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BMS Level 2 > Cell and Molecular bio > Flashcards

Cell and Molecular bio Flashcards

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1
Q

What is the structure of an alpha helix?

A

Secondary structure of a protein, in which every 3.6 AA’s a hydrogen bond forms between N-H group and C-O, creating a coil structure, turning every 3.6AA’s

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2
Q

What is the structure of a Beta sheet?

A

Hydrogen bonds between adjacent AA make up the backbone, with the AA side chains giving polarity. (aromatic AA normally found in the middle of the backbone)
The sheets are lined up either parallel or anti by hydrogen bonds between C-O and N-H groups on different backbones.

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3
Q

What are aromatic AA’s?

A

An amino acid that contains an aromatic ring e.g. Try, Phe, Trp, Val, Ile

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4
Q

Sequences at the C terminus and N terminus of proteins?

A

C terminus- COOH end

N terminus- NH2 start

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5
Q

MOst AA’s prefer a … secondary structure except for …. and …

A

alpha helix

Proline and glycine

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6
Q

Hydrogen bonds between what? How?

A

An H shared between O and N atoms, where O is negative and N positive, so where the electrode is on the right will attract the positive N (simplified)

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7
Q

Ionic/electrostatic bonding?

A

Attraction between positive and negative atoms.

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8
Q

Weak Van der Walls bonding?

A

Short range hydrophobic reactions, e.g. by polarity across atom, where electron one end and nucleus the other.

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9
Q

Disulphide bonding?

A

Chemical link between adjacent cystines, very strong even under heat

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10
Q

When folding a protein what side chains are folded inside and which are exposed? Why?

A

Hydrophobic hidden inside, and hydrophilic outside- they can form H bonds with the aqueous solution around.

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11
Q

An example of a quaternary structure?

A

2Alpha, and 2Bsubunits in haemoglobin.

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12
Q

What is tertiary structure of a protein?

A

The way the alpha helicies and beta sheets interract e.g. helix coil helix, random coil etc.

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13
Q

What is Edman degradation?

A

Add PITC to the polypeptide chain, and will bind to the terminal AA and cleave it off under acidic conditions. This AA bound forms a PTH complex, which can be run on gel electrophoresis or chromotography (HIgh performance Lipid Chromotography). The molecular weight can then be calculated by taking away the PITC. Then move onto the next AA etc, gradually sequencing

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14
Q

Drawback to the Edman Degradation method?

A

Polypeptides can’t be longer than around 30 residues to accuritely sequence, but proteins could be cleaved into smaller fragments.
Can’t if the N terminus is altered e.g. Acetylation

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15
Q

Word meaning molecule having both hydrophobic and hydrophilic parts?

A

Amphipathic

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16
Q

After found the primary sequence what next as prediction?

A

Can look for patterns and predict bonding to work out secondary structure, and even programmes online can submit the sequence of AA’s and it will find simialr proteins/ predict structures.

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17
Q

4 methods to determine a protein secondary structure accuritely?

A

CD- circular Dichroism
X-ray crystallography
Nuclear magnetic resonance
Electron Microscopy

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18
Q

What is Circular Dichroism? How?

A

Put the protein in solution and shine circular polarised light through to see the absorbtion.
Alha helices, B sheets and random coils all have characteristic shapes on the CD spectrum, so can show what percentage of protein is each but not arrangment.

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19
Q

Another use of Circular Dichroism?

A

After heating a protein and causing it to unfold, CD can be repeated at different temperatures to determine the stability of the structures by how easily they denature and if cool whether refold back.

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20
Q

3 advantages and one disadvantage to circular dichroism?

A

+Quick
+Cheap
+No limitation on size of protein
- Low resolution

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21
Q

Nuclear magnetic resonance use and method?

A

Used to understand protein dynamics.

Pure protein labelled with isotopes which are introduced into proteins by recomination in bacteria as are grown on 15N or 13C.
The spin of particles is pared so there is no spin of the nucleus, however due to uneven protons in isotope there is a slight wobble.
Depending on the local environment the protons resonate at different frequences (chemical shift)

22
Q

One advantage and three disadvantages of NMR Spectroscopy?

A
  • Very expensive
    + Good resolution 2A
  • Size limited to 50kDa
    -Quick but data analysis slow
23
Q

What is Xray Crystallography? Method?

A

First crystallise the pure protein, then an xray beam is fired at the protein. Most of the xray goes straight through but some is deflected giving a refraction pattern. From this the diffraction can be traced back to the protein structure.

24
Q

Two advantage and one disadvantage of Xray Crystallography?

A
  • Very expensive, set up £300million and £200 thousand every time used
    +crystalisation slow but analysis fast
    + HIgh resolution 1A of lower.
25
Q

Transmission electron Microscopy use? Method?

A

useful for large proteins structures.

  1. Put protein on screen and dry
  2. Add heavy metal (negative stain) e.g. lead
  3. View down EM.
26
Q

what is Cryo EM?

