Cell Biology Exam 1

Chapters 4 and 11

Amino Acid Sequence

The order of amino acids; it is unique for each protein

Primary Structure

The linear sequence of amino acids

Determination of Amino Acid Behavior

The R-group (a.k.a., side chain) of an amino acid determines both its identity and its chemical behavior

Condensation Reaction

The addition of a subunit (i.e., another amino acid) onto one end of a polymer chain, resulting in the loss of a molecule of water per subunit added

Polypeptide Backbone

The repetitive sequence N-C-C of multiple amino acids chained together

N- to C- Synthesis

The directionality of protein synthesis

Protein Folding

Hydrophobic forces (e.g., the hydrophobic side chains of many amino acids) help proteins fold into compact conformations

Urea Denaturing Experiment

Adding urea disrupts noncovalent bonds and denatures amino acids until the urea is removed; thus, we learned that proteins fold into the conformation of lowest energy

Chaperone Proteins

Assist the folding of a polypeptide chain; some can create makeshift “chambers,” isolating proteins until they correctly fold and are released

Secondary Structure

Interactions between atoms of the backbone (i.e., no side chain involvement); includes α helices, β sheets, and turns

Tertiary Structure

The full three-dimensional conformation of a protein primarily due to interactions between its R-groups

Quarternary Structure

Association of multiple polypeptide chains (i.e., it consists of more than one amino acid chain)

Number of Amino Acids per Complete Turn

3.6 amino acids per complete turn

α Helices in Lipid Bilayers

Receptors, transmembrane proteins, and transport proteins can be embedded in the cell membrane and consist of α helices; about 20 amino acids are required to span the membrane, and the R-groups that stick out of the helices are NONPOLAR

Domain

Separate regions in a tertiary structure, usually 40-350 amino acids; many proteins are composed of separate functional domains

Family

Proteins that have similar amino acid sequences and thus similar shapes

Subunit

A whole polypeptide chain; large proteins often contain more than one

Binding Site

Regions involved in the interaction between different subunits

Sickle Cell Anemia Example

A point mutation swapping Glu for Val leads to hemoglobin molecules that stick together and deform the cells; the nonpolar Val sticks to itself due to the hydrophobic effect

Protein Conformation Models

1. Backbone Model (simplest)

2. Ribbon Model

3. Wire Model (similar to Backbone Model, but the “wires” almost seem like broken benzenes in angle)

4. Space-Filling Model

Cross-Link Bonds

Bonds that hold together single filamentous molecules

Disulfide Bonds

These only form OUTSIDE of the cell; cytoplasm in the cell is a reducing environment, so the equilibrium of the disulfide reaction is shifted towards reduction in the cytoplasm

Protein Subunit Assemblage Types

Identical protein subunits can assemble into complex structures; dimers, helices, and rings can form depending on the shape/orientation of the binding site, and filaments, spherical shells, and hollow tubes can form from the exact same subunit

Protein-to-Molecule Binding

HIGHLY selective as the shape is important; ligands and proteins must fit perfectly, and they’re held by many weak, noncovalent bonds

Methods of Catalysis Encouragement via Enzymes

1. Enzymes can bind two substrates in a precise orientation to encourage a reaction between them

2. Enzymes can bind a substrate and rearrange its electrons in order to create reaction-favorable partial negative and positive charges

3. Enzymes can strain/reshape a bound substrate to force it into a more favorable transition state

Hydrolase

Enzymes that catalyze a hydrolytic cleavage reaction

Nuclease

Enzymes that break down nucleic acids by jydrolyzing bonds between the nucleotides

Protease

Enzymes that break down proteins by hydrolyzing peptide bonds between amino acids

Ligase

Enzymes that join two molecules together (e.g., DNA ligase joins two DNA strands together end to end)

Isomerase

Enzymes that catalyze the rearrangement of bonds within a single molecule

Polymerase

Enzymes that catalyze polymerization reactions such as synthesis of DNA and RNA

Kinase

Enzymes that catalyze the addition of phosphate groups to molecules (e.g., protein kinases, which bind phosphate groups to proteins)

Phosphatase

Enzymes that catalyze the hydrolytic removal of a phosphate group from a molecule

Oxido-Reductase

General name for enzymes that catalyze reactions wherein one molecule is oxidized while another is reduced (e.g., oxidases, reductases, or dehydrogenases)

ATPase

Enzymes that hydrolyze ATP

Measures of Enzyme Performance

Enzyme performance depends on how rapidly it can process its substrate; measured via V, Vmax, and Km

V

The rate of enzyme reaction

Vmax

The maximum rate a given enzyme can work at; here, all substrate binding sites are occupied

Km

The amount of substrate wherein the enzyme works at half speed; enzymes with a low Km bind tightly, and enzymes with a high Km have weak binding

