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Proteins and Enzymes- Emily > Protein Chemistry > Flashcards

Protein Chemistry Flashcards

1
Q

What happens when pH > pKa ?

A

Unprotonated is favoured. Deprotonation occurs as pH increases.

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2
Q

Which amino acids carry a charge at neutral pH?

A

Lysine, arginine, aspartic acid, glutamic acid and histidine (+0.5).

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3
Q

What are the characteristics of a peptide bond?

A

Planar and has partial double bond characteristics.

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4
Q

What are the features of alpha helices?

A

3.6 residues per turn.Angle pairs -50 to -60 degrees.Stabilised by intrahelical hydrogen bonding between the nth residue and the (n+4)th residue.

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5
Q

What are the features of beta sheets?

A

Angle pairs +180 degrees Hydrogen bonding is interstrand.The strands can either be parallel or antiparallel.

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6
Q

What are loop regions?

A

They link regions of secondary structure. The angle pairs fall within the allowed regions of a Ramachandran plot.

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7
Q

What bonds stabilise the tertiary and quaternary structure?

A

Disulphides bonds, electrostatic interactions, ionic interactions, and salt bridges.

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8
Q

How and where do disulfide bonds form?

A

In an oxidation reaction between two cysteine residues. Formed in the oxidising environment of the ER lumen, producing cystine.

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9
Q

What is a protein motif?

A

The ordered arrangement of secondary structure elements and are supersecondary structures. Often have a specific function and are conserved structures.

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10
Q

What is the helix-loop-helix motif?

A

The simplest type of motif with a specific function. From C terminus to N terminus- helix-loop-helix.

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11
Q

What is the specific function of the helix-loop-helix motif?

A

DNA binding.

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12
Q

What is the specific function of the EF hand motif?

A

Calcium ion binding, where the loop region binds the ion and the helix regions are involved in coordinating the metal.

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13
Q

What is the beta-alpha-beta helix and where is it found?

A

N-C: Beta sheet, connected by a loop to an alpha helix, connected by a loop to a beta sheet. Found in almost every structure that contains parallel beta sheets.

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14
Q

What is the alpha/beta barrel motif?

A

An 8 stranded barrel with beta strands in the centre and alpha helices on the outside.

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15
Q

Give an example of an enzyme containing an alpha/beta barrel.

A

Triosephosphate isomerase.

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16
Q

What is the leucine-rich repeat domain? Give an example of an enzyme where it is found.

A

An alpha/beta horseshoe fold with the alpha helices exposed on the outside and connected to the beta strand by loops. Found in a ribonuclease inhibitor.

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17
Q

How are proteins released from mammalian cells?

A

Using centrifugation at different g forces to separate different cell components. The suspension must be put on ice as the process produces heat which denatures proteins and activates proteases to degrade proteins.

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18
Q

How are proteins released from E.coli?

A

Using a sonicator to burst the cells, as the membranes are less robust than in mammalian cells. Freeze-thaw cycles can be used to cause osmotic shock in small volumes of E.coli.

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19
Q

How are proteins released from yeast?

A

Using a french press, as the cells have a cell wall which is more robust than a cell membrane. The system must be kept cool as the high pressure used would increase temperature, denaturing proteins and activating proteases.

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20
Q

How does a french press burst cells?

A

The cell suspension is forced through a tiny nozzle at high speed and high pressure to burst the cells. Cells then hit a metallic target at high speed to burst any remaining intact cells.

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21
Q

What is gel filtration chromatography?

A
  • Separates according to size. - the column is packed with porous beads - small molecules can pass through pores in beads so move through the column more slowly- large molecules pass through the spaces within the matrix so pass through the column more quickly and are eluted first
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22
Q

Why are recoveries from gel filtration chromatography high?

A

There are no interactions between the sample and the column.

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23
Q

What is high performance liquid chromatography?

A
  • HPLC allows high resolution separation of proteins. - uses very high pressure to force the proteins through the column.- separates proteins by size.
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24
Q

What is ion exchange chromatography?

A
  • separates proteins according to their charge and isoelectric point.- groups attached to beads in the column carry the charge. - polyanion (CM) is used for cationic exchange - polycation (DEAE) is used for anionic exchange
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25
Q

What are the limitations of ion exchange chromatography?

A

Proteins can have similar sizes and charges.

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26
Q

What is affinity chromatography?

A
  • based on the affinity of the protein of interest and another factor immobilised on the column.- can add His tag to the protein terminus, protein binds to Nickel ions on beads, adding a low concentration of imidazole removes low affinity binding proteins, adding a high concentration of imidazole removes high affinity binding proteins.
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27
Q

What are the limitations of affinity chromatography?

A

The specificity means that it only works for one protein.

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28
Q

What is a homogenous sample?

A

A pure sample containing only one protein species.

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29
Q

What is the isoelectric point?

