Lecture Notes: Protein Analysis and Plasma Membrane

SDS-PAGE and protein subunits

• Goal: separate amino acid chains and polypeptides so they aren’t folded together.

• SDS structure: contains sulfur, several oxygens, and sodium; large hydrocarbon chain with many carbons and hydrogens. Used to coat proteins with a uniform negative charge.

• Disulfide bonds in proteins can link subunits (e.g., subunit a and subunit b) into a single, larger molecule.

• Treatment with reducing agents (e.g., NCS and β-mercaptoethanol) breaks disulfide bonds, separating subunits so they run as separate bands on the gel.

• Gel electrophoresis outcome:

  • When separated, you’ll see a band for the larger subunit and a band for the smaller one.

  • If a protein has disulfide bonds, breaking them changes the band pattern; if a single-subunit protein has no disulfide bonds, SDS+β-mercaptoethanol won’t “break” it, but it still carries a negative charge.

  • Bands run toward the positive anode due to the negative charge imparted by SDS.

• Gel type: polyacrylamide gel is used for separating proteins.

• If you add SDS to multiple subunits, all will carry a negative charge and separate primarily by size.

• Concept recap: smaller polypeptides migrate faster through the gel than larger ones.

Mass spectrometry

• Mass spectrometry provides a unique protein fingerprint based on the protein’s shape/fragment pattern.

• Procedure (general): excise a protein spot from a polyacrylamide gel, then analyze the protein in a mass spectrometer.

• Output: relates abundance to mass-to-charge ratio, denoted as m/z.

• Interpretation: compare the obtained pattern to a protein database to identify the protein.

• Note: this transcript only scratches the surface of MS; the key idea is fingerprinting and database matching for identification.

X-ray crystallography

• Historical context: Rosalind Franklin’s X-ray crystallography contributed to the discovery of DNA structure; the same method is used to study proteins today.

• Process overview:

  • Grow a crystal of the protein.

  • Shoot X-rays at the crystal; X-rays diffract depending on protein shape.

  • Collect diffraction pattern on a detector.

  • Use high-powered computers to compute the 3D structure from the diffraction data.

• Outcome: obtain the three-dimensional structure of the protein.

• Conceptual note: translating diffraction patterns into a 3D model involves complex calculations; it is not a direct “image” but a computational reconstruction.

Protein domain families and functional prediction

• Concept: Some proteins consist of multiple domains; a family may be composed of single-domain proteins (e.g., red/yellow/green vs. brown/blue/orange).

• Practical implication: knowing what each domain does helps predict the possible functions when domains are linked together in multi-domain proteins.

• Rationale: domain functions can suggest how domains interact and what activities might be carried out when combined.

Differential centrifugation: organelle ordering and pellet formation

• Context: separating cellular components by centrifugation speeds to progressively pellet larger particles.

• Practical exercise described: arrange components from largest to smallest that would pellet at different speeds.

• Observed (most popular sequence): nucleus → large macromolecules → mitochondria → fragments of the endoplasmic reticulum (ER).

• Important nuance: the largest things pellet out first; with higher speeds, progressively smaller components pellet.

• Typical sequence (as described):
  1) Low speed: pellet contains whole cells, nuclei, cytoskeletons (green pellet); supernatant is relatively homogeneous.
  2) Medium speed: pellet contains mitochondria, lysosomes, peroxisomes.
  3) High speed: pellet contains rough ER fragments and other small vesicles.
  4) Very high speed: pellet contains ribosomes, viruses, and large macromolecules.

• Practical tip: rewrite the notes or draw diagrams to solidify the order of pelleting and the relative sizes.

Membranes, lipids, and phospholipids

• Core idea: membranes are made of a lipid bilayer with embedded proteins.

• Basic membrane structure:

  • Lipid bilayer consists of phospholipids with hydrophilic (polar) heads facing water and hydrophobic (nonpolar) tails facing inward.

  • Proteins are embedded in the bilayer and can be transmembrane (spanning the bilayer), partially embedded, or attached to lipids/proteins.

• Lipids: definition and examples

  • Lipids are insoluble in water but soluble in fat.

  • Example: triglyceride (triglycerol): glycerol head plus three fatty acid chains (hydrocarbon tails).

