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Biochem > Ch. 3: Nonenzymatic Protein Function and Protein Analysis > Flashcards

Ch. 3: Nonenzymatic Protein Function and Protein Analysis Flashcards

1
Q

what are the two main functions of proteins within the cell?

A
  1. supporting cellular shape and organization
  2. acting as enzymes
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2
Q

where are structural and motor proteins found? (2)

A
  1. within individual cells
  2. the extracellular matrix
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3
Q

in general terms, how are proteins involved structurally intracellulary and extracellularly?

A

INTRACELLULAR: the cytoskeleton (a 3-D web or scaffolding system for the cell) is comprised of proteins that are anchored to the cell membrane by embedded protein complexes

EXTRACELLULAR: matrices composed of proteins also support the tissues of the body (tendons, ligaments, cartilage, and basement membranes)

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4
Q

what are the 5 primary structural proteins in the body?

A
  1. collagen
  2. elastin
  3. keratin
  4. actin
  5. tubulin
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5
Q

char (3): structural proteins

A
  1. have a highly repetitive secondary structure
  2. have a super-secondary structure/motif
  3. regularity gives many a fibrous nature
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6
Q

defn: motif/super-secondary structure

A

a repetitive organization of secondary structural elements together

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7
Q

char + func (2) + location: collagen

A

char: has a characteristic trihelical fiber (three left-handed helices woven together to form a secondary right-handed helix)

func: 1. makes up most of the extracellular matrix of connective tissue
2. important in providing strength and flexibility

location: throughout the body

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8
Q

func (2): elastin

A
  1. another important component of the extracellular matrix of connective tissue
  2. main role is to stretch and then recoil like a spring, restoring the original shape of the tissue
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9
Q

defn + func (3): keratin

A

defn: intermediate filament proteins found in epithelial cells

func: 1. contribute to the mechanical integrity of the cell
2. function as regulatory proteins
3. the primary protein that makes up hair and nails

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10
Q

char (2) + func: actin

A

func: makes up microfilaments and the thin filaments in myofibrils

char: 1. the most abundant protein in eukaryotic cells
2. have a positive and a negative side (polarity that allows motor proteins to travel unidirectionally along an actin filament like a one way street)

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11
Q

func + char: tubulin

A

func: makes up microtubules

char: has polarity (negative end of a microtubule is usually located adjacent to the nucleus, the positive end is usually in the cell periphery)

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12
Q

can structural proteins have motor functions?

A

yes, some can in the presence of motor proteins (e.g. cilia and flagella of bacteria and sperm)

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13
Q

explain how motor proteins can display enzymatic activity

A

they act as ATPases that power the conformational change necessary for motor function

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14
Q

what 2 things do motor proteins have transient interactions with?

A
  1. actin
  2. microtubules
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15
Q

defn + func (3) + char: myosin

A

defn: the primary motor protein that interacts with actin

func: 1. the thick filament in a myofibril
2. involved in cellular transport
3. movement of the neck is responsible for the power stroke of sarcomere contraction

char: each myosin subunit has a single head and neck

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16
Q

defn + char (2) + func of both: kinesin and dynein

A

defn: the motor proteins associated with microtubules

char: 1. have 2 heads, at least one of which remains attached to tubulin at all times
2. have opposite polarities

func: important for vesicle transport in the cell

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17
Q

func (2): kinesin

A
  1. key role in aligning chromosomes during metaphase
  2. key role in depolymerizing microtubules during anaphase of mitosis
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18
Q

func: dynein

A

involved in the sliding movement of cilia and flagella

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19
Q

explain what it means that kinesin and dynein have opposite polarities

A

KINESINS bring vesicles toward the positive end of the microtubule

DYNEINS bring vesicles toward the negative end of the microtubule

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20
Q

diagram: stepwise activity of kinesins

A

kinesins move along microtubules in a stepping motion such that one or both heads remain attached at all times

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21
Q

defn: binding proteins

A

proteins that have stabilizing functions in individual cells and the body that act to transport or sequester molecules by binding to them

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22
Q

what are 3 common binding proteins/groups?

A
  1. hemoglobin
  2. calcium-binding proteins
  3. DNA-binding proteins (often transcription factors)
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23
Q

each binding protein has an affinity curve for its molecule of interest, what makes this curve differ?

A

this curve differs depending on the goal of the binding protein

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24
Q

what is the affinity when sequestration of the molecule is the goal? why?

A

the binding protein usually has high affinity for its target across a large range of concentrations so it can keep it bound at nearly 100 percent

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25
Q

what is the affinity for a transport protein? why?

