Cell Biology Exam 1

How do amino acids come together to create proteins?

They link through the formation of a covalent peptide bond

What are the four categories of amino acids?

• Acidic (negatively charged) polar

• Basic (positively charged) polar

• Uncharged polar

• Uncharged nonpolar

How is the folding of proteins determined?

• Steric restrictions confine the energy minima for the bond angles in polypeptides to a narrow range

• The folding is determined by many different sets of weak noncovalent bonds that form between one part of the chain and another (backbone and side chain atoms)

  • hydrogen bonds, ionic bonds (electrostatic), van der Waals attractions, and hydrophobic forces

What is hydrophobic force?

• Hydrophobic molecules, including nonpolar side chains of amino acids, tend to be forced together in an aqueous environment

  • Therefore, an important factor governing folding is the distribution of its polar and nonpolar amino acids, as nonpolars will cluster toward the inside

Parallel and antiparallel beta sheets

A is antiparallel, B is parallel

What is a coiled-coil, and what force causes it?

• Alpha helices can wrap around each other to form a particularly stable structure known as a coiled-coil

• Caused by hydrophobic forces to move nonpolar side chains towards inside

What is the primary structure of proteins?

The amino acid sequence

What is the secondary structure of proteins?

• Alpha helices

• Beta sheets

What is the tertiary structure of proteins?

Its final, 3D shape

What is the quaternary structure of proteins?

Individual proteins (subunits) binding to each other to create a larger protein complex, such as a dimer

Why are the secondary structures of proteins common?

Because they can form due to hydrogen bonds between backbone atoms

Why is the alpha helix “regularly” spaced?

Because the C=O of one amino acid bonds with the N-H of the amino acid exactly 4 amino acids away

What is special about an alpha helix with entirely hydrophobic side chains?

They can cross membranes if they have at least 20 amino acids

• Membranes are super hydrophobic as well

Protein domains vs subunits

• Domains: sub-structure of a protein that folds more or less independently (within a chain)

  • Typically encoded by a single exon

• Subunits: an individual protein within a protein complex

  • Subunits exist within protein complexes, and domains exist within subunits/proteins

Protein complex levels

• Dimer: two subunits

• Trimer: three subunits

• Tetramer: four subunits

Homodimer vs Heterodimer

• Homodimer: forms from two identical subunits, symmetrical

• Heterodimer: forms from different subunits

What is self-assembly?

The spontaneous organization of individual proteins into larger, functional structures driven by specific molecular interactions (like hydrogen bonds, hydrophobic effects, charge) and physical forces (like crowding) within a cell

What is exon shuffling/domain shuffling?

Eukaryotic proteins are often the result of exon shuffling, in which a sequence encoding a particular domain has been inserted into many different genes, creating proteins with a modular structure, which means that a protein has different regions that carry out specific types of function

• Why the same domain shows up in many different proteins

What is a protein module?

A common protein domain

What are protein families?

• Proteins can be classified into many families, each family member having an amino acid sequence and 3D conformation that resembles other members

• Each is encoded by genes derived from the same ancestral gene (homologous)

• Evolutionary process: arises primarily through gene duplication, where an extra gene copy accumulates mutations and diverges to form new functions

• Must be at least 30% identical

What are serine proteases?

A large, essential family of enzymes that cut (cleave) peptide bonds in proteins

What is a cell culture?

A homogeneous population of cells growing in a lab (the cells are all the same)

How is a cell culture made from a tissue?

Isolate and separate the cells 

• Disrupt ECM --> cell suspension  

  • Proteolytic enzymes – degrade ECM proteins (ex: collagen (collagenase)) 

  • EDTA – binds to calcium ions (Ca²⁺), which are required for cell-cell and cell-ECM junctions; EDTA breaks these contacts  

• Make homogenous (ie. One cell type) 

  • Centrifugation (isolated by size)

  • Grow in a defined medium (isolated by growth properties)  

  • Fluorescence-activated cell sorting (FACS) (isolated by light scattering and fluorescent characteristics)

• Expand cells in the lab 

  • Plastic petri dish (growth medium, bottom coated with ECM, helps give cells something to grow on/stick to  

    • Standard medium: growth factors include salts, glucose, amino acids, vitamins, and bovine serum 

    • Defined medium: contains all the above growth factors, but no serum, and includes a specific growth factor

• Make cell line (a cell clone, derived from one cell)

  • Can also make cells immortal and freeze in liquid nitrogen (permanent stock)

  • Done by providing cells with the gene that encodes the catalytic subunit of telomerase

How does fluorescence-activated cell sorting (FACS) work?

• Uses an antibody bound to the cell surface

• Identifies and sorts heterogeneous cell mixtures into distinct, highly pure populations based on specific light scattering and fluorescent characteristics

Standard medium vs Defined medium

• Standard medium: growth factors include salts, glucose, amino acids, vitamins, and bovine serum 

• Defined medium: contains all the above growth factors, but no serum, and includes a specific growth factor

What is a cell line? How can they be made immortal?

