PROTEIN STRUCTURE 2

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Introduction to Proteins and Their Domains

What Is A Protein Domain?

  • Definition: Protein domains are independently folding, stable structural units within a protein.

  • Length: Typically, they are about 50-250 amino acids long.

  • Independence: They can retain structure/function even when separated from the rest of the protein.

  • Modular Building Block: They serve as building blocks for the architecture of proteins.

  • Evolutionary Conservation: Domains are frequently conserved across different proteins.

Structural and Functional Roles of Protein Domains

  • Structural Role:

    • Provide a stable framework for a protein’s overall architecture.

    • Allow for a modular assembly of complex proteins.

  • Functional Role:

    • Each domain can have a specific job within the protein.

    • Domains can work together to perform complex functions.

Types of Domains

  • Structural Domains:

    • Defined by a specific fold.

  • Functional Domains:

    • Linked to specific biochemical activities.

  • Topological Domains:

    • Defined by their location within the protein.

  • A single protein may contain multiple types of domains combined within its structure.

Importance of Domains

  • Evolution: Domains are reused across proteins for creating new functions.

  • Bioinformatics: Identifying domains assists in predicting protein functions based on sequences.

  • Biotechnology: Domains can be engineered or swapped to create new capabilities.

  • Medicine: Many diseases arise from mutations in specific domains.

Protein Purification Techniques

General Overview

  • Each cell contains thousands of different proteins; to study a specific protein, separation is needed.

  • Methods for protein separation are applicable across all biochemicals; specialized methods are required for high purity.

Sample Preparation

  • Objective: Extract cellular contents by disrupting the cell membrane.

  • Techniques:

    • Sonication: Utilizes high-frequency sound waves to cause rapid pressure changes in liquid.

    • Shearing: Utilizes mechanical forces to physically disrupt membranes.

    • Lysis Buffer: Uses chemical detergents to solubilize membranes, aiding extraction.

Protein Separation Methods

Fractionation by Size

Centrifugation

  • Low-Speed Centrifugation (1000xG):

    • Removes unbroken cells, nuclei, and large debris.

  • Mid-Speed Centrifugation (10,000xG):

    • Isolates mitochondria and lysosomes.

  • High-Speed Centrifugation (100,000xG):

    • Isolates microsomes and ribosomes.

  • The supernatant contains soluble cytosolic proteins and small complexes.

Dialysis

  • Purpose: Use a semi-permeable membrane to retain larger proteins while allowing smaller ones to diffuse.

  • Procedure: Protein lysate is placed inside dialysis tubing, immersed in a buffer.

  • Outcome: Small proteins pass through the membrane, larger proteins remain in the bag.

Size Exclusion Chromatography

  • Mechanism: Separates molecules based on size by passing them through a column with porous beads.

  • Process: Larger molecules elute first as they cannot enter the beads' pores, while smaller molecules take longer paths and elute later.

Fractionation by Charge

Affinity Chromatography

  • Process: Uses specific binding interactions between protein and ligand on a resin.

  • Binding: The target protein binds selectively while others flow through.

  • Elution: Achieved by adding a competitive ligand or altering pH to disrupt binding.

Ion Exchange Chromatography

  • Mechanism: Separates proteins based on net surface charge at specific pH levels.

  • Cation Exchange: Utilizes a negatively charged resin to bind positively charged proteins.

  • Anion Exchange: Uses a positively charged resin to bind negatively charged proteins.

  • Elution: Achieved by using buffers that alter pH, affecting the ionic interactions.

Determining A Protein’s Amino Acid Sequence

Step 1: Separate and Purify Polypeptide Chains

  • Need for Separation: Necessary when multiple polypeptide chains are present; some proteins consist of single chains.

  • Importance of Denaturation: Denature the protein while maintaining peptide bonds intact to analyze individual chains.

Step 2: Break Interchain Disulfide Linkages

  • Significance: Disulfide linkages contribute to the 3D structure, thus need to be broken to obtain a linear polypeptide for analysis.

  • Chemical Reduction: Reactions that cleave these bonds prevent their reformation, facilitating linearization of the polypeptide.

Step 3: Determine Amino Acid Concentration

  • Instrumentation: Amino acid analysers can provide a report on amino acid composition in under 60 minutes.

  • Output: Reports are presented as percentages of each amino acid presence in the sample.

  • Sensitivity: These instruments can operate on samples of less than 1 nanomole of protein.

Step 4: Identify Terminal Amino Acids

  • N-Terminal and C-Terminal: A polypeptide has one of each; determining these can indicate the presence of multiple chains.

  • Methods: Tagging techniques can identify N-terminal residues.

  • Fragmentation: Tagging and subsequently cleaving the polypeptide can reveal terminal residues.

Step 5: Cleave Protein into Small Fragments

  • Purpose: Create a digest of protein samples to obtain various fragments due to different cleavage patterns.

  • Specific Example – Trypsin: Cuts after K (lysine) and R (arginine), resulting in specific fragments.

    • Possible configurations: Fragments produced can lead to multiple potential complete sequence configurations.

Step 6: Locate Disulfide Bridges

  • Optional Step: May be skipped to preserve disulfide bridges for analysis.

  • X-Ray Diffraction: Can be utilized to investigate the shape of fragments created with intact linkages.

Mass Spectrometry Sequencing

Overview of Mass Spectrometry (MS)

  • Function: Measures mass-to-charge ratio (m/z) of ionized molecules.

  • Applications: Can establish molecular mass, sequence, modifications, and interactions based on the setup used.

Ionisation Process

  • Conversion: Neutral molecules are converted to charged ions for manipulation via electric and magnetic fields.

Mass Analyser

  • Separation: Charged ions are categorized based on their mass-to-charge ratios, enabling analysis of protein fragments.

Types of Mass Spectrometry

“Top-Down” Mass Spectrometry

  • Function: Directly measures the mass of intact proteins, allowing for molecular weight determination and detection of isoforms or post-translational modifications.

  • Usage: Primarily used for verifying protein expression and confirming the composition of engineered constructs.

“Bottom-Up” Mass Spectrometry

  • Process: Liquid chromatography is used to separate digested polypeptides before mass spectrometry.

  • Outcome: Identifies protein identity and sequence coverage, and can reveal unexpected peptides from alternative splicing or mutations.