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SFP > Bonev - Protein structure > Flashcards

Bonev - Protein structure Flashcards

1
Q

What are two reasons as to why we want to know and understand protein structure?

A
  1. Understand their function
  2. Infer the function of unknown proteins with similar structure
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2
Q

When does the secondary structure form?

A

Spontaneously at the ribosome immediately after translation

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3
Q

How is it stabilised?

A

Hydrogen bonds between the NH and CO of the backbone

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4
Q

Where are the hydrogen bonds in the alpha helices structure?

A

Between the carbonyl oxygen of one amino acid, and the amide hydrogen of another amino acid four residues further down

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5
Q

Where are the hydrogen bonds in the beta sheets?

A

Between adjacent strands of the beta sheet. Carbonyl oxygen of one amino acid and the amide hydrogen of another amino acid on an adjacent strand.

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6
Q

How many residues are present per turn in alpha helices?

A

3.6

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7
Q

What is the angular increment per residue?

A

100º

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8
Q

What is the helical pitch?

A

5.4Aº

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9
Q

What type of domain is usually found at plasma membrane proteins?

A

Alpha helices

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10
Q

For beta sheets, which way do the side chains point?

A

Outwards

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11
Q

What do beta barrels consist of?

A

Antiparallel strands

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12
Q

How is the backbone able to change direction?

A

Due to beta-turns. These are also needed for a closed barrel strucutre.

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13
Q

Beta barrels are common folds in outer membrane proteins. What places are they found?

A

Bacteria, mitochondria, and chloroplasts

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14
Q

Proline (P) and Glycine (G) are present in many proteins with beta sheets and are found in barrels. What is their purpose?

A

Proline = found in the second position of beta-turns and facilitates the reversal of the the direction in the turn

Glycine = found in the third position of the turns, smallest amino acid which allows greatest flexibility

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15
Q

What is meant by torsional flexibility at the peptide bond?

A

It refers to the ability of the peptide bond to rotate around two specific angles, known as the phi and psi angle

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16
Q

What does the phi angle represent?

A

Represents the rotation around the bond connecting the nitrogen atom (N) of one amino acid to the alpha carbon (Cα) atom of the same amino acid

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17
Q

What does the psi angle represent?

A

Represents the rotation around the bond connecting the alpha carbon (Cα) of one amino acid to the carbonyl carbon (C) of the next amino acid

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18
Q

What is the purpose of Ramachandran plots?

A

Ramachandran plots display the possible combinations of phi and psi angles that are energetically favorable and allowed for protein structures. It can show the possible secondary structures to be produced.

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19
Q

Ramachandran plot has the psi angle on the y-axis (left), phi angle on the x-axis (bottom). Beta sheet normally top left, left a helix in middle right, and right a helix bottom left

A
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20
Q

What is the supersecondary structure made up of?

A

Multiple secondary structures. They come together and interact, often stabilising each other when they come close.

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21
Q

What is an EF hand?

A

A Ca2+ binding motif from calmodulin acting as a binding site for calcium

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22
Q

What are the four non-covalent interactions which form the tertiary structure?

A
  1. Hydrophobic interactions
  2. Electrostatic interactions
  3. Hydrogen bonds
  4. Van der Waals
23
Q

Difference between protein domain and structural motif?

A

Domains are larger and have more diverse functionalities, while structural motifs are smaller and contribute to the overall folding and stability of protein structures

24
Q

Why do proteins react differently to left and right handed polarised light?

A

Because they have a chiral centre, making them optically active

25
Q

What does Circular Dichroism measure?

A

Measures the difference in absorption between left-hand and right-hand circularly polarised light as a function of wavelength of light as it passes through optically active samples.

26
Q

What does it determine?

A

It can determine the secondary structure content. These structural elements impart characteristic patterns in the CD spectrum.

27
Q

Infrared Fourier-transform infrared (FTIR) spectroscopy and Raman spectroscopy are two techniques used to determine the secondary structure of proteins based on what?

A

Based on the vibrational modes and interactions with infrared or raman radiation. Vibrations of the atoms within the molecule.

28
Q

How does Infrared FTIR work?