A

transport proteins must be able to bind or unbind its target to maintain steady-state concentrations, and is thus likely to have a varying affinity depending on the environmental conditions

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26
Q

defn: cell adhesion molecules (CAMs)

A

proteins found on the surface of most cells (all integral membrane proteins) and aid in binding the cell to the extracellular matrix or other cells

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27
Q

what are the 3 categories of CAMs?

A
  1. cadherins
  2. integrins
  3. selectins
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28
Q

defn + func + char: cadherins

A

a group of glycoproteins that mediate calcium-dependent cell adhesion

func: often hold similar cell types together (such as epithelial cells)

char: different cells usually have type-specific cadherins (i.e. epithelial cells use E-cadherin, nerve cells use N-cadherin)

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29
Q

defn + func (3): integrins

A

a group of proteins that all have 2 membrane-spanning chains called alpha and beta that are very important in binding to and communicating with the extracellular matrix

func: 1. play a very important role in cellular signaling
2. can greatly impact cellular function by promoting cell division, apoptosis, or other processes
3. important role in host defense

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30
Q

what are 3 examples of processes which integrins are involved in?

A
  1. allowing platelets to stick to fibrinogen, a clotting factor, which causes activation of platelets to stabilize the clot
  2. white blood cell migration
  3. stabilization of epithelium on its basement membrane
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31
Q

defn + char (2) + func: selectins

A

defn: bind to carbohydrate molecules that project from other cell surfaces

char: 1. the weakest formed bonds by CAMs
2. expressed on white blood cells and the endothelial cells that line blood vessels

func: important role in host defense, including inflammation and white blood cell migration

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32
Q

what is the common purpose of the cells and proteins involved in the immune system?

A

to rid the body of foreign invaders

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33
Q

defn (2) + aka + char (3) + diagram: antibodies

A

defn: 1. the most prominent type of protein found in the immune system
2. proteins produced by B-cells that function to neutralize targets in the body, such as toxins and bacteria, and then recruit other cells to help eliminate the threat

aka: immunoglobulins (Ig)

char: 1. Y-shaped proteins made up of two identical heavy chains and two identical light chains
2. disulfide linkages and covalent interactions hold the heavy and light chains together
3. each has an antigen-binding region at the tips of the Y

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34
Q

func: antigen-binding region

A

within this region, there are specific polypeptide sequences that will bind one (and only one) specific antigenic sequence

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35
Q

outside of the antigen-binding region, what is the rest of the antibody molecule called and what is its function?

A

name: constant region

func: involved in recruitment and binding of other cells of the immune system, such as macrophages

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36
Q

defn: antigens

A

the targets of antibodies

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37
Q

what are the three outcomes of when antibodies bind to their targets (antigens)?

A
  1. neutralizing the antigen, making the pathogen or toxin unable to exert its effect on the body
  2. marking the pathogen for destruction by other white blood cells immediately (opsonization)
  3. clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages
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38
Q

defn: biosignaling

A

a process in which cells receive and act on signals

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39
Q

in what 4 capacities do proteins participate in biosignaling?

what 2 functions do proteins have to participate?

A

capacities: 1. extracellular ligans
2. transporters for facilitated diffusion
3. receptor proteins
4. second messengers

functions: 1. substrate binding
2. enzymatic activity

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40
Q

defn: ion channels

A

proteins that create specific pathways for charged molecules

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41
Q

what are the 3 main groups of ion channels?

what is the similarity and differences between the ion channel groups?

A
  1. ungated channels
  2. voltage-gated channels
  3. ligand-gated channels

have different mechanisms of opening, but all permit facilitated diffusion of charged particles

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42
Q

defn + func (2): facilitated diffusion

A

a type of passive transport

the diffusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein

func: 1. used for molecules that are impermeable to the membrane (large, polar, or charged)

  1. allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic fatty acid tails of the phospolipid bilayer
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43
Q

defn: ungated channels

A

have no gates and are thus unregulated

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44
Q

defn: voltage-gated channels

A

the gate is regulated by the membrane potential change near the channel

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45
Q

defn: ligand-gated channels

A

the binding of a specific substance or ligand to the channel causes it to open or close

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46
Q

where can we derive the kinetics of transporters such as ion channels in membranes?

A

the Km and vmax parameters that apply to enzymes are also applicable

we can derive them from the Michaelis-Menten and Lineweaver-Burk plot equations where Km refers to the solute concentration at which the transporter is functioning at half of its maximum activity

47
Q

defn: enzyme-linked receptors

A

membrane receptors that display catalytic activity in response to ligand binding

48
Q

what are the three primary protein domains of enzyme-linked receptors?

