Chapter 3 Nonenzymatic Protein Function and Protein Analysis

What are structural proteins?

-proteins with generally high repetitive secondary structure and a super-secondary structure.

-They are fibrous in nature

-They make up the cytoskeleton, anchoring proteins, and much of the extracellular matrix

What is a super-secondary structure?

a specific arrangement of multiple secondary structural elements within a protein, creating a recognizable local 3D structure

What are the primary structural proteins in the body?

Collagen, Elastin, Keratins, Actin, and Tubulin

Describe the structure of collagen and its uses.

-Trihelical fiber

-Makes up most of the extracellular matrix of connective tissue

-Provides strength and flexibility throughout the body

Describe the structure of Elastin and its uses.

-fibrous protein

-found in the extracellular matrix of connective tissue

-Primary role is stretching and recoil restoring tissues to it’s original shape

Describe the structure of Keratin and its uses.

-Intermediate filament proteins in epithelial cells

-contribute to the mechanical integrity of the cell

-function as regulatory proteins

-Makes up the hair and nails

Describe the structure of Actin and its uses.

-Thin fibrous protein

-makes up microfilaments and the thin filaments in myofibrils

-it has polarity (positive and negative side) allowing motor proteins to travel unidirectionally

Describe the structure of Tubulin and its uses.

-fibrous protein

-Makes up microtubules for structure

-Used in chromosome separation in mitosis and meiosis

-Intracellular transport with kinesin and dynein

-Tubulin has polarity: the negative end of a microtubule is usually located adjacent to the nucleus, whereas the positive end is usually in the periphery of a cell

What is the most abundant protein in a eukaryotic cell?

-Actin

What are motor proteins?

-Proteins that can turn chemical energy into mechanical energy and allow for movement throughout the cell. The interact with either actin or microtubules.

TRUE OR FALSE: Motor proteins are not enzymes

False: motor proteins can display enzymatic activity acting as ATPases that power the changes necessary for motor function.

What are examples of structural proteins that can have motor function?

-Cilia and flagella in bacteria and sperm

What are examples of motor proteins?

-Myosin

-Kinesins and Dyneins

What are myosins?

-primary motor protein that interacts with actin.

-Thick filament in myofibril

-can be involved in cellular transport

Describe the structure of myosin.

-Has a single head and neck, movement at the neck is responsible for the power stroke of sarcomere contraction

What are Kinesins and Dyneins and what is the difference?

They are motor proteins associated with microtubules. They have two heads, one of each stays attached to the tubulin at all times.

-Kinesins help with the alignment of chromosomes during metaphase and depolymerizing microtubules in anaphase of mitosis.

-Dyneins are involved with the sliding and moving of flagella and cilia.

-Both aid with vesicle transport. Kinesins bring neurotransmitter vesicles to the positive end (axon), Dyneins bring vesicles of waste or recycled neurotransmitter to the negative end.(soma)

What are binding proteins?

-Proteins that transport or sequester molecules by binding to them.

What are some examples of binding proteins?

-hemoglobin

-calcium-binding proteins

-DNA-binding proteins( often transcription factors)

Which kind of proteins usually have an affinity curve?

-Binding proteins; the affinity curve differs based on the goal of the binding protein.

-When sequestering is the goal they have high affinities

-When transport is the goal, they have high affinities when binding is necessary and lower affinities when unbinding the target. The environment changes the affinity.

What are CAMs?

-Cell adhesion molecules, they are molecules that are found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells

What are the three major families of CAMs

Cadherins, Integrins, and Selectins

What are Cadherins?

-Calcium dependent adhesion glycoproteins. They usually holds similar cells types together.

Different cells use different types of Cadherins Epithelial uses E-Cadherin Nerve cells use N-cadherin

What are integrins?

-A group of proteins that have two membrane spanning chains called α and β

-They play a role in cellular signaling and impact cellular function by promoting cell division, apoptosis, white cell migration, and stabilization of epithelium on its basement membrane.

What is an example of a specific function of integrins?

Integrin α_IIbβ₃ allows platelets to stick to fibrinogen a clotting factor that causes activation of platelets to stablize a clot.

What are selectins?

-proteins that bind to carbohydrate molecules that project from other cell surfaces. They are expressed on white blood cells and endothelial cells that line blood vessels

-They have an important role in host defense including inflammation and white blood cell migration

Which CAM has the weakest formed bonds?

Selectins

What is another name for immunoglobulin(Ig)?

-Antibody

What is the role of an antibody?

Antibodies tag pathogens and bacteria for neutralization and recruit other cells to help eliminate the threat.

What immune cell produces antibodies?

-B-cells

What are the subunits of antibodies?

-Antibodies have two identical heavy chains and two identical light chains.

What holds the heavy and light chains together?

-Disulfide bonds and noncovalent interactions

Describe the structure of an antibody.

