Chapter 3 Flashcards
What are the primary structural proteins in the body?
- Collagen
- Elastin
- Keratin
- Actin
- Tubulin
MNEMONIC: Cold Elephants Kill Angry Tigers
What is the role of structural proteins?
Structural proteins help the body maintain structure by making up the cytoskeleton of cells, anchoring proteins, the extracellular matrix, and connective tissue like tendons and ligaments.
What is Collagen?
Collagen is a characteristic, trihelical fiber. This protein makes up most of the extracellular matrix of the connective tissue. It is found throughout the body and is important in providing both strength and flexibility.
What is Elastin?
Elastin is another important component of the extracellular matrix of connective tissue. It’s primary role is to stretch and then recoil like a spring, which helps to restore the original shape of tissue after it has been deformed.
What is Keratin?
Keratin is an intermediate filament protein found in epithelial cells. Keratin contributes to the mechanical integrity of the cell and can also function as a regulatory protein. Keratin is the primary protein in hair and nails!
What is Actin?
Actin is a protein that makes up microfilaments and thin filaments in myofibrils. It is the most abundant protein in eukaryotic cells. Actin proteins have a positive side and a negative side, which allows motor proteins to travel unidirectionally along an actin filament, like a one way street. involved in cell migration
What does Tubulin make, and what is this structures 3 functions?
Tubulin is the protein that makes up microtubules. MIcrotubules are important for providing structure, chromosome separation in mitosis and meiosis, and intracellular transport with kinesin and dynein. Tubulin, like actin, has polarity: The negative end is usally closer to the nucleus and the positive end is usually in the periphery of a cell.
What is the role of motor proteins?
Motor proteins are proteins responsible for muscle contraction, cellular movement and movement of vessicles. The motor proteins act as ATPases, which means they use ATP to power the conformational changes associated necessary for motor function. Motor proteins interact with microtubules and actin filaments to exert their effects.
What is Myosin?
Myosin is the primary motor protein that interacts with actin. Myosin also acts as the thick filament in a myofibril. Canbe involved in cellular transport.
What is Kinesin?
Kinesin is one of the two motor proteins that associates with microtubules. Kinesin plays a key role in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase. Kinesin also transports cargo vesicles towards the positive end of the microtubule.
What is Dynein?
Dynein is one of the two motor proteins that interacts with microtubules. Dyneins are involved in the sliding movement of cilia and flagella. Dynein also transports cargo vesicles towards the negative end of the microtubule.
What are binding proteins?
Binding proteins are proteins with special affinity for a specific molecule of interest to provide stabilization. They are usually used to wrap up or transport things. For example, hemoglobin is a binding protein for oxygen. There are also calcium binding proteins, DNA binding proteins, and others. Sequestration=high affinity, transport=varying affinity (needs to bind and let go). calcium and magnesium commonly bound
What are cell adhesion molecules and what are the three types?
Cell adhesion molecules (CAMs) are proteins found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells. All CAMs are integral membrane proteins.
- Cadhedrins
- Integrins
- Selectins
What are cadhedrins?
Cadhedrins are a group of glycoproteins that mediate calcium dependent cell adhesion. Cadhedrins usually hold similar cell types together. For example, E-cadhedrin holds epithelial cells while N-cadhedrin holds nerve cells together.
What are Integrins?
Integrins are a group of proteins that have two membrane spanning chains referred to as alpha and beta. These chains are important in binding to and communicating with the extracellular matrix. Also, integrins play an important role in cellular signaling, and thus can greatly impact cellular function affecting cell division or apoptosis. Finally, integrins help white blood cells migrate and attach to cells.
What are Selectins?
Selectins are unique in that they bind to carbohydrate molecules that project from other cell surfaces. Selectin bonds are the weakest bonds found in Cell Adhesion Molecules. Selectins are expressed on white blood cells and endothelial cells. They play an important role in inflammation and white blood cell migration, like integrins.
What is an antibody?
An antibody, aka immunoglobulin, is a protein produced by B-cells that functions to neutralize immune system targets and then recruit other cells to help eliminate the threat.
What are the three possible outcomes when an antibody binds to an antigen?
- The antigen is neutralized, making the pathogen or toxin unable to exert its effect on the body.
