Chapter 3: Nonenzymatic Protein Function and Protein Analysis Flashcards
What is the cytoskeleton?
What is the most common protein found in the cytoskeleton?
The cytoskeleton can be thought of as a three dimensional web or scaffolding systems for the cell.
It is comprised of proteins that are anchored to the cell membrane by embedded protein complexes.
Extracellular matrix disease is composed of proteins also support the tissues of the body.
Actin is the most common protein found in the cytoskeleton.
What is collagen? What three AA are commonly found in collagen?
The three AA commonly found in collagen are proline Pro P, hydroxyproline Hyp O, and glycine Gly G.
Collagen has a characteristic trihelical fiber (three left-handed helices is woven together to form a secondary right handed helix).
Collagen makes up most of the extracellular matrix of connective tissue. Collagen provides strength and flexibility.
What is elastin?
Elastin is another important component of the extracellular matrix of connective tissue. Its primary role is to stretch and then recoil like a spring, which restores the original shape of the tissue.
What are keratins? What AA are commonly found in keratin?
Keratins are intermediate filament proteins found in epithelial cells.
Keratins contribute to the mechanical integrity of the cell and also function as regulatory proteins.
Keratin is the primary protein that makes up hair and nails.
Keratin, a fibrous structural protein, is rich in amino acids like cysteine, serine, glycine, arginine, and proline, with cysteine being particularly abundant and crucial for forming disulfide bonds that contribute to keratin’s strength and structure.
What is osteogenesis imperfecta?
Osteogenesis imperfecta is known as brittle bone disease.
Collagen, a major component of bone, forms a specific secondary helical structure based on the abundance of the amino acid glycine Gly G. The replacement of glycine with other amino acids can cause improper folding of the collagen protein and cell death, leading to bone fragility.
The most common AA found in collagen are proline Pro P, hydroxyproline Hyp O, and glycine Gly G.
What is actin? What is the most common AA in actin?
Actin is a protein that makes up micro filaments and the thin filaments in myofibrils.
Actin is the most abundant protein in eukaryotic cells. Actin proteins have a positive side and negative side; this polarity allows motor proteins to travel, directionally along and actin filament, like a one-way street.
Actin contains 376 AA, 5% Pro P 8% Gly G
What is tubulin?
Tubulin is the protein that makes up microtubules. Microtubules are important for providing structure, chromosome, separation in mitosis and meiosis, and intercellular transport with kinesin and dynein.
Tubulin has polarity. The negative end of a microtubule is usually located adjacent to the nucleus, the positive and is usually in the periphery of a cell.
In contrast to intermediate filaments, which are composed of a variety of different fibrous proteins, microtubules are composed of a single type of globular protein, called tubulin
What are the motor proteins associated with tubulin?
Kinesin and dynein.
Kinesin play roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis.
Dynein are involved in the sliding movement of cilia and flagella.
What is myosin?
Myosin is the primary motor protein that interacts with actin.
In addition to its role as a thick filament in a myofibril, myosin can be involved in cellular transport.
Movement at the neck of myosin is responsible for the power stroke of sarcomere contraction.
What are binding proteins?
Binding proteins have stabilizing functions in individual cells and the body. Proteins that act in this way transport or sequester molecules by binding to them.
Binding proteins include hemoglobin, calcium binding proteins, DNA binding proteins (transcription factors).
What are cell adhesion molecules (CAMs)? What are the three types of cell adhesion molecules (CAMs)?
Cell adhesion molecules (CAMs) are proteins, found on the surface of most cells and abiding the cell to the extracellular matrix or other cells. They are integral membrane proteins.
Cadherins
Integrins
Selectins
What are cadherins?
Cadherins are a group of glycoproteins that mediate calcium dependent cell adhesion. They hold similar cell types together, such as epithelial cells.
Cadherins are a family of transmembrane cell adhesion proteins that play a crucial role in tissue formation, homeostasis, and cell-cell interactions
Different cells usually have types specific cadherins. For example, epithelial cells use E-cadherins, nerve cells use N-cadherins.