-A “Y” shaped protein with an antigen binding region at the tips and have a specific polypeptide regions that only bind one and only one specific antigen

-The antibody have a constant region at the bottom of the antibody that is involved in the recruitment and binding of other cells of the immune system such as macrophages.

What do we call the targets of antibodies?

-Antigens

What are the three outcomes when an antibody tags an antigen?

-Neutralizing the antigen: making it unable to exert its effects on the body

-Opsonization: marking the pathogen for destruction by other white blood cells immediately

-Agglutinating: Clumping together the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macorpahges

What could permit a binding agent involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound?

-If there is a high amount of substrate around

What is biosignaling?

-A process in which cells receive and act on signals

What role do proteins play in biosignaling?

-Acting as extracellular ligands

-Transporters for facilitated diffusion

-Receptor proteins

-Second messengers

What are ion channels?

-proteins that create specific pathways for charged molecules

What are the three main groups of ion channels?

-Ungated: have no gates, always open

-Voltage-gated: regulated by membrane potential change near the channel, opens when “excited”

-Ligand-gated: open when a specific ligand is bound the channel causing it to open or close

What is facillitated difussion?

-a type of passive transport. Molecules diffuse down a concentration gradient through a pore in the membrane created by a transmembrane protein.

Why is facilitated diffusion important?

-It allows for molecules to pass into the cell through these protein channels and avoid the hydrophobic fatty acid tails in the lipid bilayer

What is an example of an ungated channel?

-Potassium channels,

-potassium passes through these channels freely, in all cells so there is an efflux of potassium unless it is at equilibrium

What is an example of voltage gated channels?

-The voltage gated sodium channels in neurons.

-They are closed under resting conditions, but membrane depolarization causes conformational change that allows them to quickly open, allow sodium in, then close quickly.

-Also expectable answer: sodium-potassium channels in the SA node; they respond to changes in voltage

What is an example of a ligand gated channel?

-the GABA neurotransmitter binds to a chloride channel and opens it

What are enzyme linked receptors?

-proteins on the surface of a cell that are activated by binding a ligand.

What are the three primary domains of enzyme linked receptor?

-a membrane spanning domain: anchors the receptor in the cell membrane.

-Ligand binding domain: binds to a ligand that induces conformational changes that activates a catalytic domain.

-Catalytic domain: the part of the enzyme that catalyzes a reaction

Explain a second messenger cascade.

-Receptor tyrosine kinases (RTKs) are composed of monomer units that dimerize upon ligand binding. The dimer is now an active form and the phosphorylate other cellular enzymes including the receptor itself. (autophosphorylation)

Explain the process that a G-protein coupled receptor (GCPR) undergoes.

GCPRs are a involved in signal transduction.

In its inactive form the α subunit is bound to GDP, and is in a complex with β and γ

Step 1: A ligand binds to the GCPR and the recptor becomes activated causing a conformational change that swaps GDP for GTP

Step 2: The α subunit bound to GTP dissociates from the β and γ subunits and goes on to alter the activity of adenylate cyclase.

(α_s-activates α_i- inhibits)

Step 3: The GTP on the α subunit gets dephosphorylated to GDP and then with go back to the β and γ subunits going back to the inactive state.

What are the subunits of the G-protein?

α,β,γ

What are the different types of G-proteins and what do they do?

-G_s: stimulates adenylate cyclase which increases levels of cAMP in the cell

_-G_i : Inhibits adenylate cyclase which decreases levels of cAMP in the cell

-G_q: activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP₂ (PIP₂ gets cleaved into DAG and IP₃, and IP₃ can open calcium channels in the ER increasing calcium in the cell.)

What do enzyme linked receptors and G proteins coupled receptors have in common? What are their differences?

They both bind ligands for activation, they both have a transmembrane portion and an extracellular domain.

-G protein is a trimer, and is a two protein complex.

-Enzyme linked has autoactivity and displays enzymatic activity.

What are some techniques for isolating proteins?

-Electrophoresis and Chroatography

Define homogenization.

-crunching, grinding, or blending of the tissue into an evenly mixed solution.

Define Centrifugation.

-after cell lysis the cell components are placed in a centrifuge tube, then in a centrifuge which spins the tube at high speeds that separate the different components of a cell from each other.

What is electrophoresis?

A technique by which compounds are subjected to and electric field that moves them according to their net charge and size.

What charge would you expect from a compound that moves toward the anode during electrophoresis?

-Negative charge, the anode is positively charged.

What charge would you expect from a compound that moves toward the cathode during electrophoresis?

-A positive charge; the cathode is negatively charged.

What is migration velocity?

-The velocity at which the compound travels in electrophoresis.

What is the formula for migration velocity? What do the letters stand for?

v=Ez/f

-V: migration velocity

-E: electric field strength

-z: net charge of molecule

-f: coefficient representing the mass and shape of migrating molecules.

Is ‘E’ directly or inversely proportional to ‘v’ in the migration velocity formula?

