Chapter 3: Nonenzymatic Protein Function and Protein Analysis

Question 1

What is the cytoskeleton?

What is the most common protein found in the cytoskeleton?

Answer 1

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.

Question 2

What is collagen? What three AA are commonly found in collagen?

Answer 2

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.

Question 3

What is elastin?

Answer 3

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.

Question 4

What are keratins? What AA are commonly found in keratin?

Answer 4

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.

Question 5

What is osteogenesis imperfecta?

Answer 5

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.

Question 6

What is actin? What is the most common AA in actin?

Answer 6

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

Question 7

What is tubulin?

Answer 7

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

Question 8

What are the motor proteins associated with tubulin?

Answer 8

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.

Question 9

What is myosin?

Answer 9

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.

Question 10

What are binding proteins?

Answer 10

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).

Question 11

What are cell adhesion molecules (CAMs)? What are the three types of cell adhesion molecules (CAMs)?

Answer 11

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

Question 12

What are cadherins?

Answer 12

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.

Question 13

What are integrins?

Answer 13

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.

Question 14

What are selectins?

Answer 14

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).

Question 15

What are immunoglobulins?

Answer 15

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.

Question 16

What is the antigen binding region? What is an antigen?

Answer 16

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.

Question 17

When antibodies bind to their antigens, they can cause one of three outcomes. What are those outcomes?

Answer 17

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.

Question 18

MCAT concept check cellular functions 3.1 page 95 question 1

How do cytoskeleton proteins differ from motor proteins?

Answer 18

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.

Question 19

MCAT concept check cellular functions 3.1 page 95 question 2

True or false. Motor proteins are not enzymes.

Answer 19

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.

Question 20

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?

Answer 20

If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrate will be bound despite a low affinity.

Question 21

MCAT concept check cellular functions 3.1 page 95 question 4

Rank them by strength

Answer 21

Cadherin stronger than integrin stronger than selectin.

Question 22

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?

Answer 22

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.

Question 23

What is biosignaling?

Answer 23

Biosignaling is the process in which cells received and act on signals.

Question 24

What is an ion channel?

What are the three varieties of ion channels?

Answer 24

Ion channels are proteins that create pathways for charged molecules.

Ungated channels
Voltage gated channels
Ligand gated channels

Question 25

What is facilitated diffusion?

Answer 25

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.

Question 26

What is an ungated channel? Provide example of an ungated channel.

Answer 26

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.

Question 27

What is a voltage gated channel? Provide example of a voltage gated channel.

Answer 27

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.

Question 28

What is a ligand gated channel? Provide example of ligand gated channel.

Answer 28

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)

Question 29

Do Km and Vmax apply to ion gates channels? What are Km and Vmax?

Answer 29

Yep. They are enzymes. Km refers to solute concentration at which the transporter is functioning at half of its Vmax.

Question 30

What are enzyme linked receptors?

Answer 30

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.

Question 31

What is a good example of an enzyme linked receptor?

Answer 31

Question 32

What is a G protein coupled receptor? Three main types (hint: cAMP and IP3)

Answer 32

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.

Question 33

How do G protein coupled receptors GPCR work? Why are they named G protein coupled receptors GPCR?

Answer 33

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).

Question 34

What are Gs, Gi, and Gq? (Hint GPCR)

Answer 34

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.

Question 35

Again. What are Gs, Gi, Gq?

Answer 35

These are not GABA receptors as the image states. They are GPCR.

Question 36

What is a heterotrimeric protein?

Answer 36

A heterotrimeric protein has three subunits. G proteins are heterotrimeric.

Question 37

Mnemonic for functions of heterotrimeric G proteins.

Answer 37

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)

Question 38

Image of trimeric G protein cycle (Gs or Gi depending on inhibiting or stimulating adenylate cyclase).

Answer 38

Trimeric, three, alpha, beta, gamma. 1. GDP with beta and gamma. 2. Then phosphorylated to GTP creating an active G protein. 3. Activated alpha changes the activity of adenylate cyclase. 4. Alpha rebinding to beta and gamma.

Question 39

MCAT concept check biosignaling page 99 question 1

Answer 39

Question 40

MCAT concept check biosignaling page 99 question 2 What type of ion channel is active at all times?

Answer 40

Ungated hannels are always open.

Question 41

MCAT concept check biosignaling page 99 question 3 How do transport kinetics differ from enzyme kinetics?

Answer 41

Question 42

The book lists two main methods of protein isolation. What are they?

