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MCAT Biochem > Non-Enzymatic Protein Function And Protein Analysis > Flashcards

Non-Enzymatic Protein Function And Protein Analysis Flashcards

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MCAT flashcards
1
Q

Generally describe the cytoskeleton

A

Three dimensional web of proteins anchored to the cell membrane by embedded protein complexes

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2
Q

What are the primary structure of proteins in the body?

A

Collagen, elastin, keratin, actin, tubulin

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3
Q

Describe the structure of structural proteins

A

Generally highly repetitive secondary structure and a super secondary structure- a repetitive organization of secondary structural elements together sometimes referred to as a motif

This regularity gives many structural proteins of fibrous nature

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4
Q

Collagen

A

Structural protein
Trihelical fiber, three left-handed helices woven together to form a secondary right hand helix
Makes up most of the extra cellular matrix of connective tissue
Found throughout the body and is important in providing strength and flexibility

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5
Q

Elastin

A

Structural protein
Important component of extra cellular matrix of connective tissue
Stretch and recoil

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6
Q

Keratin

A

Structural protein
Intermediate filament proteins found in epithelial cells
Contribute to the mechanical integrity of the cell and also function as regulatory proteins

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7
Q

Actin

A

Structural protein
Composes microfilaments, and the thin filaments in myofibrils
Most abundant protein in eukaryotic cells
Have a positive side and a negative side allowing motor proteins to travel, unidirectionally along an actin filament like a one-way street

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8
Q

Tubulin

A

Structural protein
Mixup microtubules
Micro tubules are not only important for mitosis and meiosis but also for intracellular transport with kinesin and dyein
Polarity
Negative end adjacent to the nucleus and the positive end peripheral

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9
Q

Motor protein activity

A

Act as ATPases to fund their activity and power conformational chance necessary for motor function
Have transient interactions with either actin or microtubules

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10
Q

Myosin

A

The primary motor protein that interacts with actin
Thick filament in myofibril
Can be involved in cellular transport
Each subunit has a head and neck, the neck being responsible for power stroke of sarcomere contraction

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11
Q

Kinesins and dyneins

A

Motor protein associated with microtubules
They has two heads, at least one remaining attached to tubulin at all times
Vesicle transport
Opposite polarities

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12
Q

Kinesins

A

Play key roles in aligning chromosomes during metaphase and depolymerizing microtubules during anaphase of mitosis
bring vesicles toward the positive end of the microtubule
Bring vesicles of neural transmitter to the positive end of the axon of microtubules (toward the synaptic terminal)

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13
Q

Dyneins

A

Involved in the sliding movement of cilia and flagella
Bring vesicles of waste or recycled neurotransmitter back to the negative end of the microtubule (toward the soma) through retrograde transport

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14
Q

Binding proteins

A

Transport or sequester molecules by binding to them
Hemoglobin, calcium binding proteins, DNA binding proteins, transcription factors, and others
Affinity curve

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15
Q

Affinity curve of binding proteins

A

When sequestration of a molecule is the goal, the binding protein usually has high affinity for its target a Crossing large range of concentration so you can keep it bound at nearly 100%
A transport protein is likely to have varying affinity, depending on the environmental conditions

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16
Q

Cell adhesion molecules

A

Found on the surface of most cells and aid in binding the cell to the extracellular matrix or other cells
There are a number of different types of cams
All integral membrane proteins
Adhesion molecules can be classified into three major families: integrins, cadherins, selectins

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17
Q

Cadherins

A

Cell adhesion molecules
Groups of glycoproteins that mediate calcium dependent cell adhesion
Specific cadherin types for specific tissue types (E-cadherin for epithelial and N-cadherin for nerve cell)

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18
Q

Integrin

A

Cell adhesion molecules
Group of proteins that all have two membrane spanning chains called alpha and beta
These chains are very important in the binding and communities with the extra cellular matrix
Cell signaling, apoptosis, cell division
Blood clotting, white blood cell migration, basement membrane stabilization

19
Q

Selectin

A

Cell adhesion molecule
Bind to carb molecules that project from other cell surfaces
These bonds are the weakest formed by the CAMs
Expressed on white blood cells and endothelial cells that line blood vessels
Host defense

20
Q

Immunoglobulins

A

Rid body of foreign invaders
Also called antibodies
Produced by B cells
Neutralize targets
Recruit others
Y-shaped
Made up of two heavy identical chains and two identical light chains
Disulfide linkages and noncovalent interactions hold the heavy and light chains together
Each antibody has an antigen-binding region at the tips of the Y
Antigenic sequence specific
Remaining portion is the constant region which recruits binding of other immune cells

21
Q

What happens when antibodies bind to antigens?

A

1) neutralize
2) opsonization
3) agglutinating to be phagocytized

22
Q

ion channels

A

facilitated diffusion, passive transport

used for impermeable molecules (large, polar, uncharged)

3 types: ungated, voltage-gated, ligand-gated

23
Q

ungated channels

A

no gates, unregulated
K+ leak channels all cells possess

24
Q

voltage-gated channels

A
  • regulated by membrane potential change
  • possessed by excitable cells
  • closed under resting conditions, but membrane depolarization causes protein conformation change that allows them to quickly open and then quickly close as voltage inc
  • sodium-potassium channels found in SA node, pacemaker current
25
Q

ligand-gated channels

A
  • specific ligand binding causes channel to open or close
  • NT act at LGC
26
Q

enzyme-linked receptors

A
  • 3 primary protein domains:
    1. membrane-spanning domain (anchor)
    2. ligand-binding domain (induces conformational change to activate catalytic domain)
    3. catalytic domain (second messenger cascade)