A
  1. membrane-spanning domain
  2. ligand-binding domain
  3. catalytic domain
49
Q

func: membrane-spanning domain

A

anchors the receptor in the cell membrane

50
Q

char + func: ligand-binding domain

A

stimulated by the appropriate ligand

induces a conformational change that activates the catalytic domain which often results in the initiation of a second messenger cascade

51
Q

what are 3 common classes of enzyme-linked receptors?

A
  1. receptor tyrosine kinases
  2. serine/threonine-specific protein kinases
  3. receptor tyrosine phosphatases
52
Q

defn: G protein-coupled receptors

A

a large family of integral membrane proteins involved in signal transduction that differ in specificity of the ligand-binding area found on the extracellular surface of the cell and are characterized by their 7 membrane-spanning alpha-helices

53
Q

func: heterotrimeric G protein

A

used by GPCRs to transmit signals to an effector in the cell

54
Q

what are G proteins named for?

A

their intracellular link to guanine nucleotides (GDP and GTP)

55
Q

how do GPCRs and G proteins work together?

A
  1. the binding of a ligand increases the affinity of the receptor for the G protein
  2. the binding of the G protein represents a switch to the active state and affects the intracellular signaling pathway
56
Q

what are the 3 main types of G proteins + their functions?

A
  1. Gs –> stimulates adenylate cyclase, which increases levels of cAMP in the cell
  2. Gi –> inhibits adenylate cyclase, which decreases levels of cAMP in the cell
  3. Gq –> activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP2, which is then cleaved into DAG and IP3 which can open calcium channels in the ER, increasing calcium levels in the cell
57
Q

mnemonic: functions of heterotrimeric G proteins

A

GS Stimulates

GI Inhibits

Mind your P’s and Q’s: GQ activates Phospholipase C

58
Q

what are the three subunits of the G protein?

A

alpha, beta, gamma

59
Q

what is the status of the subunits when the G protein is in its inactive form?

A

the alpha subunit binds GDP and is in a complex with the beta and gamma subunits

60
Q

what happens to the subunits when a ligand binds to the GPCR, the receptor becomes activated, and engages the corresponding G protein? + diagram

A
  1. the alpha subunit is able to dissociate from the beta and gamma subunits
  2. the activated alpha subunit alters the activity of adenylate cyclase
  3. if the alpha subunit is alphas –> the enzyme is activated, if it is alphai –> the enzyme is inhibited
  4. once GTP on the activated alpha subunit is dephosphorylated to GDP, the alpha subunit will rebind to the beta and gamma subunits, rendering the G protein inactive
61
Q

how are proteins and other biomolecules isolated from body tissues or cell cultures?

A
  1. cell lysis
  2. homogenization
62
Q

defn: homogenization

A

crushing, grinding, or blending the tissue of interest into an evenly mixed solution

63
Q

func: centrifugation

A

can isolate proteins from much smaller molecules before other isolation techniques must be employed

64
Q

what are the 2 most common isolation techniques? can they be used on both native and denatured proteins?

A
  1. electrophoresis
  2. chromatography

yes, they can be used on both native and denatured proteins

65
Q

how does electrophoresis work?

A

by subjecting compounds to an electric field, which moves them according to their net charge and size

so negatively charged compounds will migrate toward the positively charged anode and positively charged compounds will migrate toward the negatively charged cathode

66
Q

defn: migration velocity

A

velocity of the migration of charged compounds toward the charged anodes in electrophoresis

67
Q

what is migration velocity, v, directly proportional to (2) and inversely proportional to (1)? + equation

A

directly: 1. electric field strength E
2. the net charge on the molecule z

inversely: a frictional coefficient f

68
Q

what does the frictional coefficient f depend on?

A

the mass and shape of the migrating molecules

69
Q

what is the standard medium for protein electrophoresis?

A

polyacrylamide gel

70
Q

char (3): polyacrylamide gel

A
  1. a slightly porous matrix mixture
  2. solidifies at room temperature
  3. multiple samples can be run simultaneously due to the gel’s size
71
Q

how does polyacrylamide gel interact with proteins during protein electrophoresis?