- Marking the pathogen for destruction by other white blood cells, this marking function is also called opsonization.
- Clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can then be phagoctyized and digested by macrophages.
What is Biosignaling and what are three main types of proteins responsible for it?
Biosignaling is a process in which cells receive and act on signals.
- Ion Channels
- Enzyme Linked Receptors
- G-Protein Coupled Receptors
What are ion channels and what are the three types?
Ion channels are proteins that create specific pathways for charged molecules. Ion channels can have different mechanisms for opening and closing, but they all permit facilitated diffusion of charged particles.
- Ungated Channels
- Voltage Gated Channels
- Ligand Gated Channels
What is facilitated diffusion?
Facilitated diffusion is a type of passive transport in which molecules go down a concentration gradient through a pore in the membrane created by a protein. Facilitated diffusion is necessary for molecules that are impermeable to the membrane (large, polar, or charged).
What is an ungated channel?
Ungated channels have no gates, and are therefore completely unregulated. Essentially, these are just holes in the membrane through which certain molecules can pass normally. responsible for maintaining resting membrane potential
What is a voltage gated channel?
A voltage gated channel is a gate that is regulated by the membrane potential changes near the channel. (ex. neurons have voltage gated sodium channels)
What is a Ligand Gated Channel?
A ligand gated channel is a channel that is regulated by the binding of specific ligands.
What are the 5 major classes of non-enzymatic proteins?
- Structural Proteins
- Motor Proteins
- Binding Proteins
- Immunoglobins
- Biosignaling Proteins
MNEMONIC: Some Motorcycles Bind to Immune Bikers.
What is an enzyme linked receptor?
An enzyme linked receptor is a membrane receptor that displays catalytic activity in response to ligand binding. These enzyme linked membrane receptors have 3 primary domains: the membrane spanning domain, the ligand binding domain, and the catalytic domain. Enzyme linked receptors often lead to second messenger cascades.
Below is an Receptor Tyrosine Kinase (RTK) a common enzyme linked receptor. Monomer Dimerizes upon ligand binding. The dimer is the active form that phosphorylates additional cellular enzymes, including the receptor itself (autophosphorylation)
What is a G-protein coupled receptor (GPCR)?
GPCR’s are a large family of integral membrane proteins involved in signal transduction. They are characterized by their seven membrane spanning alpha helices.
How do GProtein Coupled Receptors exert their effects?
G-Protein Coupled Receptors are, as the name suggests, coupled to a heterotrimeric G protein. G proteins are named for their usage of GTP instead of ATP to power its actions.
When the appropriate ligand binds, the G protein’s affinity for the receptor increases. When the G protein binds to the receptor, it changes to the active state and can now activate nearby enzymes. Depending on the type of G protein, secondary messenger pathways will be activated or inhibited.
What are the three types of G proteins and what do they do?
- Gs stimulates adenylate cyclase, which increases levels of cAMP in the cell.
- Gi inhibits adenylate cyclase, which decreases levels of cAMP in the cell.
- Gq activates phospholipase C, which cleaves a phospholip from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3. IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell
What are the three subunits of a G protein and what do they do?
- Alpha Sub Unit: Carries GDP/GTP to and from the G protein and the nearby enzymes to catalyze.
- Beta Sub Unit: Structural component of G protein
- Gamma Sub Unit: Structural component of G protein.
What is homogenization?
Homogenization is when a tissue of interest is crushed, ground, or blended into an evenly mixed solution. This is an important preliminary step for isolating and analyzing proteins in tissues.
What is centrifugation?
Centrifugation is the process of applying centrifugal force to a solution by spinning it. Centrifugation causes solid particles in the solution to collect at the bottom of the solution.
What are the two most common isolation techniques for proteins?
- Electrophoresis
- Chromatography
What is electrophoresis and what are the three main types?
Electrophoresis is the process of generating an electric field that separates proteins based on their charge and their size. Electrophoresis cells operate as electrolytic cells, meaning the cathode is negative and the anode is positive for all methods. ANALYTICAL
1. Native PAGE
2. SDS Page
3. Isoelectric Focusing
What is polyacrylamide gel?