  • Fatty acids can be saturated (straight chains) or unsaturated (kinks due to double bonds).

• Phospholipids specifics:

  • Phospholipids have two fatty acid chains and a glycerol backbone.

  • Polar head group contains choline and phosphate, making the head hydrophilic.

  • The two hydrocarbon tails are nonpolar and form the hydrophobic interior of the bilayer.

  • Amphipathic nature (both hydrophilic and hydrophobic) drives spontaneous bilayer formation.

• Bilayer formation in water:

  • Phospholipids spontaneously form bilayers or micelles/liposomes in polar solvents (e.g., water) with heads outward and tails inward.

  • A flat bilayer is energetically unfavorable at the edges when exposed to water; it tends to close into a sealed sphere (liposomes) to shield hydrophobic tails.

• Flip-flop (transbilayer movement):

  • Lipids rarely flip between leaflets due to the high energetic barrier of moving the polar head through the hydrophobic core; can occur but at a low rate.

• Membrane dynamics within a leaflet:

  • Lateral diffusion: individual phospholipids move sideways within the same leaflet.

  • Flexion: tails bend/move within the leaflet.

  • Rotation: entire phospholipid can rotate within the plane of the leaflet.

• Membrane fluidity:

  • Membranes are flexible and behave like a fluid; lipid composition determines fluidity.

  • Shorter hydrocarbon chains increase fluidity (less tight packing).

  • Unsaturated hydrocarbon chains increase fluidity due to kinks that prevent tight packing.

  • Cholesterol modulates fluidity by inserting between phospholipids; its effect depends on location within the bilayer.

• Cholesterol specifics:

  • Cholesterol is amphipathic: both hydrophobic and hydrophilic portions.

  • Structure: a single hydrocarbon tail and a rigid ring system; it intercalates among phospholipids.

  • Cholesterol distribution affects local fluidity: center region tends to be less fluid; outer regions (near tails facing interior/exterior) can be more fluid.

• Functional significance of membrane fluidity:

  • Fluidity allows membrane proteins to move and reassemble where needed for function.

Membrane proteins: types, placement, and roles

• General idea: proteins associated with the lipid bilayer can be integral (permanently bound) or peripheral (loosely associated).

• Integral (transmembrane) proteins:

  • Extend through the lipid bilayer; portions on both sides interact with aqueous environments.

  • Often contain alpha-helical segments within the bilayer; beta-barrel structures can form pores.

  • Some span the membrane with hydrophobic regions inside the bilayer and hydrophilic regions outside.

  • Some are anchored to the cytosolic side by an amphipathic alpha helix; the helix has hydrophobic interior with hydrophilic edges facing cytosol.

  • Some are partially embedded and attached to lipids rather than spanning the bilayer.

  • Others are bound to other membrane proteins on either the extracellular or cytosolic side.

• Peripheral membrane proteins:

  • Temporarily bound and easily released from the membrane.

• Examples of membrane roles and organizations:

  • Transporters and channels move substances across the bilayer, either requiring energy (active transport) or moving down a gradient (passive transport).

  • Transmembrane proteins can form networks that connect to cytoskeletal elements (e.g., actin and spectrin in red blood cells) to maintain cell shape and mechanical properties.

  • Epithelial cells attach to the extracellular matrix via membrane proteins to maintain tissue structure.

  • Receptors detect extracellular signals and relay messages into the cell, initiating intracellular signaling cascades.

  • Enzymes at the membrane catalyze reactions near the surface by converting substrates to products.

• Integral membrane protein examples of orientation:

  • Hydrophilic portions on extracellular/cytosolic sides; hydrophobic portions within the lipid bilayer.

• Experimental examples to study membrane protein mobility:

  • Human-mouse cell fusion experiments show membrane proteins mix between two originally distinct cell membranes, indicating lateral mobility.

  • Fluorescence recovery after photobleaching (FRAP): label membrane proteins with a fluorescent dye, bleach a patch with a laser, observe recovery as unbleached proteins diffuse into the patch.