What are integrins?
Integrins are a group of proteins that all have two membranes spanning chains called alpha and beta.
Integrins are heterodimeric proteins composed of two subunits: an α (alpha) subunit and a β (beta) subunit. Both subunits are transmembrane proteins with extracellular, transmembrane, and cytoplasmic domain.
Integrins are important in binding to and communicating with the extra cellular matrix.
What are selectins?
Selectins are unique because they bind to carbohydrate molecules that project from other cell surfaces.
These bonds are the weakest formed by cell adhesion molecules (CAMs).
What are immunoglobulins?
Immunoglobulins are antibodies.
Immunoglobulins (Ig) are proteins produced by B cells that function of neutralize targets in the body, such as toxins and bacteria, and then recruit other cells to help eliminate the threat. They are Y shaped.
What is the antigen binding region? What is an antigen?
Each antibody has an antigen binding region at the tips of the Y. Within this region, there are specific polypeptide sequences that will bind with one and only one specific antigenic sequence.
The targets of antibody are called antigens.
When antibodies bind to their antigens, they can cause one of three outcomes. What are those outcomes?
Neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body.
Marking the pathogen for destruction by other white blood cells immediately; this marking function is also called opsonization.
Clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages.
MCAT concept check cellular functions 3.1 page 95 question 1
How do cytoskeleton proteins differ from motor proteins?
Cytoskeletal proteins tend to be fibrous with repeating domains.
Motor proteins tend to have ATPase activity and binding heads.
Both types of protein function in cellular motility.
MCAT concept check cellular functions 3.1 page 95 question 2
True or false. Motor proteins are not enzymes.
False. An enzyme is a protein or RNA molecule with catalytic activity. Motor proteins have catalytic activity. Motor function is generally considered non-enzymatic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity.
MCAT concept check cellular functions 3.1 page 95 question 3
What could permit a binding protein involved in sequestration to have a low affinity for its substrate and still have a high percentage of substrate bound?
If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrate will be bound despite a low affinity.
MCAT concept check cellular functions 3.1 page 95 question 4
Rank them by strength
Cadherin stronger than integrin stronger than selectin.
MCAT concept check cellular functions 3.1 page 95 question 5
When an antibody binds to its antigen, what are the three possible outcomes of this interaction?
Render the antigen nonfunctional, making the pathogen or talks, and unable to exert its effect on the body.
Mark the pathogen for destruction by other white blood cells (opsonization)
Agglutinate the antigen and antibody into large and soluble protein complex is that can be phagocytized and digested by macrophages.
What is biosignaling?
Biosignaling is the process in which cells received and act on signals.
What is an ion channel?
What are the three varieties of ion channels?
Ion channels are proteins that create pathways for charged molecules.
Ungated channels
Voltage gated channels
Ligand gated channels
What is facilitated diffusion?
Facilitated diffusion is a type of passive transport.
A transmembrane protein creates a pore through which diffusion of molecules can go down a concentration gradient.
What is an ungated channel? Provide example of an ungated channel.
Ungated channel have no gates and are therefore unregulated.
For example, all cells possess ungated potassium channels. This means there will be a net reflux of potassium ions through these channels unless potassium is at equilibrium.
What is a voltage gated channel?
Provide example of a voltage gated channel.
Voltage gated channels have a gate which is regulated by the membrane potential change near the channel.
For example, many excitable cells such as neurons possesses voltage gated sodium channels.
The channels are closed under resting conditions, but membrane depolarization causes a protein that allows them to quickly open and then quickly close as the voltage increases.
What is a ligand gated channel?
Provide example of ligand gated channel.
Ligand gated channel requires the binding of a specific substance or ligand to the channel to cause it to open or close.
Neurotransmitters act as ligand gated channels at the postsynaptic membrane. (GABA: gamma-aminobutyric acid binds to a chloride channel and opens it)
Do Km and Vmax apply to ion gates channels? What are Km and Vmax?
Yep. They are enzymes. Km refers to solute concentration at which the transporter is functioning at half of its Vmax.
What are enzyme linked receptors?