Feedback Inhibition

A method by which protein function can be regulated by downstream products

Inhibitors

Reduce the activity of an enzyme; negative regulation

Activators

Increase the activity of an enzyme; positive regulation

Allosteric Regulation

When a regulatory molecule binds to an enzyme someplace other than the active site; can serve to inhibit OR to activate

*Allosteric regulation always involves conformational change

Phosphorylation

Controls protein activity by causing conformational change; one of the most important protein control mechanisms in nature as 1/3 of proteins are regulated by this process

*Can turn “on” or “off” a process

Phosphorylated Amino Acids

1. Serine

2. Threonine

3. Tyrosine

GTP-Binding Proteins

Proteins that are switched on/off by binding and hydrolyzing a GTP molecule; molecule is “on” upon binding GTP to the G protein, and it only turns off when GTP is hydrolyzed into GDP

ATP Hydrolysis

Allows motor proteins to produce directed movement in cells

Post-Translational (Enzyme) Modification

Greater than 100 types (e.g., acetylation, glycosylation, ubiquitination, phosphorylation, etc.); can activate/deactivate, define protein location or fate, and can modify multiple sites on certain proteins

Homogenization

Gentle mechanical procedures that rupture the plasma membranes of cells so that their contents are released

Homogenate / Extract

The “thick soup” resulting from homogenization

Homogenization Methods

1. Breaking apart cells with high-frequency sounds (ultrasound)

2. Using mild detergent to make holes in plasma membrane

3. Forcing cells through small holes using high pressure

4. Shearing cells between close-fitting rotating plunger and thick walls of a glass vessel

Centrifugation

The most widely used procedure to separate a homogenate into different parts, or fractions

*Modern centrifuges have speeds up to 100k RPM and produce forces as high as gravity x 600k

Supernatant

The smaller, less dense components of a homogenate post-centrifugation

Differential Centrifugation

Allows you to isolate different cellular components based on size and density; each step is the result of increasing speeds of centrifugation

Differential Centrifugation Results by Pellet

1. Pellet 1 - Whole cells, nuclei, cytoskeletons

2. Pellet 2 - Mitochondria, lysosomes, peroxisomes

3. Pellet 3 - Closed fragments of endoplasmic reticulum, other small vesicles

4. Pellet 4 - Ribosomes, viruses, large macromolecules

Velocity Sedimentation

Uses a sucrose gradient to top separate cellular components based on size and their sedimentation rates; as the sediments in the tube post-centrifugation separate, the tube is punctured in the bottom and allowed to drain into different tubes that move automatically below it

Equilibrium Sedimentation

Allows you to isolate different cellular components based on their buoyant density Equi; once the bands have separated based on density, they are gathered using the same draining method as velocity sedimentation

Column Chromatography

A method of fractionating proteins where a micture of proteins in solutions is applied to the top of a cylindrical column filled with a permeable solid matrix immersed in solvent; choice of matrix allows proteins to be separated according to their charge, hydrophobicity, size, or binding ability to certain groups

Ion-Exchange Chromatography

Ion-exchange columns are packed with small beads carrying either positive or negative charges; association between protein and matrix depends on pH and ionic strength of solution

Gel-Filtration Chromatography

Gel-filtration columns separate proteins according to their size; protein molecules small enough to enter the columns tiny porous beads are filtered out more slowly than larger protein molecules that cannot enter the beads

Affinity Chromatography

Affinity columns contan a matrix covalently coupled to a molecule that interacts specifically with the protein of interest; proteins can subsequently be released via pH change or concentrated salt solution, and they emerge highly purified

Electrophoresis

When an electric field is applied to a solution containing protein molecules, the proteins migrate at a speed reflective of their size and net charge

SDS Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

Individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and thus migrate as negatively charged SDS-protein complexes through a slab of porous polyacrylamide gel; reducing agents like mercaptoethanol prevent disulfide linkages to allow movement

Isoelectric Focusing

Proteins are electrophoresed in a narrow tube of polyacrylamide gel in which a pH gradient is established by a mixture of special buffers; proteins move to a location corresponding to their isoelectric point and stay there

Two-Dimensional Gel Electrophoresis

Combines Gel Electrophoresis and isoelectric focusing in order to resolve more than 1000 proteins in a two-dimensional protein map

Antibody

Protein that bind very tightly to their target antigens; produced in vertebrates as a defense against infection

*Humans produce billions of types, each with different binding sites

Parts of Antibodies

1. 4 Polypeptide chains with hypervariable antigen-binding sites at their ends:

   1. 2 heavy chains (dark green in image)

   2. 2 light chains (light green in image)

2. Variable domains

   1. Constant domains (gray in the image)

   2. Variable light (Vl) and Variable heavy (Vh) domains (blue in the image)

*Altering the length or amino acid sequence of the variable domains provide different antibodies

B Lymphocyte (B Cells)

Class of white blood cells responsible for antibodies; upon a B Cell’s antibody binding, it is stimulated to divide and to secrete large amounts of the same antibody in soluble form

