Protein Structure Flashcards Preview

BMS 242 - Advanced Cell and Molecular > Protein Structure > Flashcards

Flashcards in Protein Structure Deck (59)
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1
Q

What is the backbone structure of all proteins?

A

N-C-C-N

2
Q

What gives the function of a protein?

A

The different side chains attached

3
Q

How many different R groups are there?

A

20

4
Q

What stays the same and what changes throughout different proteins?

A

Backbone structure stays the same

Side chains change

5
Q

What is the secondary structure of a protein?

A

Organisation of the side chains

6
Q

What are the 3 possible secondary structures of a protein?

A

Alpha-helix
Beta-pleated sheet
Random coil

7
Q

How is the secondary structure of a protein held together?

A

By weak hydrogen bonds

8
Q

What happens to H bonds in a protein?

A

Continuously breaking and reforming without enzymatic activity

9
Q

What is the tertiary structure of a protein?

A

Globular structure, in which the individual structural elements (a-helix, beta-pleated sheet, random coil) pack together

WITHIN a protein and BETWEEN subdomains of a protein

The entire structure of 1 protein

10
Q

What is the quaternary structure of a protein?

A

Multimeric protein of many proteins interacting together

many tertiary structures

11
Q

What must be considered when considering the activity of the protein and why?

Example

A

ALL of the structures of the protein (primary–> quaternary) as a feature in one structure may impact the activity of another

Example: A protein in the primary structure may be able to be phosphorylated, which could impact how proteins interact with each other in the multipmeric quaternary structure

12
Q

What are the hydrophobic amino acids?

A
Alanine (Ala)
isoleucine (Ile)
Leucine (Leu)
Methionine (Met)
Phenylalanine (Phe)
Valine (Val)
Glycine (Gly)
Proline (Pro)
13
Q

What are hydrogen bonds?

A

Weak bonding between H and O or H and N

14
Q

What are ionic bonds?

A

Electrostatic forces between positive and negative charges

15
Q

What are Van der Walls?

A

Very weak, short range interactions between molecules

16
Q

What are the 4 different representations of a secondary protein structure?

Describe them

A

1) Backbone
- Skeleton of the C-N backbone

2) Sticks
- Shows ALL the connections between all of the atoms
- Back-bone and side chains

3) Space-filling
- Shows protein in a globular manner and all the pockets for interaction with other molecules

4) Ribbon
- Describes the motif
- Flattened

17
Q

Which representation of a protein is more true to life?

A

The space-filling representation

18
Q

What is important for function in a protein?

A

Structure/shape

19
Q

What is Src?

A

A multidomain tyrosine kinase

20
Q

What is Src involved with?

A

Cancer

21
Q

What are the 4 domains of Src?

A
  • SH2
  • SH3
  • Small kinase domain
  • Large kinase domain
22
Q

How are the structure and functions of the domains in a multi-domain complex related to each other?

A

They normally have in dependant structures and functions

But all work together to achieve the task of the protein

23
Q

How can domains in a protein show a common ancestor?

A

Same domains - through evolution they are conserved and paired with other domains to achieve different functions

24
Q

What happens if take one domain from a tyrosine kinase and substitute in a different domain from another tyrosine kinase?

A

Function still occurs

25
Q

What are 3 methods that can be used to work out the primary structure of a protein?

How are these methods used?

A

1) Predict from the DNA sequence
2) Obtained directly by amino-acid sequencing (EDMAN DEGRADATION)
3) Mass spectrometry

Methods are used to compliment each other - not used in isolation

26
Q

What is Edman degradation normally used for?

A

To sequence a short bit of a protein sample, to allow this to be used to search a database to identify the rest of the protein

27
Q

What is the process of Edman degradation?

A

Cycle
Using PITC:
1) Attaches to the end of the protein through a chemical reaction (the UNFIXED END)

2) Solution is acidified - causes the terminal AA to drop off - is washed off from the protein
3) AA which is washed off is analysed using HPLC separation (High Performance Liquid Chromatography)

28
Q

In Edman degradation, how many AA are identified per cycle?

A

1

29
Q

What sized proteins does Edman degradation work for?

A

Proteins up to 60 amino acids in length

30
Q

Describe the graph produced by HPLC

A
  • Numbers along the bottom of the graph are the rounds of Edman cycle
  • Peaks on the graph are AA released
31
Q

In HPLC, how are the AA detected?

A

By their absorbance of light at 269nm

32
Q

What is needed for HPLC to be effective?

A

A very purified protein

33
Q

How can the tertiary structure of a protein be identified from a primary structure?

A

FIRST, predict secondary structure from the primary structure using:

  • Biophysical modelling
  • Compare primary structure to KNOWN proteins (likely to fold in a similar way AND have a similar function)
34
Q

What is ‘biophysical modelling’?