A

Sample absorbs infrared and each characteristic of the secondary structure will have unique vibrational modes and absorption frequencies. Bonds stretch, bend, or vibrate

29
Q

At what wavelength does alpha helices absorb the polarised light in circular dichroism?

A

208nm and 222nm

30
Q

What about for beta sheets?

A

Maximum around 200nm

31
Q

Information about secondary structures absorbing wavelength of 190 or less, is found out by using what type of radiation?

A

Synchrotron radiation

32
Q

Where does the Amide 1 band typically occur in FTIR?

A

1600cm-1700cm

33
Q

What is an advantage of FTIR?

A

Can study membrane proteins

34
Q

What is a disadvantage of FTIR?

A

Sample must be dehydrated to minimise absorption by water because water absorbs infrared very strongly. Raman spectroscopy can be used to study hydrated samples though.

35
Q

What can Analytical ultracentrifugation (AUC) be used for?

A

Estimate protein size, allows protein complexes to be confirmed in vitro, shape, and mass

36
Q

What is the basic principle of AUC?

A

Relises on sedimentation, where particles, including proteins, are subjected to a high centrifugal force, causing them to sediment at different rates based on their size and shape

37
Q

So, what are the two things AUC measures?

A

Sedimentation velocity and protein diffusion. Smaller proteins sediment more slowly, whilst larger complexes diffuse more slowly

38
Q

What does X-ray cyrstallography determine?

A

The 3D structure of proteins at the atomic level

39
Q

What is the simple principle of it?

A

It involves the crystallisation of a protein and then defracting x-rays through the crystal, then analysing them

40
Q

What type of maps are obtained?

A

Electron density maps because within the nucleus of atoms, they do not move and can determine the structure

41
Q

Why are X-rays used?

A

The wavelength is around 1.54º which is the typical length of a bond within the protein so it can determine the position of individual atoms

42
Q

What are the strengths of x-ray crystallography? (3)

A
  1. Total structure determination at atomic level of detail
  2. The most productive structural technique
  3. Once crystals of good quality are obtained, structure determination is a fairly well established process and can be automated
43
Q

What are the weaknesses of x-ray crystallography? (5)

A
  1. Crystals can be obtained from most soluble proteins; Mebrane proteins remain a challenge
  2. Protein dynamics is frozen
  3. Structures may appear distorted due to multiple proteins observed and within contact
  4. Heavy metal labelling required
  5. Radiation damage of the sample, greater energy+intensity of the beam leads to more damage for the protein
44
Q

What is the first step in NMR?

A

A recombinant protein is expressed and then labelled with stable isotopes

45
Q

Why is it labelled with stable isotopes?

A

Labeling the protein with stable isotopes, such as carbon-13 and nitrogen-15, allows the positions of specific atoms within the protein to be tracked and analyzed using NMR spectroscopy. By labeling the protein with these isotopes, the signals from the labeled atoms can be distinguished from the signals of the regular atoms in the protein. This makes it easier to determine the positions of the labeled atoms and generate a 3D model of the protein structure.

46
Q

What is the next step?

A

Data analysis - obtaining enough constraints (distances and torsion angles) to determine full structure at atomic resolution

47
Q

Alpha helix:
Hn - Hn couplings at i, i+1 residues
Hn - Ha couplings at i, i+3 residues

A
48
Q

Beta sheet:
Hn - Ha couplings at i, i+1 residues

A
49
Q

What are three benefits of using NMR to determine secondary structure?

A
  1. Total structure determination
  2. Proteins are studied in near native state in aqueous solutions
  3. Provides information on protein dynamics
50
Q

What are three negatives of NMR?

A
  1. Molecular weight limit of 20kDa meaning only small proteins can be determined
  2. Often requires stable isotope labelling which is expensive
  3. Studying membrane proteins is challenging - need detergents
51
Q

What does Cryo TEM not use?

A

Staining

52
Q

What can it study?

A

Soluble, large protein complexes like membrane proteins

53
Q

The sample is flash-frozen in liquid ethane

A
54
Q

What is meant by polymorphism of a protein and how is this a negative in Cry TEM?

A

Polymorphism refers to the ability of a protein or other molecule to adopt multiple conformations, which can lead to variability in the images obtained by cryo-TEM

SFP (14 decks)

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