A

Liquid Nitrogen is used to freeze the specimen, and the difference in electron density is used to visualise the protein.

27
Q

How can SDS PAGE be used to show protein- protein interractions?

A

When run, the two differenent proteins will have different molecular weights so will run at different speeds therefore producing different bands.

28
Q

How do Leucine zippers interract with DNA?

A

2 alpha helices with leucine hydrophobic side chains which bind the two helices together. They create an X shape which straddles the DNA major groove and contact DNA bases with hydrogen bonds.

29
Q

What is the structure of a zinc finger?

A

Zinc in the centre interracts with 2 histadines of the coiled alpha helix, and 2 cystines of the B sheet.

30
Q

In gel electrophoresis larger mw’s move ……, and in gel filtration larger mw’s move …

A

slower- are reatrded by the gel

faster- as they are too big to get caught in the beads unlike the smaller mw’s.

31
Q

how is a zinc finger arranged tetrahedrally?

A

With a central zinc and bound to 4 other atoms. Can bind to secondary structures like Beta sheets.

32
Q

How can zinc fingers be made more structurally stronger?

A

By binding 3 in a row (or more) e.g. as only bind to two nucleotides each, they then bind to 6 in total as they are then bound together.

33
Q

How do zinc fingers bind to DNA?

A

Throgh Histadine or Arginine, they bind to 2 nucleotides to guanines.

34
Q

How could a zinc finger be separated from DNA?

A

Affinity chromotography.
Silica bead with complimentary DNA that it will bind to.
No salt buffer and non bound will be eluted, but add medium to high and the bound proteins will be.

35
Q

What is DNA footprinting used for?

A

Show location of DNA interracting proteins

36
Q

How does DNA footpriting work?

A
  1. Radiactive End-labelled (32P) DNA treated with Dnase1 which will cleave the many strands randomly.
  2. Run these samples on PAGE electrophoresis.
  3. This creates a ladder of DNA strands, however if a protein is bound then DNA cant be cut here, so creating a ‘DNA footprint’, showing the location the protein is bound.
37
Q

How are the SDS strands visuallised?

A

autoradiography- using radiation of end tag on the DNA to visuallise the strands.

38
Q

What is an EF- Hand?

A

Helix loop helix structural domain that binds the two alpha helices by calcium (or magnesium)

39
Q

What is a SH2 domain?

A

A protein domain that allows proteins contraining this domain to bind to phosphorylated tyrosine residues of other proteins ionically.
e..g in the Protein tyrosine kinase receptor.

40
Q

What is a SH3 domain?

A

Ar protein domain that uses an aromatic amino acid stack to interdigitate between the prolines of a ligand (P-x-x-P)
This hydrophobic stack contains aromatic tyrosine and tryptophans.

41
Q

How are SH2, SH3 and PH domains used in the signalling cascade of insulin binding?

A
  1. Insulin binding activates the receptor tyrosine kinase, which autophosphorylates.
  2. This activates the IRS1 docking protein, causing it’s phosphorylation. (this has a PH domain that binds to phosphoinositide binding to the P.M
  3. This phosphorylation then enables the binding to Grb2 adapter protein through a SH2 domain.
  4. The Sos then interracts with this adapter protein through a SH3 domain which is now activated and causes downstream signalling, along with a scaffold protein also bound by another SH2 domain to G3b2.
42
Q

What is a PH domain?

A

Its involved in membrane binding of proteins, e.g through a Phosphotidylinositol
The protein interracts with the charged head of the phospholipid.

43
Q

What is phosphotidylinositol?

A

PI- a phosphate and fatty acid structure with an inositol ring structure.
PIP and PIP2 = PI phosphorylated once, twice etc

44
Q

Leucine zipper types?

A

Homodimers- bind symmetrical alpha helix sequences or heterodimers bind different

45
Q

What is the helix-turn- helix binding motif?

A

Often made of two alpha helices that interract on the DNA. The recognition helix inserts into the major groove and makes specific contacts with the DNA and joined by a short turn of Amino acids

46
Q

The recognition sequence for helix-turn-helixes are..

A

palindromic

47
Q

What is a helix-loop-helix?

A

Related to a leucine zipper but the loop between the long alpha and short alpha chains in each dimer allows flexibility.

48
Q

Why would DNA binding proteins bind as dimers?

A

Increases strength (synergy)

49
Q

What 4 domains do transcription factors have?

A

DNA binding domain, protein binding domain, activation domain and regulatory domain

50
Q

What does EMSA stand for?

A

Electrophoretic Mobility Shift Assay.

51
Q

What happens in EMSA?

A

Radioactively label one end of the DNA, and mix this probe with protein of interest. Run this on gel electrophoresis and if the protein doesn’t interact then it will move further faster, however if it is then it will move more slowly, and this can be seen and shows interaction. Do not heat up the sample, unlike above, so the protein isn’t released.

BMS Level 2 (7 decks)

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