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Biological Chemistry I > Chapter 5: Proteins, Primary Structure > Flashcards

Chapter 5: Proteins, Primary Structure Flashcards

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1
Q

One of the keys to deciphering the function of a given protein is to?

A

understand its structure

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2
Q

what makes proteins different from nucleic acids?

A

proteins don’t have uniform, regular structures because of the 20 different amino acids with different chemical and physical properties

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3
Q

what is a polypeptide

A

multiple amino acids attached to each other via peptide/covalent bonds/disulfide bonds

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4
Q

largest known polypeptide ?

A

titin, helps arrange the repeating structures of muscle fibers

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5
Q

what is the primary structure of a protein ?

A

the sequence of the amino acids
the first stage of protein formation
simplest form
determines final structure
generally non functional except for insulin

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6
Q

at least how many residues make a polypeptide chain

A

40 with the vast majority containing between 100-1000 residues
average is 355

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7
Q

what’s unique about insulin

A

has 2 polypeptide chains (A and B)
an additional disulfide is formed within the A chain.
disulfide bonds between cysteine
only 51 a.a.

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8
Q

how does size affect a proteins optimization of biochemical processes?

A

Forty residues appears to be near the minimum for a polypeptide chain to fold into a discrete and stable shape that allows it to carry out a particular function.
-Polypeptides with well over 1000 residues may approach the limits of efficiency of the protein synthetic machinery. The longer the polypeptide(and the longer its corresponding mRNA), the greater the likelihood of introducing errors during transcription and translation.

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9
Q

The characteristics of an individual protein depend more on its _____than on its amino acid composition

A

amino acid sequence

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10
Q

the first step in understanding protein function?

A

protein purification

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11
Q

purifying a protein helps identify what?

A

the amino acid sequence and structure

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12
Q

what conditions at all stages of the purification process must be controlled to keep a protein stable and free from damage?

A

-pH
-temperature
-presence of dergadative
-adsorption to surfaces
-long term storage
Once a protein has been removed from its natural environment, it becomes exposed to many agents that can irreversibly damage it. These influences must be carefully controlled at all stages of a purification process.

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13
Q

what is used to identify a target protein at every stage of the purification process to make sure you’re isolating the right protein?

A

ASSAY

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14
Q

assays must be?

A

specific for the target protein, highly sensitive, and convenient to use

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15
Q

Determines whether the protein is present/a means of identifying a protein based on a unique property of the protein?

A

an assay

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16
Q

2 types of assays?

A

-enzyme assays
-immunoassays (ELISA replaced by RIA)

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17
Q

what is ELISA

A

an enzyme-link immunosorbent assay that use antibodies to detect the presence of antigens

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18
Q

first two steps of protein purification

A

Ist step - get it out of the cell and into the solution
2nd step- keep pH, temperature, and othe conditions controlled
2nd step- make sure protein is present through assays
3rd step- find out fractionation purification process that is dependent on protein characteristic

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19
Q

Among the most straightforward protein assays are those for enzymes that catalyze reactions with readily detected products, because

A

the rate of product formation is proportional to the amount of enzyme present.

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20
Q

what is used to assist in detecting product in enzyme proteins

A

Substances with colored or fluorescent products

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21
Q

a coupled enzymatic reaction.

A

If no such substance is available for the enzyme being assayed, the product of the enzymatic reaction may be converted, by the action of another enzyme, to an easily quantified substance.

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22
Q

Describe the steps of ELISA

A
  1. attachment of antibody to polystyrene plate
    2.the solution containing protein is applied/binded to the antibody
  2. a second protein-antibody complex with the addition of an enzyme is attached to the existing protein-antibody complex
    4.the amount of substrate converted to product indicates the amount of protein present/ the enzyme converts substrate to produce colored product
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23
Q

during ELISA the intensity if color corresponds to the amount of?

A

compound in a given sample

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24
Q

which process is more sensitive Immunoassays or enzyme assay

A

immunoassays

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25
Q

what is RIA

A

radioimmunoassay, A protein in a complex mixture can be detected by its binding to its corresponding antibodies.the protein is indirectly detected by determining the degree to which it competes with a radioactively labeled standard for binding to the antibody.