-Directly proportional

Is ‘v’ directly or inversely proportional to ‘f’ in the migration velocity fomrula?

-inversely

What is the standard medium for electrophoresis and why?

-Polyacrylamide gel. This get is a slightly porous matrix mixture and allows smaller, or more charged particles to move faster and larger or neutral particles to move slower.

If there is a smaller electric field would you expect charged particles to move quickly or slowly in an electrophoresis assay?

Charged particles will move quicker than no charged particles but they will move slower than they would if the electric field were larger.

What is PAGE useful for?

-analyzing proteins in their native states. It allows for the comparison of proteins based on size and charge, they can further be analyzed by other techniques

Why is SDS PAGE so useful?

-This is a PAGE assay where SDS is added. The SDS binds to proteins denaturing it, and also making it very negatively charged. This means when exposed to an electric field the proteins will migrate solely based on their mass and the strength of the electric field. The bands can be stained for visualization.

What does SDS stand for?

Sodium-dodecyl sulfate

Explain how Isoelectric focusing works?

-Isoelectric focusing exploits the acidic and basic properties of amino acids by separating on the basis of isoelectric point (pI)

-Proteins are placed in a gel mixture with acidic gel at the positive anode and basic gel at the negative cathode and neutral charge in the middle.

-When exposed to an electric field the proteins will move based on their charges (positive to the cathode and negative to the anode). Once the hit a pH that is equal to their pI they will stop moving.

What is a zwitterion?

an amino acid that is electrically neutral.

What is chromatography?

-Chromatography refers to a variety of techniques that require the homogenized protein mixture to be fractionated through a porous mixture.

Why would you want to use chromatography over electrophoresis?

-if you are separating a large amount of protein

In chromatography the sample must be placed on a solid stationary phase called what?

-Stationary phase or adsorbent.

The mobile phase in chromotography does what?

Helps run the mobile phase through the stationary phase, this process is called elute.

What would you expect to happen to components that have a high affinity for the stationary phase?

-They will barely migrate or will migrate slowly.

What is the term that refers to the amount of time spent in the stationary phase.

-retention phase

What is partitioning in chromatography?

-the separation of the components based on the retention time

What are the different types of column chromatography?

-Ion-exchange: separates proteins out by charge

-Size-Exclusion: separates proteins out by size

-Affinity: separates proteins out by affinities

What is column chromatography?

-Chromatography that uses a column filled with either silica or alumina beads as an absorbent, and gravity moves the solvent and compounds down the column.

-The beads are usually polar and have small pores that slow down the smaller proteins. The large ones travel quickly through the column because they don’t get trapped in the pores.

Can column chromatography be used be used for more than just proteins?

-Yes, other macromolecules such as nucleic acids can undergo column chromatography

What are the fractions for in column chromatography?

The solvent drips into columns and the columns spin at certain intervals so that different sized compounds will be separated.

In ion exchange chromatography the beads are coated with charged substances, why is that?

-So that compounds with the opposite charge will bind to them.

How do you remove the proteins that have been stuck to the beads once the solute has passed through?

A salt gradient is used to elute the charged molecules that have stuck to the column

Who does affinity chromatography work?

-the beads in the column are coated with receptors or antibodies that can specifically bind to the protein. Once the protein is retained we use another antibody to be eluted to wash the column and tag the bead bound receptor and frees the protein from the column.

What are techniques that can be used to determine protein structure?

• X-ray crystallography

• Nuclear magnetic resonance (NMR)

Explain how x-ray crystallography?

-Proteins must be isolated and crystallized. The proteins are the ‘sliced’ then and electron density is measured.

What is the most reliable and common method for determining protein structure?

-x-ray crystallography, 75 percent of the protein structures known today were certified through this method.

How do we determinee amino acid composition?

-Hydrolysis of proteins and then subsequent chromatographic analysis.

Small proteins are best analyzed by what?

Edman degradation; it cleaves sequence of proteins up to 50-70 amino acids. It removes the N-terminal amino acid of the protein which can be analyzed via mass spectroscopy

For larger proteins Edman degradation cannot be used, what can you do?

-Digestion with chymotrypsin, trypsin, and cyanogen bromide can be done, they selectively cleaves proteins at specific amino acid residues creating smaller fragments, that then can be analyzed using Edman degradation.

What is a downfall of using trypsin and chymotrypsin in amino acid composition?

-they cleave at disulfide bonds and salt bridges this means you cannot identify where they were in the sequences

How can we identify protein activity?

-Monitoring protein activity when it reacts with a known substrate and compares it to a standard

-What is UV spectroscopy?

-measures the absorption of ultraviolet and visible light by samples.

What are some calorimetric assays that can be done to determine protein activity?

-BCA assay, Lowry reagent, Bradford assay.

What is the Bradford assay?

a colorimetric method used to measure the concentration of protein in a sample by observing the color change of a dye, Coomassie Brilliant Blue G-250, when it binds to proteins in a solution, with the color change directly proportional to the protein concentration