A

Predicting the folding and energy state of this folding using a computer programme

Then, selecting for the LOWEST ENERGY state (as this is what proteins fold into)

35
Q

If two proteins have a similar primary structure, what can this predict?

A

That they fold in a similar way and have similar functions

36
Q

What can protein databases provide?

A

Predictions for:

  • Protein - protein interactions
  • Sequence alignment
  • Domain composition
  • Post-translational modifications
  • Structure

By showing what proteins the protein of interest is related to (likely to have similar features)

37
Q

What is Circular Dichrosim used for?

A

To determine the secondary structure of a protein and to give information about the tertiary structure

38
Q

What is the process of CD to work out the secondary structure of a protein?

A
  • Use far-UV light (190-250nm)
  • Different protein structures (beta-sheet, a-helix, random coil) absorb DIFFERENT WAVELENGTHS of light, to give a characteristic shape to the CD spectrum
39
Q

What is the wavelength of far-UV light? (used in CD)

A

190-250nm

40
Q

What is the shape of the CD spectrum of a alpha-helice?

A

W shaped

2 peaks at:

1) 210nm
2) 250nm

41
Q

What is the shape of the CD spectrum of a beta-sheet?

A

Looks like the ‘sine-wave’

Peak at:
215nm

42
Q

In CD, what happens if there is a mix of different structures (b-sheets, a-helice) in the protein?

A

There will be a mixture of different absorptions

43
Q

How readout does CD give and not give?

A

GIVES the % for each secondary structure type in the protein

DOESN’T GIVE any information about the arrangement of the amino acids

44
Q

If the %’s of the CD readout change in different conditions, what does this show?

A

Shows how the protein reacts to different conditions - is the protein stable?

45
Q

How can the tertiary structure be identified using CD?

A

Can ‘melt’ the protein, this disassembles the tertiary structure into a secondary structure (as tertiary structure is more sensitive to temperature change, will keep secondary structure for longer)

This can be done over a temperature range - to follow protein unfolding

46
Q

What signals in CD are sensitive to the overall tertiary structure of the protein?

A

Aromatic amino acids and disulphide bonds

47
Q

What is X-ray crystallography used for?

A

To determine the tertiary structure of a protein

48
Q

What is the process of X-ray crystallography?

A

1) Concentrate very pure protein to super concentrated state - to form a crystal of protein
2) Fire high energy and focussed beam of X-ray through the crystal
3) Most of the X-rays pass through, but some are deflected, producing a DIFFRACTION PATTERN
4) Can trace the diffraction pattern back to the structure of the protein

49
Q

What is the background behind NMR?

A
  • In most atoms, there is no overall spin of the nucleus, at the spin of the subatomic particles (protons and neutrons) are paired against each other)
  • HOWEVER, some atoms can be INCOPORATED into the protein
  • In these atoms, there is an uneven number of protons and neutrons - causing a slight wobble of the nucleus, as the spin is NOT balanced
  • The wobble of these atoms produce a VIBRATION
50
Q

Which atoms produce a wobble-spin?

A

H^1
C^13
N^15
isotopes

51
Q

How are the radioactive isotopes, which produce a wobble spin, incorporated into the protein?

What does this produce?

A
  • By growing bacteria in a RADIOACTIVE MEDIUM, where the sole nutrient is N^15 and/or C^13
  • Producing recombinant proteins, which every atom is singly or doubly labelled with N^15 or C^13
52
Q

What is the process of NMR?

A
  • Incoporate radioactive N^15 and C^13 into every atom
  • Expose the protein to high specific frequency in high magnetic field
  • The different protons resonate (vibrate) at a different, predictable frequencies
  • Enables to count the number of H, C and N in the protein of interest
  • Analysed by a computer programme, which analyses the data at different conditions , to produce a prediction of the protein structure
53
Q

What ‘chemical shift’ and what is it dependant on?

A

The frequency of vibration of protons, which is predictable

Dependant on the local environment of the protein

54
Q

How does the computer programme in NMR produce a prediction of the protein structure?

A

Iteratively, arriving at several possible structures and representing them as an ensemble

55
Q

Why is electron microscopy limited for protein analysis?

A

Can only see the larger structures (cannot figure out the protein structure)

So can only work out the what the larger complexes of proteins look like

56
Q

What is the process of EM?

What makes this process easier? (examples)

A

Use image analysis of a preserved specimen to build up an average structure of the protein, by taking lots of pictures from different angles

The more ordered and symmetrical the structure, the easier the averaging process
(eg. actin - helical symmetry, viruses - radial symmetry)

57
Q

In EM, what is used to preserve the specimen?

A

Negative stain or vitreous ice (cryo-EM)

58
Q

What methods can be used to work out the secondary structure of a protein?

A

Biophysical modelling

Circular dichroism

Predictions from the primary structure (compare to other proteins)

59
Q

What methods can be used to work out the tertiary structure of a protein?

A

Circular dichroism (melt the protein)

Protein databases

Electron microscopy

Xray crystolography