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26
Q

The concentration of a substance in solution can be measured by?/Protein Concentrations Can Be Determined by

A

absorbance spectroscopy

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27
Q

what is absorbance spectroscopy’s and its equation

A

A solution containing a solute that absorbs light does so according to the BeerLambert law,
A=log(I 0/ I)=εcl
-I 0 is the intensity of the incident light at a given wavelength λ,
-I is its transmitted intensity at λ,
-ε is the absorptivity (alternatively, the extinction coefficient) of the solute at λ,
-c is its concentration
-l is the length of the light path in centimeters.

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28
Q

polypeptides absorb strongly in what region and why?

A

UV around wavelength 200-400 usually at 280 nm
because their aromatic side chains (those of Phe, Trp, and Tyr) have particularly large extinction coefficients in this spectral region

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29
Q

polypeptides do not absorb what? do they have color

A

visible light around wavelength 400-800 nm
are colorless
Nevertheless, if a protein has a chromophore that absorbs in the visible region of the spectrum, this absorbance can be used to assay for the presence of the protein in a mixture of other proteins.

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30
Q

what about proteins is used to separate them over other proteins?

A

their properties

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31
Q

what helps simplify the selection of fractionation procedures?

A

knowing something about the target protein or the proteins it can be separate them

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32
Q

advantages and disadvantages of detecting proteins near 280 UV?

A

Advantage: This method will give you the absolute concentration of your protein; the colorimetric methods described further down will give you concentrations relative to a standard protein.
Disadvantage: to quantify the specificities protein, the protein needs to be pure. Your protein solution has to be free of other compounds absorbing radiation at 280 nm., can only have that one protein and no contamination from other proteins

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33
Q

why do the aromatics have absorbance in the UV region 280?

A

because they have a conjugation of pie bonds and its aromaticity

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34
Q

which of the three aromatics have an insignificant/negligent absorbance?

A

phenylalanine

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35
Q

what are the 3 aromatics

A

tryptophan and tyrosine

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36
Q

which aromatic has the most absorbance?

A

Tryptophan

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37
Q

when dealing with a protein that has tryptophan and tyrosine

A

you can quantify it by measuring the absorbance at 280
A=280, when they’re present the absorbance is larger

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38
Q

what to know about spectrophotometric that has far UV? advantages and disadvantages ?!

A

extinction coefficient e at 205 nm:
~ 3,000 M-1cm-1 per peptide bond
Advantage: Independent of the amino acid composition of your protein.
Disadvantage: Many other compounds absorb at 205 nm, including most buffers. It would be best to measure in pure water.

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39
Q

what is a biuret test?

A

a chemical test used for detecting the presence of a peptide bond
-another form of protein concentration determination

40
Q

how is biuret obtained?

A

by heating 2 moles of urea at 180 degrees Celsius

41
Q

how to know if protein is present during biuret test

A

if solution turns purple

42
Q

proteins are purified by what kind one procedures? what is its purpose

A

fractionation procedure
not necessarily to minimize the loss of the desired protein, but to eliminate selectively the other components of the mixture so that only the required substance remains.

43
Q

what two assays stem from the biuret assay?

A

lowry and bca

44
Q

difference between lowry/bca assay from biuret assay

A

lowry and bca assay are very sensitive detecting low concentrations unlike the briquet assay that detects high concentration

45
Q

difference between lowry and bca assay

A

-BCA: Step 1 is biuret asay
Step 2 is 2 bca reagents
Violet color
-In a similar fashion to the BCA assay the Lowry assay relies on the reaction of copper ions, produced from the interaction of copper(II) sulfate with proteins present in the sample, with a further reagent. In this case Folin–Ciocalteu reagent with the sample becoming blue in colour when higher levels of protein are present.

46
Q

What makes the lowry and bca assay more sensitive than biuret?

A

hat the formed BCA/copper complex absorb light much more strongly than those generated by the biuret assay alone greatly increasing the assays sensitivity

47
Q

what reagent and color does lowry assay use

A

Folin–Ciocalteu reagent

48
Q

what is the Bradford assay

A

Based on dye coomassie brilliant blue that is brown, binds to protein,
then turns blue
absorbs light at 595 nm
this is slightly less sensitive than bca but simple and faster to perform

49
Q

what are the 5 protein characteristics used to determine the purification procedure?

A

solubility, ionic charge, polarity, size, and binding specifity

50
Q

the purpose of purification

A

The idea is not necessarily to minimize the loss of the desired protein, but to eliminate selectively the other components of the mixture so that only the required substance remains.
-intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms.