Enzyme linked receptors are membrane enzymes with a membrane spanning domain, a lingand binding domain, and catalytic domain.
Enzyme linked receptors result in second messenger cascade.
What is a good example of an enzyme linked receptor?
What is a G protein coupled receptor?
Three main types (hint: cAMP and IP3)
GPCR are integral membrane proteins involved in signal transduction, characterized by 7 membrane spanning alpha helices.
Gs: STIMULATES adenylate cyclase and increase cAMP
Gi: INHIBITS adenylate cyclase and decreases cAMP.
Gq: activates phospholipase C, PIP2 to IP3. IP3 opens calcium channels, increases calcium level in cells.
How do G protein coupled receptors GPCR work?
Why are they named G protein coupled receptors GPCR?
They utilize HETEROTRIMERIC (three subunits) G protein. The binding of a specific ligand increases the affinity of the receptor for a a G protein. The binding of a G protein is a switch to the active state and affects the intracellular signaling pathway.
They are named so due to their inteacellular link to GUANINE NUCLEOTIDES (GDP and GTP).
What are Gs, Gi, and Gq? (Hint GPCR)
Gs: stimulates adenylate cyclase, increases cAMP (that then amps protein kinase a that increases protein phosphorylation)
Gi: inhibits adenylate cyclase, decreases cAMP.
Gq: phospholipase C cleaves a PIP2, making an IP3 that opens calcium channels which increase calcium in the cell.
These are not GABA receptors like the image says. They are GPCR.
Again. What are Gs, Gi, Gq?
These are not GABA receptors as the image states. They are GPCR.
What is a heterotrimeric protein?
A heterotrimeric protein has three subunits.
G proteins are heterotrimeric.
Mnemonic for functions of heterotrimeric G proteins.
Gs Stimulates (stimulates adenylate cyclase and therefor cAMP signaling, increases phosphorylation)
Gi: Inhibits (inhibits adenylate cyclase, inhibit cAMP signaling, decrease phosphorylation)
Gq: mind your Ps and Qs: GQ activates Phopholipase C (cleaves to a PIP2, making IP3, increasing calcium in the cell)
Image of trimeric G protein cycle (Gs or Gi depending on inhibiting or stimulating adenylate cyclase).
Trimeric, three, alpha, beta, gamma.
- GDP with beta and gamma.
- Then phosphorylated to GTP creating an active G protein.
- Activated alpha changes the activity of adenylate cyclase.
- Alpha rebinding to beta and gamma.
MCAT concept check biosignaling page 99 question 1
MCAT concept check biosignaling page 99 question 2
What type of ion channel is active at all times?
Ungated hannels are always open.
MCAT concept check biosignaling page 99 question 3
How do transport kinetics differ from enzyme kinetics?
The book lists two main methods of protein isolation. What are they?
Electrophoresis and chromatography
What is homgenization? Centrifugation?
Homogenization is processing of biomolecules by crushing, grinding, blending etc. into an evenly mixed solution.
Centrifugation can then isolate proteins from much smaller molecules before other isolation techniques are employed.
What is electrophoresis?
What the kinds of electrophoresis are mentioned in the book?
Electrophoresis works by subjecting compounds to an electric field which moves them according to their charge and size.
Negatively charged compound will migrate to the positively charged ANODE.
(NEGATIVE TO THE positive ANODE)
Positively charged compounds will migrate to the negatively charged CATHODE.
(POSITIVE TO negative CATHODE)
What is polyacrymalide gel?
Polyacrymalide gel is the standard medium for protein electrophoresis. It is slightly porous and solidifies at room temperature.
Faster through the medium if it is small highly charged or placed in a large electric field.
Slower through the medium when bigger and more convoluted, electrically neutral, or placed in a small electric field.
Which way to negative charged molecules migrate in gel electrophoresis? Positive?
Negative moves to the positively charged anode.
Positive moves to the negatively charged cathode.
Which way to anions move? Cations?
Anions move to the anode.
Cations move to the cathode.
What is Native PAGE gel electrophoresis? Can you recover the functional native protein from Native PAGE?