A

The pH where the amino acid is a zwitterion and its R group is neutral.

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30
Q

What is SDS-PAGE used for?

A

Monitoring the progress of a purification procedure. To check the purity of the final product. To give a rough idea of the molecular weight of a protein (using molecular weight markers).

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31
Q

What is the SDS used for in SDS-PAGE?

A

To denature proteins, forming nascent proteins- by disrupting hydrogen bonds and hydrophobic interactions.

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32
Q

What are β-mercaptoethanol and DTT? Why would they be used in SDS-PAGE?

A

These chemicals are reductancts which are used in SDS-PAGE to break any disulfide bonds in the proteins.

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33
Q

What could multiple bands on an SDS-PAGE gel suggest?

A

They could be due to contamination or multiple subunits.

34
Q

Name two stains that could be used to visualise proteins on an SDS-PAGE gel?

A

Coomassie blue stain and silver stain.

35
Q

How does Coomassie blue stain proteins?

A

Binds to lys residues in a protein, giving a blue band in the gel.

36
Q

How does silver stain proteins?

A

Silver ions bind to the negative R groups in Asp and Glu residues, the sulfhydryl group in Cys and the imidazole ring in His.

37
Q

When is a western blot used?

A

To detect proteins that have been separated by SDS-PAGE.

38
Q

Explain the western blot technique.

A

Transfer from gel to nitrocellulose. Probe with an antibody specific to the protein of interest. Probe with a second antibody which binds to the first antibody and is linked to an enzyme. Add an enzyme-substrate chemiluminescent signal or a fluorescent signal.

39
Q

What is isoelectric focussing?

A

Separation of proteins according to their charge, through a gel.

40
Q

Explain the isoelectric focussing technique.

A

An ampholyte solution is incorporated into a gel. A stable pH gradient is established, using an electric field. The protein solution is added and the electric field is reapplied. After staining, proteins are shown to be distributed along the pH gradient according to their isoelectric points.

41
Q

What are the limitations of isoelectric focussing?

A

It is difficult to separate proteins with similar isoelectric points.

42
Q

What is 2D gel electrophoresis used for?

A

To separate proteins by both charge and mass.

43
Q

What is the relationship between mobility and charge/mass in 2D gel electrophoresis?

A

Mobility is directly proportional to charge. Mobility is inversely proportional to mass.

44
Q

What is 2D gel electrophoresis made up of?

A

An isoelectric focussing gel placed on an Polyacrylamide gel for SDS-PAGE. Splitting first by isoelectric point and then by mass.

45
Q

What does the extinction coefficient define?

A

How strongly a substance absorbs light at a given wavelength.

46
Q

Why is the extinction coefficient different in folded and unfolded proteins?

A

Due to the accessibility of Tyr/trp residues, which would be more accessible in an unfolded protein.

47
Q

What is a Lowry assay?

A

Peptide bonds chelate copper (II) ions and reduced to copper (I) ions, this reduces phosphomoylbdic-tungstic acid in the Lowry reagent. This gives a distinctive blue colour, where the darkness of the colour is proportional to the number of peptide bonds present in the sample. Quantified by measuring absorbance at 760nm.

48
Q

How is the amino acid composition of a protein detected?

A

1) hydrolysis of the protein into its constituent amino acids.
2) separation of the amino acids in the mixture.
3) quantification of the individual amino acids.

49
Q

How is a protein hydrolysed?

A

Protein dissolved in 6M HCl in evacuated ampoule and heated to 110 degrees for 24 hours. The sample is then separated into its constituent amino acids using a cation exchange column.

50
Q

How are the constituent amino acids of a protein separated?

A

Using a cation exchange resin with sulfonated polystyrenes in the column. The order of elution is; negatively charged residues, hydrophobic residues, positively charged residues.

51
Q

How are the constituent amino acids of a protein detected?

A

Using ninhydrin, which binds to the amino acid, producing a purple/blue compound. The absorbance of the compound produced at each elution peak is proportional to the amino acid concentration.
OR
Amino acids are reacted with a fluorescent compound to give a fluorescent complex- more sensitive method.

52
Q

What are the limitations of the method for determining amino acid constituents?

A

Some amino acids cannot react with the compounds used for detection. Some amino acids are destroyed by the harsh hydrolysis method. Trp must be detected using a separate method. Ser, thr, Tyr tend to get degraded so need to be protected using a phenol or a thiol.

53
Q

How are disulfide bonds cleaved?

A

By reductive acetylation.

1) reduction by mercaptoethanol in an excess of thiol, reduction leaves free SH groups.
2) acetylation using excess iodoacetic acid prevents reoxidisation of the -SH groups

54
Q

Where does trypsin cleave?

A

Cleaves the protein after a lysine or arginine residue.

55
Q

Where does chymotrypsin cleave?

A

Cleaves after tyrosine, tryptophan, phenylalanine and leucine residues.