Polyacrylamide gel is the standard medium for protein electrophoresis. This gel is a slightly porous mixture which solidifes at room temperature. The gel acts like a seive, allowing smaller, more charged particles to easily pass while slowing down larger, less charged particles.
Describe the factors (and equation) that determine the migration velocity of a protein?
v = Ez/f
Where E is the electric field, z is the net charge on the protein, and f is a frictional coefficient that is based on the protein’s size and weight. From this equation, it can be said that high electric fields in the gel (E), high charges on proteins (z) and small proteins (lower f) move fastest in polyacrylamide gel.
Describe Native PAGE?
Native PAGE is when you place proteins in a PAGE Gel and generate the electric field with no other modifications to the proteins. This means that proteins will migrate according to their size and charge.
This can be problematic, because a small protein with a low charge might end up moving about as fast as a big protein with a high charge.
However, this can be useful, because the proteins were not modified and therefore retain their function and can be recovered.
Native PAGE is best used on proteins that are known to be similar in either size or charge, so that the relative rates of velocity can be used to separate them based on their dissimilar characteristic (if the proteins have similar charge, then using Native PAGE helps determine the relative masses. Vice Versa.)
Describe SDS-PAGE (what two things does SDS do)
Sodium Dodecyl Sulfate (SDS) PAGE is useful because it separates proteins based only on their relative molar masses. This is accomplished through the addition of SDS to the proteins before running them on the gel. SDS serves two purposes. One, it disrupts noncovalent bonds in the proteins, denaturing them without breaking the primary structure. Two, it imparts on the proteins massive nonpolar groups that essentially mask the actual charge of the protein and instead leave it with a constant negative charge.
Because all proteins are carrying more or less the same negative charge, the relative velocities of migration are dependent entirely on the masses of the proteins.
What is Isoelectric Focusing?
Isoelectric Focusing is an electrophorytic method that exploits the basic and acidic properties of amino acids by seperating them on the basis of their isoelectric point.
Instead of a normal PAGE, the gel in the cell will have a pH gradient where the pH is low (positive charge) near the anode and the pH is high (negative charge) near the cathode.
At a pH below its pI, a protein will have a positive charge. This will cause it to migrate towards the cathode. However, as the protein moves towards the cathode, it will move into higher pH gel. Eventually, the gel’s pH will be equal to the protein’s pI. At this point the protein has no charge, and will move no longer.
Protein moves towards PI
Note, this does not allow us to separate proteins based on mass, only on pI.
What are the 4 most common forms of chromatography?
- Column Chromatography
- Ion Exchange Chromatography
- Size-Exclusion Chromatography
- Affinity Chromatography
What is chromatography?
Chromatography is a tool that uses physical and chemical properties to separate and identify compounds from a complex mixture. Fundamentally, chromatographic methods involve the passing of homogenized protein mixutres through matrices of various sorts. Depending on the type of matrix used, different types of proteins will move through it quickly.
What is the stationary phase in chromatography?
The stationary phase, otherwise known as adsorbent, is the stationary medium onto which the proteins are originally loaded.
What is the mobile phase in chromatography?
The mobile phase is the gas or liquid that is ran through the chromatograph to pull the sample through the stationary phase, thus allowing it to elute.
How do the mobile and stationary phase work together to support chromatography?
The mobile and stationary phase will be different enough such that different types of proteins will bind to them differently. Proteins that have a high affinity for the mobile phase will quickly move through the chromatograph and elute first. Proteins that have a high affinity for the stationary phase will slowly move through the chormatograph and elute last.
What is retention time (chromotography)?
The retention time is the amount of time a compound spends in the stationary phase. High retention times indicate high affinity for the stationary phase.
What is partitioning?
Partitioning is the separation of components by nature of their relative affinities for the stationary phase.
Describe Column Chromatography
In column chromatography, a column is filled with silica(polar) or alumina(polar) beads as the stationary phase. A solvent of almost any type can be chosen as the mobile phase. Proteins are placed in the solvent of choice and poured into the column. Gravity moves everything down the column. As everything moves down the column, the polar proteins will have a higher affinity for the stationary phase, increasing their retention time.
Describe Ion Exchange Chromatography?