Native PAGE is PolyAcrylamide Gel Electrophoresis (PAGE) is a method for analyzing proteins in their native states.
Native PAGE is useful to compare molecular size and charge if proteins known to be similar in size from other analytic methods like SDS PAGE or size exclusion chromatography.
You can reviver the native functional protein as long as not stain has been used as stain had a tendency to denature the proteins.
What is SDS PAGE?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) separates proteins based on their molecular mass.
The SDS detergent disrupts all noncovalent interactions, binding to proteins and creates a large chain with net negative charges.
What does SDS do in SDS PAGE? What are the only variables that affect the molecules velocity in SDS PAGE?
The SDS detergent disrupts all noncovalent interactions, it binds to proteins and creates a large chain with net negative charges.
The only variables effecting their velocity are the electric field E and the frictional coefficient f (which depends on mass).
What is migration velocity of electrophoresis?
What kind of cell does electrophoresis utilize?
Which way to anions go? Cations?
Electrophoresis uses an electrolytic cell (deltaG>0, Ecell<0).
Remember that anions always move toward the anode and cations always move toward the cathode.
What is a Dalton?
What is the average molar mass of one amino acid in Daltons (Da) and gram/mol?
Protein atomic mass is typically expressed in Daltons (Da), an alternative to molar mass (g/mol).
One Dalton (Da) = 1 gram/mol
Average AA is 100 Da, or 100g/mol
What is isoelectric focusing? Recap isoelectric point (pI) and zwitterion.
pI is the pH at which the protein or AA is electrically neutral, with an even number of positive and negative charges, for AA this is called a zwitterion.
Isoelectric focusing is separating proteins based on their isoelectric point.
How does isoelectric focusing work?
Proteins are placed in a gel with a pH gradient:
Acidic at positive (anode)
Basic at negative (cathode)
Neutral in the middle
And then exposed to an electric field.
The protein of AA will stop when pH=pI (when the pH of the gel equals the isoelectric point of the molecule).
Mnemonic to remember the relative acidity of isometric focusing technique.
Anode is isoelectric focusing: A+
Anode has Acidic gel and (+) charge.
We associate acids with protons, which carry positive charge, and thus the anode is positively charged.
We associate bases with negative charged hydroxide ions, which gives us the negatively charged cathode.
When will a protein or AA stop when exposed to isoelectric focusing? What designation does the anode have? Cathode?
A protein or AA will stop moving when pI=pH.
The anode is positive acidic. The cathode is negative basic.
What is chromatography?
Chromatography refers to a number of techniques that require a homogenized protein mixture to be fractionated through a porous mixture.
Components that have high affinity for the stationary phase will barely migrate, components that have a low affinity for the stationary phase will migrate much more quickly.
All chromatography is about the affinity of a substance for the mobile and stationary phase, except for size exclusion chromatography.
When is chromatography preferred over electrophoresis?
Chromatography is preferred over electrophoresis when large amounts of protein are being separated.
What are the four kinds of chromatography in the book?
What is column chromatography?
What qualities of a compound are considered for column chromatography?
What can be changed about column chromatography to help elute the protein of interest?
Column chromatography: a column is filled with silica or alumina as an absorbent, and gravity moves the solvent and compounds down the column.
Both size and polarity have a role in determining how quickly a compound move through the polar silica or alumina beads.
The less polar the compound, the faster it can elute through the column (short retention time)
The more polar the compound, the slower it will elute through the column (fast retention time)
Solvent, polarity, pH, or salinity can easily be changed to help elute the protein of interest.
What is ion exchange chromatography?
Ion exchange chromatography: the beads in the column are coded with charged substances, so they attract or bind compounds that have an opposite charge.
For instance, a positively charged column will attract and hold a negatively charged protein as it passes through the column, either in increasing its retention time or retaining it completely.
What is size exclusion chromatography? Why will small molecules move slower down a size exclusion chromatograph?
Size exclusion chromatography: the beads used in the column contained tiny pores of varying sizes. The pores allow small compounds to enter the beads, thus slowing them down.