Cell clones derived from a single cell that are all genetically homogeneous

• Can also make cells immortal and freeze them in liquid nitrogen (permanent stock)

• Done by providing cells with the gene that encodes the catalytic subunit of telomerase

• Immortal cell lines usually derived from tumors (tumor cell lines)

What is fractionation of cells and in what ways can it be done?

• Cell lysis, disrupting the cell membrane to get cell extract (cell components)

  • Homogenization: shearing force mechanically disrupts the membrane

    • Sonication

    • Pestle in cell suspension (high-frequency sound waves)

    • Osmotic lysis (hypotonic solution)

What is fractionation of cell extract and in what ways can it be done?

• Done on cell extract (rather than cell as a whole) to iolate organelles and protein complexes

  • Ultracentrifugation (differential centrifugation)

    • Separates cell components by size and density based on the spin speed

    • Pellet is created at the bottom, and then you centrifuge the supernatant again (liquid above pellet)

  • Velocity centrifugation (sedimentation)

    • Provides a finer degree of separation (e.g., protein complexes), separating further by size and shape

    • Utilizes a solution that contains a gradient of sucrose or salt (for example, the top may be 5% while the bottom is 25%)

      • The larger and rounder components go to the bottom (no pellet)

      • Poke a hole at the bottom and let it drip through

  • Equilibrium sedimentation

    • Provides the most fine degree of separation, separating by DENSITY rather than size and shape, can separate the same protein complexes

    • Also uses a sucrose or salt gradient, but is much higher (but more like 25% at top and 75% at bottom)

    • The denser ones move to the bottom

Velocity centrifugation

• Provides a finer degree of separation (e.g., protein complexes). separating further by size and shape

• Utilizes a solution that contains a gradient of sucrose or salt (for example, the top may be 5% while the bottom is 25%)

  • The larger and rounder components go to the bottom (no pellet)

  • Poke a hole at the bottom and let it drip through

Equilibrium sedimentation

• Provides the most fine degree of separation, separating by DENSITY rather than size and shape, can separate the same protein complexes

• Also uses a sucrose or salt gradient, but it is much higher (but more like 25% at the top and 75% at the bottom)

• The denser ones move to the bottom

What is fractionation of proteins and in what ways can it be done?

Separates complex protein mixtures into simpler, distinct fractions based on physical and chemical properties like size, charge, solubility, and affinity

• Column chromatography

  • Rate of passage through the column depends on interaction with the beads

  • The surface of the beads are designed to interact with proteins in some particular ways (such as with charges), three ways they can interact:

    • Ion exchange — beads interact with the charge of a protein

    • Gel filtration — beads have pores of a certain size, which trap proteins of a certain size

    • Affinity — beads have a conjugate that binds to the protein of interest, specific affinity to a conjugate

      • Such as an antibody (Ab) against the protein of interest

      • Antibody that binds to “affinity tag”

      • DNA - gene promoter sequence your protein binds to (for transcription factor)

Ion exchange chromatography

Beads interact with the charge of a protein

Gel-filtration chromatography

Beads have pores of a certain size, which trap proteins of a certain size

Affinity chromatography

Beads have a conjugate that binds to the protein of interest, specific affinity to a conjugate

• Such as an antibody (Ab) against the protein of interest

• Antibody that binds to “affinity tag”

• DNA - gene promoter sequence your protein binds to (for transcription factor)

What is an affinity tag?

• Used in affinity chromatography when there is no antibody for the protein

• Allows for the use of an antibody that binds to the epitope tag

What is immunoblotting (western blotting) used for?

Identifies specific proteins by exposing all the proteins on the gel to a specific antibody that has been labeled with a radioactive isotope or fluorescent dye

What is two-dimensional gel electrophoresis used for?

Separates complex protein mixtures based on two independent properties: isoelectric point and molecular weight

What is mass spectrometry?

Used to identify unknown compounds, quantify known materials, and elucidate the chemical structure of molecules by measuring the mass-to-charge ratio of ionized particles

Resolving power

The ability of an optical instrument (microscope, telescope) or imaging system to distinguish and separate the images of two closely spaced objects

• Human eyes — 0.2 mm

• Light microscope — 0.2 um

• Electron microscope — 2 nm (uses electron beam instead of lightwaves)

(Don’t need to know specific numbers)

Amplitude vs Wavelength

Bright field microscopy (light microscope method)

• Because cells are transparent:

  • Fix cells: chemically treat cells, making holes in the membrane to make them more permeable to dyes and antibodies (Ab)

  • Stain cells: dyes and antibodies

Phase contrast microscopy (light microscope method)

Converts phase differences in light into brightness (amplitude) differences

• Light enters the specimen in phase, but waves travel at different rates through the sample depending on density, exits specimen in different phases

  • The microscope converts these differences in wavelengths into brightness differences

• Increases contrast

Fluorescence microscopy (light microscope method) & parts of a fluorescent microscope

