Protein Analysis and DNA Structure

Protein Analysis and Separation Techniques

Cell Lysis

• Purpose: To break open cells and release their contents.

• Result: The broken-up cellular material in solution is called the lysate (from 'lyse' meaning 'to break').

• Methods of Cell Lysis:

  • Ultrasound (Sonication): Pulses are sent into the sample, agitating and breaking open fragile cells.

  • Syringe/Mechanical shearing: Cells are forced through a tiny channel, which is small enough to cause them to break.

  • Mortar and Pestle: Physical mashing of cells, similar to kitchen use.

Centrifugation of Lysate

• Purpose: To isolate out desired components by spinning the lysate extremely fast.

• Mechanism: Denser components are pulled to the bottom, forming a pellet, while the liquid (supernatant) remains at the top.

• Key Principle: The speed of centrifugation determines what steps/components can be collected, allowing for isolation of different organelles or membranes at varying speeds.

• Advantage over Gravity:

  • Gravity alone would cause heavy pieces to settle but would not create a dense, compact pellet.

  • A dense pellet formed by centrifugation ensures that when the liquid is removed, the desired solid components remain tightly packed at the bottom, preventing them from mixing back into the liquid.

• Understanding Outcome: It's crucial to know what components will fall to the bottom (pellet) and what will remain at the top (supernatant) after centrifugation.

Protein Separation

• Once a dense pellet containing various cellular components (including proteins) is obtained after high-speed centrifugation, the next step is to separate the specific proteins of interest.

• Methods (Examples): Separation can be based on properties like charge, size, etc.

• Antibody Binding: Scientists may use antibodies to bind to specific proteins for isolation, often in combination with other methods.

Imaging Techniques for Protein Structure

• Purpose: To determine the three-dimensional (3D) structure of isolated proteins.

• Three Key Techniques:

  • X-ray Crystallography:

    • History: Used since the $1930$s, considered a 'gold standard' for protein structure determination.

    • Process: A beam is sent through a protein structure (often a crystal), and the atoms in the protein molecules scatter the beam, resulting in a diffraction pattern. This pattern is then used to reconstruct the protein's 3D structure.

  • Nuclear Magnetic Resonance (NMR): (Mentioned as an option for structure identification).

  • Cryo-electron Microscopy: (Mentioned as an option for structure identification).

• Exam Relevance: For the class, a general understanding of these techniques (e.g., knowing one method to identify protein structure) is sufficient, rather than an in-depth knowledge of their assays.

Introduction to Chapter 5: DNA and the Central Dogma of Biology

Roadmap for Chapter 5

• DNA Structure and Organization: Understanding its fundamental components and how it's arranged.

• DNA, RNA, and Protein: Exploring their roles and interrelationships.

• DNA Replication: How DNA copies itself.

• Developmental Processes: Understanding how biological information is used for development.

• Pathologies and Diseases: Identifying where errors in these processes can lead to medical conditions.

• Engineering Perspective: As engineers focused on human health, the goal is to understand where and how things go wrong to develop solutions.

The Central Dogma of Biology

• Definition: The fundamental principle that genetic information flows from DNA to RNA to protein.

• Schematic Representation:

  • $DNA \rightarrow RNA \rightarrow Protein$

• Process Overview:

  • DNA (double helical) 'unzips' and replicates to form RNA.

  • RNA sequences are then 'read' to synthesize proteins.

• Future Lecture Details: The specific steps, transcription factors, markers, and partners involved in DNA unwinding and other processes will be covered in more detail.

DNA Structure

• Double Helical Structure: DNA is a double helix.

• Components: Formed by two complementary strands of nucleotides.

• Bonding: The two strands are held together by hydrogen bonds between specific nitrogenous bases.

• Nitrogenous Bases:

  • Adenine (A)

  • Thymine (T)

  • Guanine (G)

  • Cytosine (C)

• Complementary Base Pairing:

  • Adenine (A) always pairs with Thymine (T).

  • Guanine (G) always pairs with Cytosine (C).

• Antiparallel Strands: The two complementary strands run in opposite directions:

  • One strand runs from the $5'$ (five-prime) end to the $3'$ (three-prime) end.

  • The other strand runs from the $3'$ (three-prime) end to the $5'$ (five-prime) end.

  • Importance of Directionality: Crucial for understanding DNA function and problem-solving.

• Number of Hydrogen Bonds between Base Pairs:

  • Guanine (G) and Cytosine (C) form $3$ hydrogen bonds.

  • Adenine (A) and Thymine (T) form $2$ hydrogen bonds.

• Nucleotide Linkage: Individual nucleotides within a single strand are linked by phosphodiester bonds.

  • These bonds connect the $3'$ hydroxyl (-OH) group of one sugar to the $5'$ phosphate group of the next sugar.

DNA Length and Organization

• Length in Eukaryotic Cells: DNA is extremely long.

  • Analogy: Approximately $24$ miles of thread in a tennis ball.

• Genes in Eukaryotic Cells: A typical eukaryotic cell contains about $30,000$ genes (example given, actual number varies).

• Coding vs. Non-coding Regions:

  • Within a DNA strand, there are regions that code for different proteins (genes).

  • There are also extensive non-coding regions that are not specific to any known protein.

  • Historical View: These non-coding regions were previously referred to as 'junk DNA.'

  • Current Understanding: Research increasingly shows that these regions have various functions, even if they don't directly code for proteins.

• Bacterial DNA Comparison: Bacteria typically have a single, circular DNA strand, contrasting with the linear, multiple strands in eukaryotes.