-PAGE

    • Combining SDS-PAGE and native-PAGE techniques can reveal if a protein is a homotrimer (composed of three identical polypeptide chains).

    Mass Spectrometry

    • Mass spectrometry (MALDI-TOF) ionizes proteins, analyzes their mass-to-charge ratios, and provides crucial information about molecular mass in a vacuum.

    Analytical Ultracentrifugation

    • Used to determine protein mass and shape under high centrifugal acceleration (sedimentation).
    • The sedimentation coefficient is linked to the protein's mass, shape, and the solution's properties.

    Light Scattering

    • Techniques for determining protein mass and size (in solution) using light scattering:
      • Measure light scattered from the sample to determine protein concentration and molecular mass.
      • The intensity of scattered light varies with the angle and provides structural information.
      • SEC-MALS combines size-exclusion chromatography (SEC) with multiangle light scattering (MALS) to provide a direct read-out of protein mass per fraction.

    Spectroscopic Techniques

    • Various spectroscopic techniques explore light-matter interactions, including:
      • Absorption spectroscopy (UV/Vis)
      • Circular Dichroism (CD)
      • Fluorescence spectroscopy.

    Protein Absorption Spectroscopy

    • Used to determine protein concentration, based on the absorption of light by the protein.
    • Specific wavelengths relate to specific amino acid components.
    • The absorbance is proportional to the concentration and provides information to determine the concentration.

    Circular Dichroism (CD)

    • Used to determine the secondary structure (α-helix, β-sheet, random coil) of proteins, based on their interaction with polarized light.
    • CD measurements can determine secondary structure percentages.

    Fluorescence Spectroscopy

    • Analyzes the emission of light by proteins after excitation.
    • Often used to study protein-ligand interactions; fluorescence intensity is related to concentration and interactions.

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    Protein Characterisation Lecture - PDF

    Description

    This quiz explores various techniques for characterizing proteins, focusing on methods such as SDS-PAGE, mass spectroscopy, and chromatography. Participants will learn about the determination of polypeptide chain composition and molecular mass, which are fundamental to understanding protein properties. Test your knowledge on the critical questions and techniques in protein characterization.

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