Aggregates

Collections of antigens (can be foreign molecules, viruses, and/or bacteria) that are cross-linked by antibodies and ingested by phagocytic cells or killed by special proteins in the blood

Monoclonal Antibodies

Made by harvesting antibody-bearing B cells from an immunized animal the fusing them with a tumor cell to make a hybridoma

Increasing Antigen Detection Sensitivity

Can increase sensitivity greatly by using multiple layers of antibodies; this “sandwich” method allows smaller numbers of antigen molecules to be detected

Western Blot

An antigen of interest is separated from other molecules via electrophoresis before being incubated with labeled antibodies that bind to the antigen; allows the position of the antigen to be determined

*Separation → Transfer → Staining → Visualization

Methods of Determining Proteins’ 3-D Shape

1. X-Ray Crystallography

2. NMR Spectroscopy

3. Cryo-Electron Microscopy

Labeled Cell (just look at it)

Amphipathic

Molecules with body hydrophilic AND hydrophobic regions

Phospholipid Components

1. Phosphate-containing hydrophilic head

2. Two hydrophobic hydrocarbon tails, one of which is saturated (no double bonds) and one of which is unsaturated (1+ double bonds)

3. A kink in one of the tails which makes it harder for them to pack together and keeps membranes more fluid

Membrane Lipids

1. Phosphatidylcholine (more common)

2. Phosphatidylserine (a phospholipid)

3. Cholesterol (a sterol)

4. Galactocerebroside (a glycolipid)

Membrane Lipid Spheres

Energetically favorable conformation when in water; requires no energy to do so, keeps hydrophobic pieces away from water, and allows the membrane to self-heal

Lipid Bilayer Factors

1. Length of tail → shorter tails are more fluid

2. Number of double bonds → more double bonds are more fluid

3. Amount of cholesterol → cholesterol reduces fluidity

4. Temperature → higher temperatures increase fluidity

Scramblase

Catalyzes the transfer of phospholipids from one cytosolic layer to another in a RANDOM fashion; occurs in the ER

Flippase

Catalyzes the transfer of phospholipids from one cytosolic layer to another in a specific fashion; occurs in the Golgi Apparatus

*Can flip phospholipids, but NOT glycolipids

Golgi Apparatus

Where the asymmetric destribution of the membrane bilayer occurs; lipids are also modified here (e.g., glycosylation)

Membrane Composition in Humans

Approximately 50% protein; however there are ~50x as many phospholipids than proteins (phospholipids are much smaller)

Main Function of Membrane Proteins

Transport: getting molecules (e.g., ions, sugars, metabolites, etc.) across the membrane when they would ordinarily not be able to

*Can also serve as anchor proteins

Membrane Protein Examples

1. Transporters (e.g., Na+ Pump)

2. Ion Channels (e.g., K+ Leak Channel)

3. Anchors (e.g., Integrins)

4. Receptors (e.g., Platelet-Derived Growth Factor (PDGF) Receptor)

5. Enzymes (e.g., Adenylyl Cyclase)

Integral Membrane Proteins

1. Transmembrane

2. Monolayer-Associated

3. Lipid-Linked

Peripheral Membrane Protein

Protein-Attached

Most Common Transmembrane Protein Form

α Helices containing at least 20 amino acid residues

Hydropathy Plot

Plot that predicts trans-membrane domains; peaks are positive values that indicate hydrophobic segments of the protein

Channel Formation

Multipass transmembrane proteins can form channels

Detergent Use with Membrane Associate Proteins

Solubilizing these proteins in detergent allows their study; the proteins are brought into the solution as protein-detergent complexes

Synthetic Phospholipid Bilayer

Can be used to functionally reconstitute membrane proteins

Cell Cortex

The meshwork of proteins that lies just underneath and connects to the cell membrane; reinforces the plasma membrane

Fluid Mosaic Model

Combined mouse and human cells; initially cell membranes remained unmixed, but they steadily mixed together after time and incubation

*Membrane determined to be a fluid body!

Membrane Domain

Functionally specialized regions of a given cell’s membrane that have restricted movement

Methods of Restricting Lateral Mobility of Plasma Membrane

1. Tether to cell cortex inside the cell

2. Tether to extracellular matrix molecules outside the cell

3. Tether to proteins on the surface of another cell

4. Use diffusion barries that restric proteins to a particular membrane domain

Glycolipids

Many of the lipids on the exterior face of the lipid bilayer; these have sugars attached to their heads

Glycoproteins

Many of the membrane proteins; contain one or more short chains of sugars linked together called Oligosaccharides

Proteoglycans

Contain one or more long chains of sugars linked together called polysaccharides

Carbohydrate Layer (Glycocalyx)

The sugar layer on the outermost exterior of the cell formed by glycolipids, glycoproteins, and proteoglycans; protects the cell from physical damage and attracts water molecules