Introduction to Cell Biology & Biochemistry

Protein Structure

Part 3 Overview

  • Structure-function relationship: Myoglobin and Haemoglobin

  • Protein denaturation

  • Microscopy techniques:

    • Widefield microscopy

    • Confocal microscopy

  • Analysis of protein structure methods:

    • Electron microscopy

    • NMR (Nuclear Magnetic Resonance)

    • X-ray crystallography

Structure – Function Relationship

Myoglobin

  • Definition: Myoglobin is a protein that consists of a single polypeptide chain.

  • Structural Characteristics:

    • Lacks quaternary structure

    • Features a very high affinity for oxygen, even at low oxygen pressure

  • Function:

    • Acts as an effective storage protein due to its ability to bind oxygen tightly.

Haemoglobin

  • Definition: Haemoglobin is a protein that contains four polypeptide chains, thus exhibiting a quaternary structure.

  • Cooperativity:

    • When oxygen binds to one subunit of haemoglobin, it induces a conformational change in the structure.

    • This change enhances the affinity for oxygen in the remaining subunits—a phenomenon known as cooperativity.

  • Function:

    • Efficient transport protein due to the cooperative binding mechanism.

  • Models of Cooperativity:

    • Concerted Model (MWC): Proposes that all subunits change from one state to another simultaneously.

    • Sequential Model (KNF): Suggests that the binding of substrate changes the conformation of protein gradually, one subunit at a time.

Saturation Curves:

  • Myoglobin exhibits a hyperbolic saturation curve for O₂ binding.

  • Haemoglobin demonstrates a sigmoidal saturation curve for O₂ binding due to its cooperative binding nature.

  • Graph Representation:

    • The x-axis represents partial pressure of oxygen (PO₂) in kPa.

    • The y-axis shows saturation of the protein with O₂ (%).

Protein Denaturation

Denatured State

  • Definition: The state of a protein where its secondary, tertiary, or quaternary structure is altered.

  • Structural Changes:

    • Disruption of non-covalent bonds such as hydrogen bonds, ionic bonds, and hydrophobic interactions.

    • Disruption of disulfide bonds.

    • Primary structure remains intact as peptide bonds are unaffected.

  • Consequences:

    • Loss of biological activity.

Native State

  • Characteristics:

    • Most stable form of the protein.

    • Higher solubility in aqueous environments.

    • Exhibits biological activity.

    • Polar groups are typically oriented outward; hydrophobic groups are inward.

Agents of Denaturation

  • Factors Leading to Denaturation:

    • Changes in pH levels

    • Variations in salt concentration

    • Temperature fluctuations (higher temperatures can weaken hydrogen bonds)

    • Presence of reducing agents

    • Chaotropic agents that disrupt hydrogen bonds

    • Detergents

Reversibility

  • Reversible Process:

    • Denaturation of proteins can be reversible but is not always the case.

Microscopy Techniques

Light Microscope

  • Application: Used for visualizing cellular structures.

  • Techniques:

    • Nissl staining for highlighting cell bodies.

    • Anti-GFAP (Glial Fibrillary Acidic Protein) immunostaining for specific visualization of glial cells.

Confocal Laser Scanning Microscope

  • Functionality: Enables 3D visualization of microscopic specimens.

  • Importance:

    • Facilitates analysis of spatial relationships between cellular structures that conventional microscopy might overlook.

Microscopy Resolution

  • Definition of Resolution: The resolution of an optical microscope is the shortest distance between two points on a specimen that can still be resolved as distinct entities by the observer or camera.

  • Resolution Values:

    • Lower resolution values indicate better clarity in microscopy.

  • Key Factors:

    • Wavelength of light ($ ext{λ}$) used is critical.

    • Numerical Aperture (NA) is determined by the features and quality of the microscope.

Limitations of Light Microscopy

  • Wavelength Scale of Microscopy:

    • Radio: 10³ m

    • Microwave: 10^{-2} m

    • Infrared: 10^{-5} m

    • Visible light: 0.5×10^{-6} m

    • Ultraviolet: 10^{-8} m

    • X-rays: 10^{-10} m

    • Gamma rays: 10^{-12} m

  • Resolution Estimation:

    • The resolution of light microscopy is significantly limited by the wavelength of visible light (~500 nm).

Advanced Techniques for Protein Analysis

Electron Microscopy (EM)

  • Types:

    • Scanning Electron Microscopy (SEM):

      • Ideal for detailed 3D surface observations.

      • Has lower resolution than Transmission Electron Microscopy (TEM).

    • Transmission Electron Microscopy (TEM):

      • Requires very thin samples.

      • Capable of observing smaller structures at higher resolution.

  • Advantages of EM:

    • Excellent for imaging large assemblies, for example, viral capsids.

    • Can capture numerous images rapidly.

    • However, lacks resolution for individual proteins.

X-ray Crystallography

  • Definition: Analysis through X-ray diffraction from crystalline samples.

  • Advantages:

    • Capable of yielding very high resolution data.

    • No upper size limitation for molecules studied.

  • Disadvantages:

    • Requires crystalline samples which are not always obtainable.

Nuclear Magnetic Resonance (NMR)

  • Technique Overview:

    • A sample is subjected to a powerful magnetic field.

    • Radio waves are transmitted through the sample, and nuclei of individual atoms absorb these waves differently.

    • The distinct absorption is affected by the local chemical environment of the nuclei.

    • Data obtained can ascertain distances and relative positions of atoms in a molecule, useful for 3D structural analysis.

  • Strengths:

    • Particularly effective for small to medium proteins, ligands, and mobile regions.

    • Solution-based technique, which eliminates the need for crystallization.

  • Limitations:

    • Not suitable for studying large proteins or complexes.