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Life Science 3 > L3: Protein Function and Techniques > Flashcards

L3: Protein Function and Techniques Flashcards

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1
Q

3 types of Protein Modifications

A
  1. Disulfide-bond formation
  2. Group Addition
    • protein phosphorylation
    • protein glycosylation
    • protein ubiquitination
  3. Protein modification
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2
Q

How do proteins work to regulate cell functions?

A

By binding to other molecules (ligands or substrates), small molecules (ions or sugars), or large molecules (proteins, DNA, and RNA)

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3
Q

A protein’s function determines its ____

A

function:

  • structural proteins
  • enzymes
  • transcription factors
  • signal transduction proteins
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4
Q

Proteins with similar function can be grouped together called _____

A

Protein families

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5
Q

Protein Families

A

Members of the same protein family that usually have similar structures

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6
Q

Members of protein family have similar _____ and _____

A

structure and function

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7
Q

Key cell regulators

A

Enzymes

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8
Q

Suffix “-ase”is usually appended to its name

A

Enzymes

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9
Q

4 examples of enzymes

A
  1. Protease———–degrade protein
  2. Kinase————–add phosphate group
  3. Phosphatase—–remove phosphate group
  4. Ribonuclease—–RNase: degrade RNA
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10
Q

Proteins that catalyze cellular reactions

A

Enzymes

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11
Q

Substrates Products

A

tadahhhhh

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12
Q

Enzymes

A
  • converts substrate to product without changing itself
  • speed up reactions by factors of 10^6 or more at relatively low temperature
  • highly specific and function by lowering the activation energy of the reaction
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13
Q

How is enzymatic activity measured?

A

Through Vmax and Km

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14
Q

Vmax

A
  • maximum rate of reaction

- the bigger the Vmax, the more active the enzyme

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15
Q

Km

A
  • substrate concentration of 1/2Vmax

- the smaller the Km, the higher the enzyme substrate affinity (better the enzyme)

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16
Q

Antibodies as Molecular Tools

A
  • produced in response to the injection of foreign materials (Antigens)
  • bind tightly to specific antigens with > 10^-9 M affinity
  • can distinguish among proteins that differ by only a single amino acid
  • can be used to determine if a particular protein is present in a complex mixture of proteins
  • can be used to purify a particular protein away from the other components of a complex mixture
  • can be used to neutralize pathogens
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17
Q

How do we study proteins in living systems?

A
  1. Separation
    • separating different cellular components and proteins
    • centrifugation, chromatography, SDS-PAGE
  2. Detection (immunological techniques)
    • detecting a single protein from a mixture of proteins
    • ELISA, Immunoblot, Immunoprecipitation
  3. Sequence and Structural analysis
    • Mass spectometry, X-ray crystallography
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18
Q

Protein purification

A

to study structure and properties of proteins in interest, it often must be isolated in a pure form

19
Q

4 steps of Protein separation/purification

A
  1. break-up of cells
    • physical disruption, Detergent
  2. Centrifugations
    • differential centrifugation
    • rate-zonal centrifugation
  3. Chromatography
    • ion exchange
    • gel filtration
    • affinity column
  4. Gel Electrophoresis
    • SDS-PAGE
    • 2D gel
20
Q

Breaking up cells and tissues

A
  1. ULTRASOUND
    • break cells with high frequency sound
  2. DETERGENT
    • use mild detergent to make holes in plasma membrane
  3. FRENCH PRESS
    • force cells into small hole using high pressure
  4. HOMOGENIZE
    • when carefully applied, homogenization leave most of the membrane-bounded organelles intact
21
Q

Differential Centrifugation

A
  • particles that sediment rapidly are separated from slower sedimenting materials that remain in the supernatant (using centrifugal force)
  • rapidly sedimenting material is collected in the pellet
  • separates a mixture of particles that differ greatly in size
22
Q

Rate-zonal or velocity centrifugation

A
  • sucrose gradient is used to provide density stability during centrifugation
  • particles of similar density sediment based primarily on mass and shape
  • provides a finer degree of separation with sucrose density gradient
23
Q

Column Chromatography

A
  • cylindrical columns filled with water permeable solid of different properties
  • different properties behave different in different columns –> allows separation
24
Q

Column Chromatography (types)