Answer 42

Electrophoresis and chromatography

Question 43

What is homgenization? Centrifugation?

Answer 43

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.

Question 44

What is electrophoresis? What the kinds of electrophoresis are mentioned in the book?

Answer 44

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)

Question 45

What is polyacrymalide gel?

Answer 45

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.

Question 46

Which way to negative charged molecules migrate in gel electrophoresis? Positive?

Answer 46

Negative moves to the positively charged anode. Positive moves to the negatively charged cathode.

Question 47

Which way to anions move? Cations?

Answer 47

Anions move to the anode. Cations move to the cathode.

Question 48

What is Native PAGE gel electrophoresis? Can you recover the functional native protein from Native PAGE?

Answer 48

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.

Question 49

What is SDS PAGE?

Answer 49

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.

Question 50

What does SDS do in SDS PAGE? What are the only variables that affect the molecules velocity in SDS PAGE?

Answer 50

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).

Question 51

What is migration velocity of electrophoresis?

Answer 51

Question 52

What kind of cell does electrophoresis utilize? Which way to anions go? Cations?

Answer 52

Electrophoresis uses an electrolytic cell (deltaG>0, Ecell<0). Remember that anions always move toward the anode and cations always move toward the cathode.

Question 53

What is a Dalton? What is the average molar mass of one amino acid in Daltons (Da) and gram/mol?

Answer 53

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

Question 54

What is isoelectric focusing? Recap isoelectric point (pI) and zwitterion.

Answer 54

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.

Question 55

How does isoelectric focusing work?

Answer 55

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).

Question 56

Mnemonic to remember the relative acidity of isometric focusing technique.

Answer 56

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.

Question 58

When will a protein or AA stop when exposed to isoelectric focusing? What designation does the anode have? Cathode?

Answer 58

A protein or AA will stop moving when pI=pH. The anode is positive acidic. The cathode is negative basic.

Question 59

What is chromatography?

Answer 59

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.

Question 60

When is chromatography preferred over electrophoresis?

Answer 60

Chromatography is preferred over electrophoresis when large amounts of protein are being separated.

Question 61

What are the four kinds of chromatography in the book?

Answer 61

Question 62

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?

Answer 62

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.

Question 63

What is ion exchange chromatography?

Answer 63

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.

Question 64

What is size exclusion chromatography? Why will small molecules move slower down a size exclusion chromatograph?

Answer 64

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.

Question 65

Draw gamma-aminobutyric acid. Why is this special? The image is wrong. Why? Fix it if you can.

Answer 65

GABA is the main inhibitory neurotransmitter in your brain, stopping the chemical messages from passing from nerve cell to nerve cell.

Question 66

What is affinity chromatography? What may be a draw back of affinity chromatography?

Answer 66

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.

Question 67

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?

Answer 67

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).

Question 68

MCAT concept check protein isolation 3.3 page 109 question 2 What are the relative benefits of native PAGE compared to SDS-PAGE?

Answer 68

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.

Question 69

MCAT concept check protein isolation 3.3 page 109 question 3 What are two potential drawbacks of affinity chromatography?

Answer 69

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.

Question 70

MCAT concept check protein isolation 3.3 page 109 question 4 True or false. In size exclusion chromatography, the largest molecules elute first.

Answer 70

True. The small pores in size exclusion, chromatography trap smaller particles, retaining them in the column.

Question 71

How can protein structure be determined experimentally?

Answer 71

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.

Question 72

How do we analyze AA composition? What is Edman degradation? How do we digest larger proteins for AA analysis?

Answer 72

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.

Question 73

MCAT concept check 3.4 protein analysis page 111 question 1 Why are proteins analyzed after isolation?

Answer 73

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.

Question 74

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?

Answer 74

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.

Question 75

MCAT concept check 3.4 protein analysis page 111 question 3 True or false. The Edman degradation proceeds from the carboxy (C-) terminus.

Answer 75

False. The Edman degradation proceeds from the amino (N-) terminus.

Question 76

MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 1

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Question 77

MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 2

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 3

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 4

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 5

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 6

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 86 question 7

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 8

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 9

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 10

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 11

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 12

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 13

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 14

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MCAT mastery chapter 3 Nonenzymatic protein function and protein analysis page 87 question 15

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Question 91

What is the most abundant protein in eukaryotes?

Answer 91

Actin. Forms Microfilaments and plays critical role in cell structure, movement, and division.

Question 92

What is adenylate cyclase?

Answer 92

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.

Question 93

What is cAMP?

Answer 93

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.