51
Q

what is a protein assay purpose

A

The purpose of the protein assay is to determine the amount or concentration of a specific protein or an array of different proteins in a sample.

52
Q

what are the 5 protein characteristics that determine the purification procedure?

A

solubility, ionic chargwe, polarity, size, and binding specificity

53
Q

what separates protein by their solubility

A

salting out

54
Q

describe the salting out process

A

usually protein molecules are surrounded by water molecules which keeps them soluble. The layer of water molecules around the protein is known as the hydration layer. this layer keeps the protein in the soluble form. The ddition of ammonium sulfate removes the hydration layer around the protein. As the protein fails to interact with water molecules the protein now precipitates. Once the protein is precipitated it is dissolved in appropriate buffer and further purified by various chromatography technique

55
Q

saltin in vs salting out

A

More salt concentration= decrease in solubility salting out
Less salt concentration=increase in solubility salting in

56
Q

the most commonly used reagent for salting out? Why?

A

Ammonium sulfate, (NH4)2SO4,because its high solubility (3.9 M in water at 0°C) allows the preparation of solutions with high ionic strength.

57
Q

Where are proteins least soluble?

A

When at their isoelectric point
The pH may be adjusted to approximate the isoelectric point (pI) of the desired protein because a protein is least soluble when its net charge is zero.

58
Q

What is ion exchange chromatography

A

In ion exchange chromatography, charged molecules bind to oppositely charged groups that are chemically linked to a matrix such as cellulose or agarose.
-separation of proteins based on charge

59
Q

anions bind to? cations bind to?

A

Anions bind to cationic groups on anion exchangers, and cations bind to anionic groups on cation exchangers

60
Q

what is hydrophobic interaction chromatography and its other name

A

technique to separate and purify proteins depending on their polarity/surface hydrophobicity
-In a nutshell, HIC (also known as ‘salting out’) separates protein molecules using the properties of hydrophobicity. In this method, proteins containing both hydrophilic and hydrophobic regions are applied to an HIC column under high salt buffer conditions.
-most hydrophobic content interacts with the column the best, least hydrophobic components elute first, hydrophobia contents remain in comulm
The salt in the buffer (usually ammonium sulfate) reduces the solvation of sample solutes and exposes the hydrophobic regions along the surface of the protein molecule. This facilitates the adsorption of these hydrophobic regions to the hydrophobic areas on the solid support and precipitates (crystallizes) proteins out of the solution.
-Samples should be in a high salt concentration to promote hydrophobic interaction.
-reverse phase chromatography

61
Q

Gel Filtration Chromatography Separates Molecules According to?

A

size and shape

62
Q

during Gel Filtration Chromatography do the large or small molecules elute first and why?

A

the large elute first because the small molecules are so small they enter the beads slowing down their path whereas the large molecules don’t enter the beads and leave quicker

63
Q

Affinity Chromatography?

A

is based on specific binding interactions using ligand
beads are made with the specific chemical attached and the protein with affinity for the attached group will be retained

64
Q

What is His-Tag

A

A stretch of 6-10 consecutive histidine residues will bind to nickel (Ni2+) or cobalt (Co2+) ions. The His tag (His6-10) is usually inserted at the N- or C- terminus of a protein. The chromatography column contains immobilized Ni2+ or Co2+ ions. As there are no (or very few) natural proteins which carry 6 or more consecutive His ions, this method is highly specific.

65
Q

How do you elute a His-tagged protein from the Ni- (or Co-) column?

A

add the solution of imidazole

66
Q

what is electrophoresis?

A

the migration of ions in an electric field

67
Q

sds-page separates proteins according to their?

A

mass/weight

68
Q

how does sds-page work?

A

SDS denatures(unfolds) the protein and 1 molecule of SDS binds for every 2 amino acids
uses polyacrylamide gel

69
Q

how is sds used?

A

its a detergent added to denature proteins

70
Q

what does PAGE do

A

Polyacrylamide gel electrophoresis (PAGE) of proteins is typically carried out in polyacrylamide gels with a characteristic pore size, so the molecular separations are based on gel filtration (size and shape) as well as electrophoretic mobility (electric charge). However, electrophoresis differs from gel filtration in that the electrophoretic mobility of smaller molecules is greater than the mobility of larger molecules with the same charge density. The pH of the gel is high enough (usually about pH 9) so that nearly all proteins have net negative charges and move toward the positive electrode when the current is switched on. Molecules of similar size and charge move as a band through the gel.