Large compounds can’t fit into the pores, so they will move around them and travel through the column faster.
It is important to remember that small compounds are slowed down and retained longer, which may be counterintuitive, because they get caught up in the pores of the beads.
Draw gamma-aminobutyric acid. Why is this special?
The image is wrong. Why? Fix it if you can.
GABA is the main inhibitory neurotransmitter in your brain, stopping the chemical messages from passing from nerve cell to nerve cell.
What is affinity chromatography?
What may be a draw back of affinity chromatography?
Affinity chromatography. Beads are coated with high affinity for a selected protein.
Coat the beads with a receptor that binds a certain antibody to the protein, for example.
A draw back is that the eluant used to elute the protein of interest will be bound to the protein. If for example the eluent was an inhibitor of an enzyme it would be difficult to remove without denaturing the protein.
MCAT concept check protein isolation 3.3 page 109 question 1
What separation methods can be used to isolate a protein on the basis of isoelectric point?
Isoelectric focusing and ion exchange chromatography both separate proteins based on charge.
The charge of a protein in any given environment is determined by its isoelectric point (pI).
MCAT concept check protein isolation 3.3 page 109 question 2
What are the relative benefits of native PAGE compared to SDS-PAGE?
Native PAGE allows a complete protein to be recovered after analysis. It also more accurately determines the relative globular size of proteins.
SDS-PAGE Can be used to eliminate conflation from mass to charge ratios.
MCAT concept check protein isolation 3.3 page 109 question 3
What are two potential drawbacks of affinity chromatography?
The protein of interest may not elute from the column because its affinity is too high, or it may be permanently bound to the free receptor in the eluent.
MCAT concept check protein isolation 3.3 page 109 question 4
True or false. In size exclusion chromatography, the largest molecules elute first.
True. The small pores in size exclusion, chromatography trap smaller particles, retaining them in the column.
How can protein structure be determined experimentally?
X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy.
X ray crystallography method has identified 75% of the known protein structures. It measure electron density on a high resolution scale.
How do we analyze AA composition?
What is Edman degradation?
How do we digest larger proteins for AA analysis?
To determine the primary structure of a protein, sequential digestion of the protein with specific cleavage enzymes is used.
Edman degradation uses cleavage to sequence proteins of up to 50 to 70 amino acids then analyzed via mass spectroscopy.
For larger proteins, digestion with chymotrypsin, trypsin, and cyanogen bromide (a synthetic reagent) may be used.
MCAT concept check 3.4 protein analysis page 111 question 1
Why are proteins analyzed after isolation?
Protein isolation is generally only the first step in an analysis. The protein identity must be confirmed by amino acid analysis or activity. With unknown proteins, classification of their features is generally desired.
MCAT concept check 3.4 protein analysis page 111 question 2
What factors would cause an activity assay to display lower activity than expected after concentration determination?
Contamination of the sample with detergent or sodium dodecyl sulfate (SDS, a detergent) could yield an artificially increased protein level, leading to lower activity than expected because the protein concentration was calculated as higher than its actual value.
Alternatively, the enzyme could have been denatured during isolation and analysis.
MCAT concept check 3.4 protein analysis page 111 question 3
True or false. The Edman degradation proceeds from the carboxy (C-) terminus.
False. The Edman degradation proceeds from the amino (N-) terminus.
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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 15
What is the most abundant protein in eukaryotes?
Actin. Forms Microfilaments and plays critical role in cell structure, movement, and division.
What is adenylate cyclase?
Adenylate cyclase (AC), also known as adenylyl cyclase, is an enzyme that catalyzes the conversion of ATP to cyclic AMP (cAMP) and pyrophosphate, a key process in cellular signaling.
Recall Gs and Gi, the G protein coupled receptors which stimulate or inhibit adenylate cyclase and therefor cAMP, respectively.
What is cAMP?
cAMP, or cyclic adenosine monophosphate, is a crucial second messenger molecule involved in intracellular signaling pathways, acting as a bridge between extracellular signals and cellular responses.