A
  1. Ion exchange chromatography
    • separate according to charge
  2. Gel filtration chromatography (or sizing column)
    • separate according to size
  3. Affinity chromatography
    • separate according to ability to bind to ligands (affinity)
25
Q

Ion exchange chromatography

A
  • separate molecules according to charge
  • (+) – come through column first –> (-) bind the beads and be removed later
  • (-) – opposite
26
Q

Gel Filtration Chromatography

A
  • separates molecules according to size
  • larger molecules migrate faster in gel filtration column
  • smaller molecules migrate slower
    • take longer to travel through pores of beads
27
Q

SDS-PAGE

A
  • sodium dodecyl sulfate polycrylamide gel electrophoresis
  • separated according to Molecular Weight on polyacrylamide gels
    • only separate proteins with large differences in Molecular Weight
28
Q

Immunoblot (Western blot)

A
  • can detect specific protein within a crude extract based on the ability of an antibody to selectively bind the protein of interest
29
Q

Immunoblot (Western blot) - 2

A
  1. separate protein in an SDS-PAGE gel
  2. Blot protein bands to a nitrocellulose membrane
  3. incubate membrane with Ab1 that recognizes the antigen. then Ab2 or protein A that recognizes Ab1
  4. visualize antigen by either radioactivity label of Ab2 or protein A, or a colorimetric reactions catalyzed by the marker enzyme conjugated to Ab2 or protein A
30
Q

Chromatin Immunoprecipitation

A

can detect protein association with DNA in cell

31
Q

Proteins can also be detected using _____ to specific proteins and secondary antibodies that have a _____ _____

A

antibodies, flourescent tag

32
Q

Amino acids in membrane are _____

A

hydrophilic

33
Q

Amino acids in cytoplasmic domain are _____ …….

A

positively charged and interact with negatively charged and interact with negatively charged head groups

34
Q

Isolation of membrane proteins require ……….

A

screening of detergents to find the optimal conditions to isolate proteins in tact from membrane

35
Q

Isoelectric focusing (IEF) gel electrophoresis

A
  • based on electric charge, which is determined by the numbers of acidic and basic residues but not the molecular weight of a protein
  • charge proteins will migrate through the pH gradient until they reach their pI (isoelectric point)–pH at which the net change of the protein is 0
  • 2 unrelated proteins having similar masses are likely to have identical charges
    • low pH = (+) protein
    • high pH = (-) protein
    • isoelectric point = protein has no net charge
      • -> no longer migrates in electric field
36
Q

2D gel electrophoresis

A
  • 1st dimension: Isoelectric focusing (IE) –> separation by charge
      • proteins migrate in a pH gradient until they rich a point where pH = isoelectric point (0 net charge) of protein
  • 2nd dimension: SDS-PAGE –> separation by size
    • -proteins migrate according to size
37
Q

3 Protein sequence analysis

A
  1. chemical method – Edman degradation
  2. mass spectrometry
  3. deduction from DNA sequence
38
Q

Edman degradation

A
  • only good fro small peptides and non-modified peptides
    1. react with phenylisothiocyanate on free amino group
    2. acid cleavage
    3. amino acid analysis
    4. repeat many times
39
Q

Mass spectrometry (MS)

A
  • fast and accurate way to measure molecular weight (MW)

- predicts ratio of mass to charge which determines MW

40
Q

Mass spectrometry (4 steps)

A
  1. laser desorption/ionisation
    • atom ionized by knocking one or more electrons off to give a (+) ion
    • MS always works with (+) ions
  2. acceleration
    • ions are accelerated so they all have the same kinetic energy
  3. deflection
    • ions are deflected by magnetic field according to their mass
    • lighter = more they are deflected
    • amount of deflection depends on number of (+) charges on ion (how many e- were knocked off in the 1st stage)
    • more ion is charged = more deflected
  4. detection
    • beam of ions passing through machine is detected electrically
41
Q

Proteomics

A
  • study of all proteins produces in cell or species under a given set of conditions
  • ex: 2D gel
    • electrophoresis coupled with MS to identify MW of protein
42
Q

X-ray Chrystallography

A
  • X-ray diffraction patters of protein crystals allow determination of protein structure to atomic resolution
  • yields an electron density map that illustrates the position and types of atoms
43
Q

Why study protein structure?

A
  • analysis of 3D structure of HLH type transcription factor helps in understanding hot protein dimer binds to DNA

Life Science 3 (9 decks)

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