In this method, the beads (stationary phase) in the column are coated with charged substances, either negative or positive. Based on the charge of the beads, positive and negative proteins will have higher or lower retention times when compared to one another. For example, if the beads are coated with positive ions, negatively charged proteins would be expected to elute slowly. Variably saline eluent
Sometimes proteins can have such a high affinity for the stationary phase they get stuck in the column. These proteins can be displaced with a salt gradient, which floods the column with positive or negative ions that displace the proteins bound to the beads.
Describe Size Exclusion Chromatography
In this method, the beads used in the column contain tiny mazes called pores inside of themselves of varying sizes. Small proteins that can enter the beads have to go through the pores, which makes them take a lot longer to get through the column. Large proteins can’t fit into the pores, and so they just go around and elute faster.
What is Affinity Chromatography?
In this method, the beads are coated with a receptor or an antibody that only binds a specific protein. When a mixture of proteins is poured through an affinity chromatograph, the protein that matches the receptor or antibody will get stuck in the column while all the other proteins elute out.
Once the protein of interest is isolated in the column, molecules called free targets are flushed through the column to compete with and displace the proteins from the beads, thus allowing them to elute.
specific pH or salinity can also disrupt ligand binding
One drawback of this technique is that the free target used to flush the protein out can sometimes bind very strongly to the protein of interest, which slightly weakens the purpose of trying to isolate the protein
After a protein is isolated, what is the next step of analysis?
To study the protein’s structure, activity, concentration, and amino acid composition.
What are the ways the structure of a protein can be determined?
- Nuclear Magnetic Resonance Spectroscopy (NMR)
- X-ray Crystallography
How does X-ray crystallography work?
In X-Ray Crystallography, proteins or nucleotides are isolated and made to crystalize. Once crystalized, x rays are fired through the crystal. A film behind the crystal catches the defracted rays. These rays are then mapped and used to determine electron density. From the electron density of the molecule, a 3D shape can be determined. 75% of known protein structures today were analyzed through this method!
How does one determine the primary structure of a protein?
In order to determine the actual order of the amino acids, they can’t just be randomly hydrolzyed and separated. Instead, sequential and specific digestion of the protein in question is conducted using specific cleavage enzymes.
What method is used to determine the primary structure of small proteins?
For small proteins (50-70amino acids), primary structure is best identified using the Edman Degradation. The Edman Degradation removes individual amino acids from the N-terminus one at a time. These amino acids can be immediately sent to a mass spectrometer to determine their mass and thus identity.
What method is used to determine the primary structure of large proteins?
For large proteins, chymotrypsin, trypsin, and cyanogen bromide are used to selectively cleave the protein at specific amino acid residues, which creates small fragments that can be analyzed by elecrophoresis or the edman degradation. Based off which enzyme was used and what the fragment looks like, you can deduce the primary structure.
How does one analyze the activity of a protein?
Protein activity is generally determined by monitoring a knonw reaction with a given concentration of substrate and comparing it to a standard. Reactions with a color change are particularly convenient because you can put the protein in the reaction and see if the color changes any faster or slower.
What are the 4 main colorimetric ways to determine the concentration of a protein in a sample?
- UV Spectroscopy
- Bradford Protein Assay
- Bicinchroninic acid (BCA) assay
- Lowry reagent assay
How does UV spectroscopy allow us to determine the concentration of protein in a sample?
Each protein has a certain energy of light it absorbs. By passing different type of light through the sample and seeing how much of it is absorbed, one can determine the concentration of the protein. The higher the concentration of the protein the more of its characteristic light it absorbs.
This method is very easy, but is also highly sensitive to contamination as most proteins absorb similar energies of light.
What is the Bradford Protein Assay?
The Bradford Protein Assay mixes a protein in a solution with Coomassie Brilliant Blue Dye. The dye is protonated and green-brown in color before it mixes with the proteins. This dye will give up its protons when it binds to amino acid groups, turning blue in the process. The more protein there to interact with the dye, the longer it stays blue and the more absorbance of the characteristic wavelength of coomassie blue.
To actually conduct a Bradford Protein Assay, one generates data points by putting known concentrations of protein into the Bradford Protein Assay and measuring the absorbance. Once you have enough data points, you can make a line of best fit and use that line to estimate the concentration of an unknown protein given how much absorbance is occuring in the solution.