A fluorescent molecule absorbs light of a specific wavelength (λ) and then emits light of a longer wavelength (λ)

• Fluorescent molecules (GFP) absorb light at 460 nm and emit light at 520 nm

Parts of the microscope:

• Barrier filters: transmit (allow through) specific wavelengths

  • Excitation wavelength — one that we excite the fluorescent molecule with

  • Emitted wavelength — one that the fluorescent molecule emits

• Beam splitting mirror (dichroic): transmits light above a specific wavelength and reflects light below

  • Outcome: bright image (1 color) against dark background

What is indirect immuno-fluorescence (immuno-staining)

• What you’re actually seeing is indirectly binding to the protein (why it's considered indirect), but allows you to alter level of fluorescence

Deconvolution microscopy (a computational method)

• Computational, post-processing technique that improves the resolution, contrast, and signal-to-noise ratio of fluorescence images by removing out-of-focus light and blurring

• Uses the standard fluorescence microscope

• Collects a z-stack of images (a collection of images taken at focal planes (depths) along the z-axis of the cell)

Confocal microscopy

• Also removes out-of-focus light, but unlike deconvolution microscopy, does this through optic techniques rather than computational ones

• Only allows light through in a very specific path

• Uses a laser (allows for a specific wavelength), e.g., using 460nm for GFP

• 2 confocal pinholes (exclude out-of-focus light)

• Dichroic mirror

• Uses z-stack method: produces “optical sections” in a z-stack

• Detector

Electron microscopy

• Uses electron beam

• Magnetic coils (focus the electrons)

• Scanning EM gathers an image of the surface of the sample

What are the functions of cell membranes?

• Define cells (plasma membrane/PM) and compartments/organelles

• Are barriers, help create ion gradients (which are needed for things like ATP synthesis, nerve impulses)

• Membrane protein functions (roughly 30% of proteins are in membranes)

What are the properties of membranes?

• Made of lipids (phospholipids, glycolipids, cholesterol), proteins, and carbs

• They are dynamic (fluid bilayer, 5mm thick)

• Impermeable to most H20 soluble molecules

• Lipids are amphipathic/amphiphilic, one side (head) is polar (hydrophilic) and one side (tail) is nonpolar (hydrophobic)

What are the types of lipids covered in this course?

• Phospholipids

  • Phosphoglycerides

    • phosphatidyl-ethanolamine

    • phosphatidyl-serine

    • phosphatidyl-choline

    • phosphatidyl-inositol

  • Sphingolipids

    • sphingomyelin

• Glycolipids

• Cholesterol

What are phosphoglycerides?

• The primary structural lipids found in eukaryotic cell membranes, consisting of a glycerol backbone esterified to two fatty acids, a phosphate group, and an alcohol

• Form the lipid bilayers

• The double bonds make the tails kinked (unsaturated fatty acid)

What are glycolipids?

• Have carbohydrate (sugar) on the polar head (added in golgi lumen)

  • These carbohydrates are only present on the heads in the outer leaflet of the plasma membrane (not the cytosolic leaflet)

  • Use different sugars, and different sugars attached to each other in chains

• Tend to be in “lipid rafts”

• Function in cell-cell junctions

What is cholesterol?

• Rigid ring structure

Is bilayer formation spontaneous? Why?

• Spontaneous in solution due to two properties of the lipids

  • Shape of the lipid

  • Amphiphilic

What is a liposome?

Spherical vesicles made from synthetic lipid bilayers in a lab, used to study membrane fluidity

Characteristics of bilayer fluidity (how can lipids move within the bilayer?)

• Through experiments with liposomes, found that there is:

  • Rapid lateral diffusion of lipids (lipids switch places with neighboring lipids in the same leaflet)

  • Rotation of lipids (flexion) is common

  • Flip-flop (moving from one leaflet to another) is very rare, energetically unfavorable

In what cases would there be increased membrane fluidity? And how?

Increased fluidity (useful in colder climates/cold adaptation): increased fluidity increases temperature to counteract a cold environment

• Lipids with shorter tails (can move around more easily)

• Lipids with kinked tails/unsaturated fatty acid chain

• More cholesterol

What is lateral asymmetry?

• Lateral asymmetry: non-uniform distribution of lipids and proteins within the plane of a single leaflet

  • Lipid raft: a patch of membrane with a specific function (such as for cell signaling)

    • They are immobile — attached to ECM proteins or the cytoskeleton

    • Components:

      • Lipids have longer tails → thicker membrane

      • Transmembrane proteins have long hydrophobic alpha-helix

      • More cholesterol, more glycolipids, and more glycoproteins

What is a lipid raft?

• A patch of membrane with a specific function (such as for cell signaling)

  • They are immobile — attached to ECM proteins or the cytoskeleton, contributes to membrane asymmetry

  • Components:

    • Lipids have longer tails → thicker membrane

    • Transmembrane proteins have long hydrophobic alpha-helix

    • More cholesterol, more glycolipids, and more glycoproteins