A
  1. proteins travel through this matrix in relation to their size and charge
  2. the gel acts like a sieve, allowing smaller particles to pass through easily while retaining large particles
72
Q

what 3 things would make a molecule move faster through the gel? what 3 things would make a molecule move slower through the gel? (or not at all)

A

faster: 1. small
2. highly charged
3. placed in a large electric field

slower: 1. bigger and more convoluted
2. electrically neutral
3. placed in a small electric field

73
Q

defn: PAGE

A

polyacrylamide gel electrophoresis

a method for analyzing proteins in their native states

74
Q

diagram: electrophoresis

A
75
Q

what is PAGE limited by?

A

the varying mass-to-charge and mass-to-size ratios of cellular proteins because multiple different proteins may experience the same level of migration

76
Q

can the functional native protein be recovered from the gel after electrophoresis in PAGE?

A

only if the gel has not been stained because most stains denature proteins

77
Q

for what is PAGE most useful?

A

to compare the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography

78
Q

defn: SDS-PAGE

A

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

separates proteins on the basis of relative molecular mass alone

79
Q

how does SDS-PAGE work? (4)

A
  1. starts with the same premise as PAGE, but adds SDS, a detergent that disrupts all noncovalent interactions
  2. it binds to proteins and creates large chains with net negative charges, thereby neutralizing the protein’s original charge and denaturing the protein
  3. as the proteins move through the gel, the only variables affecting their velocity are E, the electric field strength, and f, the frictional coefficient, which depends on mass
  4. after separation, the gel can be stained so the protein bands can be visualized and the results recorded
80
Q

what unit is protein atomic mass expressed in?

A

daltons (Da) = g/mol

81
Q

defn: isoelectric point (pI)

A

the pH at which the protein or amino acid is electrically neutral, with an equal number of positive and negative charges

82
Q

defn: zwitterion

A

the electrically neutral form of individual amino acids

83
Q

for most amino acids, what groups are protonated or deprotonated for the zwitterion form?

A

amino group protonated

carboxyl group deprotonated

side chain electrically neutral

84
Q

what are the 2 amino acids that are exceptions to the above zwitterion formation pattern? when does their zwitterion form?

A

arginine and lysine (basic side chains)

amino group deprotonated (thus electrically neutral)

nitrogenous side chain protonated

carboxyl group deprotonated

85
Q

how is the pI determined for polypeptides?

A

primarily determined by the relative numbers of acidic and basic amino acids

86
Q

defn: isoelectric focusing

A

exploits the acidic and basic properties of amino acids by separating on the basis of pI

87
Q

how does isoelectric focusing work? (4)

A
  1. the mixture of proteins is placed in a gel with a pH gradient (acidic gel at the positive anode, basic gel at the negative cathode, and neutral in the middle)
  2. an electric field is generated across the gel
  3. positively charged proteins migrate toward the cathode, negatively charged proteins migrate toward the anode
  4. as the protein reaches the portion of the gel where the pH is equal to the protein’s pI , the protein takes on a neutral charge and will stop moving
88
Q

mnemonic: remembering anodes and charges in isoelectric focusing

A

Anode in isoelectric focusing: A+

Anode has Acidic (H+-rich) gel and a (+) charge

89
Q

defn + basic premise: chromatography

A

a variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix

the more similar the compound is to its surroundings (polarity, charge, etc.), the more it will stick to and move slowly through its surroundings

90
Q

what is one of the main reasons why chromatography is so valuable?

A

the isolated proteins are immediately available for identification and quantification

91
Q

when is chromatography preferred over electrophoresis?

A

when large amounts of protein are being separated

92
Q

process (5): chromatography

A
  1. place the sample onto a solid medium (the stationary phase or adsorbent)
  2. run the mobile phase through the stationary phase which will allow the sample to run through the stationary phase (elute)
  3. depending on the relative affinity of the sample for the stationary and mobile phases, different substances migrate through at different speeds (those that have a high affinity for the stationary phase will barely migrate, those that have a high affinity for the mobile phase will migrate much more quickly)
  4. varying retention times of each compound in the solution results in separation of the components within the stationary phase (partitioning)
  5. each component can then be isolated individually for study
93
Q

defn: retention time

A

the amount of time a compound spends in the stationary phase

94
Q

process (5) + diagram: column chromatography

A
  1. a column is filled with silica or alumina beads as an adsorbent and gravity moves the solvent and compounds down the column
  2. as the solution flows through the column, both size and polarity determine how quickly the compound moves through the polar beads (the less polar the compound, the faster it can elute)
  3. the solvent drips out the column’s end, and different fractions that leave the column are collected over time
  4. each fraction contain bands that correspond to different compounds
  5. the solvent can then be evaporated and the compounds of interest kept
95
Q

what 3 things can be easily changed in column chromatography to help elute the protein of interest?