RTK dimerization and autophosphorylation

RTP

serine/threonine-specific protein kinases

27
Q

GPCR

A
  • 7 membrane a-helices
  • Gs: stim adenylate cyclase, inc cAMP
  • Gi: inhibit adenylate cyclase, dec cAMP
  • Gq: activate phospholipase C, cleaves a phospholipid from membrane to form PIP2 ~> cleaved into DAG and IP3 ~> IP3 opens calcium channels in the ER inc calcium levels in cell
  • a inactive and bound to b and g but when ligand binds, GDP replaced with GTP and a dissociates, does its thing as mentioned before, and then becomes inactive and bound again after GTP dephosphorylated
28
Q

what do we do before isolation techniques employed?

A

homogenization and centrifugation

29
Q

electrophoresis

A
  • isolation technique
  • electric field moves compounds according to net charge and size
  • neg will migrate to pos anode and pos with migrate to neg anode

v = Ez/f
v: migration velocity
E: electric field strength
z: net charge
f: frictional coefficient

  • medium is polyacrylamide gel which is slightly porous, solid at room temp, movement dependent on size and charge, large particles easily retained
  • small highly charge particles move faster or placed in large electric field

-multiple samples can run simultaneously

30
Q

native PAGE

A
  • analyze protein in native state
  • functional native protein recovered from gel after electrophoresis only if it hasn’t been stained
  • limited by mass-to-charge or -size rations of cellular proteins
31
Q

SDS PAGE

A
  • separates proteins on basis of relative molecular mass alone
  • SDS detergent disrupts all noncovalent interactions by binding to proteins and creating large chains w net negative charges neutralizing and denaturing the protein
  • E and f affect velocity and after separation gel can be stained so that protein bands can be visualized and the results recorded
32
Q

isoelectric point

A

the pH at which the protein or amino acid is electrically neutral

zwitterion

exceptions: arg and lys bc of basic side chains so their zwitterion occurs when the amino group is deprotonated

for polypeptides the isoelectric point is primarily determined by the relative numbers of acidic and basic amino acids

33
Q

isoelectric focusing

A

the mixture of proteins is placed in a gel w a pH gradient

an electric field is then generated across the gel

pos proteins will migrate towards cathode and neg toward anode

as protein reaches portion where pH is equal to its pI, protein takes on a neutral charge and stop moving

34
Q

chromatography

A

sample placed on stationary phase

mobile phase runs through and elutes sample

diff substances move at diff speed

affinity of sample observed

amount of time spent in stationary phase by sample is called retention time

causes compounds to partition

35
Q

column chromatography

A
  • column filled w silica or alumina beads
  • gravity moves everything through beads
  • size and polarity determine speed of compound moving through polar beads
  • less polar, shorter retention time
  • solvent polarity, pH, or salinity can easily be changed to help elute the protein of interest
  • solvent drips out fraction by fraction
  • solvent can be evaporated and compounds of interest kept
  • can also collect nucleic acids
36
Q

ion-exchange chromatography

A

the column are coated w charged substances so they attract or bind compounds that have an opposite charge

a pos column will attract and hold a neg protein as it passes through the column either inc its retention time or retaining it completely

after all other compounds have moved through column a salt gradient is used to elite the charged molecules that have stuck to the column

37
Q

size-exclusion chromatography

A
  • porous beads
  • small compounds enter the beads thus slowing them down
  • large compounds move through the beads quickly
38
Q

affinity chromatography

A
  • customize columns to bind any protein of interest by creating a column w high affinity for that protein
  • accomplished by coating beads w a receptor that binds the protein or a specific antibody to the protein; protein retained in column
  • nickel and histidine tags
  • once protein retained it can be elites w free receptor, target, or antibody
  • can be different to remove substance from eluent
39
Q

x-ray crystallography

A
  • measure electron density on an extremely high resolution scale and can be used for nucleic acids
  • x-ray diffraction pattern is generated in this method
40
Q

Edman degradation

A
  • amino acids composition
  • sequential digestion of protein w specific cleavage enzymes is used
  • sequences up to 50-70 amino acids
  • selectively and sequentially removes N-terminal amino acids of protein, which can be analyzed via MS
  • synthetic reagent cleaves at specific amino acid residues
  • positions cannot be measure using this method bc of disulfide and salt bridges being broken
41
Q

activity analysis

A
  • rxns w color change have particular applicability bc microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest
42
Q

concentration determination

A
  • spectroscopy
  • proteins have aromatic side chains and thus can be analyzed w UV spectroscopy w/o treatment (sensitive to contaminants)
  • also cause colorimetric changes w specific rxns, particularly BCA, Lowry, and Bradford assays
43
Q

Bradford Protein Assay

A
  • mixes a protein in solution w Coomassie Brilliant Blue dye
  • dye is protonated and green-brown in color prior to mixing w proteins
  • dye gives up protons upon binding to aa groups turning blue in the process
  • ionic attractions between dye and protein stabilize this form of dye; thus inc protein concentrations are reacted w the Bradford reagent and then absorbance is measured to create a standard curve
  • the unknown sample is then exposed to the same conditions and concentration is determined based on standard curve
  • very accurate when only one type of protein is present in solution bc of variable binding of Coomassie dye
  • this assay is limited by the presence of detergent in the sample or by XS buffer

MCAT Biochem (12 decks)

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