71
Q

what is capillary electrophesis and why is it useful

A

Capillary Electrophoresis Rapidly Separates Charged Molecules.a technique in which electrophoresis is carried out in very thin capillary tubes

72
Q

what is an isoelectric point

A

A protein has charged groups of both polarities
immobile in an electric field

72
Q

what is an isoelectric point

A

A protein has charged groups of both polarities
immobile in an electric field

73
Q

Isoelectric Focusing and 2D gel electrophoresis

A

Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01. Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI value and then further separated by molecular weight through SDS-PAGE.
Two-dimensional gel electrophoresis or 2D-PAGE is the primary technique for proteomics work. It separates the complex mixture of samples using two different properties of the proteins. In the first dimension, proteins are separated by the pI value and in the second dimension by the relative molecular weight.

74
Q

2d dimensional électrophoresis does what

A

Resolves Complex Mixtures of Protein

isoelectric focusing (IEF), which separates proteins in the first dimension according to their isoelectric point, followed by electrophoresis in a second dimension in the presence of sodium dodecyl sulfate (SDS), which separates protein

75
Q

what does ultracentrifugation do

A

separates Macromolecules by mass

76
Q

Purpose of ultracentrifugation

A

a high-speed centrifuge able to separate out colloidal and other small particles and used especially in determining the sizes of such particles or the molecular weights of large molecules
Ultracentrifugation is a specialized technique used to spin samples at exceptionally high speeds. method of separating molecules having different densities

77
Q

The rate at which a particle sediments in the ultracentrifuge is related to?

A

Its mass

78
Q

the first step of protein sequencing and why?

A

identify and separate subunits/ determining the number of polypeptide chains

79
Q

The primary structures of a given protein from related species closely .

A

resemble one another

80
Q

Sequence Comparisons Provide Information on ?

A

Protein Structure and Function.In general, comparisons of the primary structures of homologous proteins (evolutionarily related proteins) indicate which of the protein’s residues are essential to its function, which are less significant, and which have little specific function

81
Q

phylogenetic tree?

A

The amino acid or DNA sequences of homologous proteins can be analyzed by computer to construct a phylogenetic tree, a diagram that indicates the ancestral relationships among organisms that produce the protein.

82
Q

proteins with similar functions have similar

A

sequences

83
Q

Protein Families Can Arise through

A

Gene Duplication

84
Q

the process of protein sequencing?

A
  1. separate the subunits/polypeptide chains by breaking the disulfide bond
  2. break the individual subunits into smaller peptides
    3.determine the sequence
    4.Use the two sets of overlapping peptide sequences to reconstruct the sequence of each polypeptide.
85
Q

disulfide bonds are cleaved using what?

A

2-mercaptoethanol or another mercaptan

86
Q

what compound prevents the reformation of disulfide bonds

A

iodoacetate

87
Q

Polypeptide chains can be cut into smaller pieces by proteases. After which amino acids does the protease trypsin cut?

A

arginine and lysine

88
Q

which enzyme(protease) is the most useful in cleaving the polypeptide

A

trypsin

89
Q

what procedures determine the amino acid sequence

A

Edman Degradation and mass spectrometry

90
Q

what is Edman degradation

A

determines the amino acid sequence by Removing a Peptide’s N-Terminal Amino Acid Residue

91
Q

what is Edman degradation

A

determines the amino acid sequence by Removing a Peptide’s N-Terminal Amino Acid Residue

92
Q

how does mass spectrometry determine AA sequence

A

Mass spectrometry can identify amino acid sequences from the mass-to-charge ratios of gas-phase protein fragments
electrospray ionization (ESI)
also identifies a protein

93
Q

where are protein sequences stored

A

databases

94
Q

What are some advantages of sequencing peptides by mass spectrometry rather than by Edman degradation?

A

Mass spectrometry can be used to sequence peptides with chemically blocked N-termini (which prevents Edman degradation)

95
Q

Explain why polypeptides have such variable sequences?

A

Because there are 20 different amino acids. For a protein with n residues, there are 20^n possible sequences. Even a small polypeptide has an enormous amount of possibilities.

Biological Chemistry I (12 decks)

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