A

solvent:
1. polarity
2. pH
3. salinity

96
Q

process (2): ion-exchange chromatography

A
  1. the beads in the column are coated with charged substances, so they attract or bind compounds that have an opposite charge
  2. after all other compounds have moved through the column, a salt gradient is used to elute the charged molecules that have stuck to the column
97
Q

process (3): size-exclusion chromatography

A
  1. the beads used in the column contain tiny pores of varying sizes which allow small compounds to enter the beads, thus slowing them down
  2. large compounds can’t fit into the pores, so they’ll move around them and travel through the column faster
  3. the size of the pores may be varied so that molecules of different molecular weights can be fractionated
98
Q

can size-exclusion and ion-exchange chromatography be used together?

A

yes, a common approach in protein purification is to use an ion-exchange column followed by a size-exclusion column

99
Q

main purpose + process (3): affinity chromatography

A

main purpose: we can customize beads to bind any protein of interest by creating a column with high affinity for that protein

  1. coat beads with a receptor that binds the portein or a specific antibody to the protein such that the protein is retained in the column
  2. once the protein is retained in the column, it can be eluted by washing the column with a free receptor (or target or antibody), which will compete with the bead-bound receptor and ultimately free the protein from the column
  3. eluents can be created with a specific pH or salinity level that disrupts the bonds between the ligand and the protein of interest
100
Q

what are 3 common stationary phase molecules in affinity chromatography?

A
  1. Nickel (used in separation of genetically engineered proteins with histidine tags)
  2. antibodies or antigens
  3. enzyme substrate analogous (mimic the natural substrate for an enzyme of interest)
101
Q

what is the only drawback to the elution step in affinity chromatography?

A

the recovered substance can be bound to the eluent

102
Q

what are the 2 methods by which protein structure can be determined? which is the more reliable and common method?

A
  1. X-ray crystallography (more reliable and common)
  2. nuclear magnetic resonance (NMR) spectroscopy
103
Q

how does X-ray crystallography work? (2)

A
  1. measures electron density on an extremely high-resolution scale and can also be used for nucleic acids
  2. an x-ray diffraction pattern is generated in this method and the small dots in the diffraction pattern can then be interpreted to determine the protein’s structure
104
Q

how can the amino acids that compose a protein be determined?

A

by complete protein hydrolysis and subsequent chromatographic analysis

HOWEVER, the random nature of hydrolysis prevents amino acid sequencing

SO, sequential digestion of the protein with specific cleavage enzymes is used

105
Q

defn + use + process: Edman degradation

A

use: used for small proteins

defn: uses cleavage to sequence proteins of up to 50 to 70 amino acids

process: selectively and sequentially removes the N-terminal amino acid of the protein (can be analyzed via mass spectroscopy)

106
Q

how is protein activity generally determined? (3)

A
  1. by monitoring a known reaction with a given concentration of substrate and comparing it to a standard
  2. activity is correlated with concentration but is also affected by the purification methods and the conditions of the assay
  3. reactions with a color change have particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest
107
Q

how is concentration determined?

A

almost exclusively through spectroscopy

108
Q

why can proteins be analyzed with UV spectroscopy without any treatment?

A

because proteins contain aromatic side chains

109
Q

what is a downside to UV spectroscopy?

A

it is particularly sensitive to sample contaminants

110
Q

what are the bicinchoininic acid (BCA) assay, the Lowry reagent assay, and Bradford assay? which of these 3 is the most common and why?

A

colorimetric changes with specific reactions caused by proteins

Bradford assay is the most common because of its reliability and simplicity in basic analyses

111
Q

how does the Bradford protein assay work? (6) + diagram

A
  1. mixes a protein in solution with Coomassie Brilliant Blue dye
  2. the dye is protonated and green-brown in color prior to mixing with proteins (acidic form)
  3. the dye gives up protons upon binding to amino acid groups, turning blue in the process (basic form)
  4. ionic attractions between the dye and the protein then stabilize this blue from of the dye (so increased protein concentrations correspond to a larger concentration of blue dye in solution)
  5. samples of known concentration are reacted with the Bradford reagent and then absorbance is measured to create a standard curve
  6. the unknown sample is then exposed to the same conditions and the concentration is determined based on the standard curve
112
Q

when is a Bradford assay good to use? when not? why?

A

very accurate: when only one type of protein is present in solution

not good: when more than one protein is present because of variable binding of the Coomassie dye with different amino acids

113
Q

what is the Bradford protein assay limited by?

A

the presence of detergent in the sample or by excessive buffer

Biochem (12 decks)

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