56
Q

How does cyanogen bromide cleave peptides?

A

Cleaves at the C terminal side of Met by converting methionine to homoserine lactone.

57
Q

Why are fewer fragments produced from cyanogen bromide cleavage than from enzymatic methods?

A

Only one codon corresponds to Met so there is a low frequency of it in proteins.

58
Q

How can 2D gel electrophoresis be used to separate fragments?

A

By using different cleavage methods to ensure that the different size fragments are of different charges.

59
Q

What are the 3 stages of mass spectrometry?

A

1) protein sample is ionised
2) electrical field accelerates ions through the flight tube
3) lightest ions arrive at the detector first
- the time taken for fragments to travel through the flight tube depends on their molecular weight.

60
Q

What are the limitations of using mass spectrometry to analyse proteins?

A

Some proteins do not travel through the flight tube very well.

61
Q

When is N-terminus sequencing used?

A

If mass spectrometry is not suitable for the analysis of a protein.

62
Q

How does Edman degradation work?

A

Specific removal of the N-terminal amino acid. Identification of the amino acid derivative that is formed in the reaction. Another cycle occurs to remove the next N-terminal amino acid until the C-terminus of the peptide is reached.

63
Q

What are the limitations of Edman degradation?

A

Low efficiency.
Proteins must be cleaved into smaller fragments before sequencing.
Post-translational modifications affect Edman degradation.
N-terminal amino group must be available to react with the PITC.

64
Q

How does glycosylation affect Edman degradation?

A

Means that the PTH derivative is unstable and harder to detect using HPLC.

65
Q

How does acetylation affect Edman degradation?

A

This usually occurs at the N-terminus of a peptide, therefore a protein must be deacetylated before Edman degradation.

66
Q

How does phosphorylation affect Edman degradation?

A

The derivative must be converted into a different form.

67
Q

How do disulfide bonds affect Edman degradation?

A
  • can produce an unclear signal if two polypeptide chains are joined by a disulfide bridge as there would be 2 N termini.
  • disulfide bonds in the same polypeptide chain must be broken before Edman degradation otherwise the derivative will not be released.
68
Q

When is C terminal sequencing used?

A

To check if the C-terminus is intact.

69
Q

What methods are used in C-terminal sequencing?

A

Hydrazinolysis, repetitive degradation and carboxypeptidase enzyme method.

70
Q

What does NMR rely on?

A

The magnetic properties of atomic nuclei which are incorporated into a protein during production. Relies on the absorption of energy to indicate the difference in energy between two states.

71
Q

What happens if the energy difference between energy states is low in NMR?

A

High [P] is used, as if a small concentration was used it would be hard to detect the difference. The same energy difference is given, it is just easier to detect when more protein is used.

72
Q

How can the difference in energy be measured using resonance in NMR?

A

The sample is irradiated by radiation of a frequency that matches the energy difference.

73
Q

How does NMR work?

A

Energy differences give information about the chemical surroundings of different chemical nuclei. Flow of electrons around a magnetic field nucleus generates a small local magnetic field opposing an applied field. Nuclei in different environments will resonate at slightly different radio frequencies.

74
Q

How can NMR be used to detect protein structure?

A

Nuclei in the protein core will resonate at different radio frequencies to exposed nuclei. Can detect 3D structure if information about how spin nuclei affect neighbouring nuclei.

75
Q

What state are proteins in prior to NMR?

A

NMR measurements are taken for many protein molecules, so that information is recorded for all conformational states of a protein.

76
Q

What is the limitation of using NMR to detect protein structure?

A

It is only suitable for proteins up to 50kDa.

77
Q

What is X-Ray Crystallography?

A

Best method for obtaining high resolution structural data. That relies on the fact that X-Rays have about the same wavelength as the length of covalent bonds, meaning that proteins diffract X-Rays.

78
Q

What are the limitations of X-Ray crystallography?

A

High amounts of protein are required as need to screen for optimum crystallisation conditions. Difficult to obtain protein crystals. It produces a single snapshot from a structure- cannot show all conformational states of a protein.

79
Q

How does X-Ray crystallography work?

A

Electrons in the protein scatter the X-Ray. The amplitude of a wave scattered by an atom is proportional to the number of electrons. Each molecule in the crystal adds to the signal produced. All protein molecules in the crystal must be in the same conformation.

80
Q

Why is there a dense ice ring in the centre of a diffraction pattern from X-Ray crystallography?

A

Crystals are frozen before X-Ray crystallography.

81
Q

How are loop regions stabilised?

A

By the aqueous environment- as the loops contain polar/uncharged residues.

82
Q

How can you tell if a purification method is effective?

A

Purify a protein using multiple purification methods, and measure the specific activity after each purification. The method that causes the biggest increase in specific activity is the most effective.

Proteins and Enzymes- Emily (14 decks)

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