Protein mobility and membrane dynamics: experimental evidence

• Membrane protein mobility demonstrated by:

  • Human-mouse hybrid cell fusion: after fusion, red (mouse) and blue (human) membrane proteins mix, indicating lateral mobility across the membrane.

  • FRAP: recovery curves show proteins move back into bleached regions over time, confirming lateral diffusion.

Carbohydrates on the cell surface

• Carbohydrates are present on the cell surface and contribute to the glycocalyx.

• Types of carbohydrate attachments:

  • Glycolipids: carbohydrate portions attached to lipids in the membrane.

  • Glycoproteins: carbohydrate portions attached to transmembrane proteins.

  • Carbohydrates can also be attached to proteins that then interact with membrane proteins.

• Conceptual visualization: glycolipids have a lipid anchor with attached sugar units; glycans are often depicted as hexagonal sugar units.

Synthesis of key themes and connections

• Structural organization of membranes underpins function: lipid bilayer provides a hydrophobic core that controls permeability; proteins provide transport, signaling, and structural roles.

• The amphipathic nature of phospholipids drives self-assembly into bilayers and liposomes, ensuring compartmentalization in cells.

• Fluidity and mobility are essential for proteins to reach their functional locations and to respond to cellular needs.

• Post-translational and post-assembly modifications (e.g., disulfide bonds, lipid attachments) alter protein interactions and localization.

• Experimental approaches (SDS-PAGE, mass spectrometry, X-ray crystallography, differential centrifugation, FRAP) offer complementary insights: size/peptide composition, identity, structure, and dynamic behavior.

Study strategies and exam expectations (as discussed in the transcript)

• Exam format: paper-based, primarily multiple choice with 1–2 short-answer questions.

• Covered chapters/focus: roughly chapters 1, 2, 4, 11, and 12, with some skipping around; refer to the syllabus for exact scope.

• Practice approaches:

  • Rewrite notes and draw diagrams to reinforce memory.

  • Do textbook exercises for additional practice questions; appearance on the exam may resemble similar questions.

  • Use tutor or class reviews to clarify any unclear concepts.

• Mentimeter prompts (memory focus):

  • Definition of a lipid and how lipid composition affects bilayer fluidity.

  • The roles of the plasma membrane in information reception, import/export, and membrane expansion.

Key definitions and quick references

• Lipids: soluble in fat, insoluble in water; example triglyceride with glycerol head and three fatty acid chains.

• Phospholipid: lipid with two fatty acid chains, glycerol backbone, and a polar head group (choline + phosphate); amphipathic.

• Amphipathic: molecule with both hydrophilic and hydrophobic parts.

• Liposome: closed bilayer structure formed by phospholipids in polar solvents.

• Diffusion modes within membranes: lateral diffusion, flexion, rotation.

• Mass spectrometry: outputs a pattern related to the mass-to-charge ratio, m/z, used to identify proteins by database matching.

• X-ray crystallography: technique to determine the 3D structure of proteins via diffraction patterns and computational reconstruction.

• Differential centrifugation: sequentially centrifuging at increasing speeds to pellet larger components first (e.g., nucleus, mitochondria, ER fragments, ribosomes).

• Integral membrane proteins: span the bilayer; can be alpha-helical or beta-barrel; can be anchored to cytosolic or extracellular sides or attached to lipids.

• Peripheral membrane proteins: loosely bound to membrane; easier to release.

• Glycolipids vs glycoproteins: carbohydrates attached to lipids or proteins, contributing to cell recognition and signaling.

Connections to real-world relevance

• Understanding membrane composition and dynamics helps explain drug delivery, membrane permeability, and the design of therapeutics targeting membrane proteins.

• The concept of membrane fluidity relates to physiological conditions and diseases linked to lipid composition (e.g., cholesterol’s effect on membrane properties).

• Structure determination by X-ray crystallography underpins drug design by revealing active sites and binding pockets.

• Mass spectrometry is a powerful tool in proteomics for identifying proteins in complex mixtures, enabling discovery and profiling in health and disease.

Ethical, historical, and practical notes

• Rosalind Franklin’s contributions to X-ray crystallography highlight the historical importance of crystallography in molecular biology.

• The integration of multiple techniques reflects an ethical commitment to corroborate findings across independent methods for robust conclusions.