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

Flashcards

Polypeptide Chains

Chains of amino acids that make up proteins.

SDS-PAGE

A technique to separate proteins based on their molecular mass by denaturing them.

Native-PAGE

A method to separate proteins without denaturing them, preserving their structure.

Isoelectric Focusing

A technique to determine the surface charge of proteins at different pH levels.

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Mass Spectrometry

An analytical technique to measure the mass-to-charge ratio of ions.

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Homotrimer

A protein composed of three identical polypeptide subunits.

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Analytical Ultracentrifugation

A technique to analyze soluble proteins' movements when spun at high speeds.

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MALDI-TOF

A specific type of mass spectrometry technique that ionizes proteins for analysis.

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Fluorescence Spectroscopy

A technique to measure fluorescence emission after absorbance of light.

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Absorbance

The process by which a substance takes in light energy, raising its energy state.

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Emission

Light released by a substance as it returns to a lower energy state after excitation.

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Tryptophan Fluorescence

The intrinsic fluorescence property of tryptophan due to its aromatic structure.

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Paracetamol Binding

The binding of paracetamol to human serum albumin affects fluorescence characteristics.

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Spectroscopic Techniques

Methods used to study the interaction between matter and electromagnetic radiation.

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Planck's Constant

A fundamental constant equal to 6.626 x 10^-34 J*s, relating energy and frequency.

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Energy Equation

The equation E=hν relates energy (E) to frequency (ν) using Planck's constant (h).

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Protein Absorption Spectroscopy

A technique to measure protein concentration by the absorption of light at specific wavelengths.

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Absorbance Formula

The equation A = log10(Io/I) calculates absorbance (A) based on incoming (Io) and transmitted light (I).

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Molar Extinction Coefficient (ε)

A measure of how strongly a substance absorbs light at a specific wavelength, in M^-1cm^-1.

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Circular Dichroism

A spectroscopic technique that measures the difference in absorption of left and right circularly polarized light.

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Amino Acid Absorbance Peaks

Specific wavelengths (lmax) where amino acids like tryptophan and tyrosine strongly absorb UV light.

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Sedimentation Coefficient (s)

A measure of how fast a particle sediments in a centrifuge, influenced by mass and shape.

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Protein Mass (m)

The total weight of a protein molecule, crucial for calculating sedimentation coefficient.

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Partial Specific Volume (⊽)

Volume occupied by a protein molecule per unit mass, affecting its buoyancy.

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Solution Density (𝜌)

Mass per unit volume of the solution, influencing sedimentation of proteins.

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Frictional Coefficient (f)

A measure of resistance a protein encounters as it moves through a solution.

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Rayleigh Scattering

Elastic scattering of light where there's no change in light's wavelength after interaction with particles.

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Multiangle Light Scattering (MALS)

Technique that measures scattered light at multiple angles to determine protein size and mass.

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SEC-MALS

A combined method of size-exclusion chromatography and light scattering for protein analysis.

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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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