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Protein structure and function > structural bio of proteins > Flashcards

structural bio of proteins Flashcards

1
Q

In a peptide bond rotation can occur. What bond do Phi and psi bonds rotate around?

A

Phi is around the NC bond.

Psi is around the CC bond.

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2
Q

A ramachandran plot has 4 segements and can be used to read regular secondary strucutres. What segements show what?

A

top left - beta strands
top right - left handed helix (rare)
bottom left- Right handed helix (common)

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3
Q

what are common secondary strucutres found in proteins?

A

Beta sheets and alpha helices

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4
Q

what is a domain?

A

an autonomous folding protein unit. doesnt have to be contiguous. E.g MHC molecule

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5
Q

What is a module?

A

A domain with a contiguous sequence.

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6
Q

What is a repeat?

A

A unit that does not fold in isolation. several copies are needed.

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7
Q

Antibodies are made of IG domains? What are the two main variants of them?

A

C1 and C2.

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8
Q

How does the polarisation of light allow us to study the secodnary structure of moleucles?

A

Circular dichroism. orientation of light is altered by molecules that have chiral centeres.

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9
Q

electron microscopy (EM) ?

A

Images of single particles is possible (limit resolution ~20A)

Averaging or crystalline samples resolution ~4A) needs extensive image processing

Routine use of EM on “ metal stained” samples for increased contrast

Without metal stain but sample freezing leads to fewer artefacts

Whole cells can be imaged and distribution of proteins analysed inside the cell

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10
Q

What are major limitations of EM?

A

radiation damage. Electrons dmage the sample as they go through it.

In transmission EM very thin samples are needed

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11
Q

How are samples prepared for Cryo EM?

A

Flash freeze samples ( eg liquid ethane)

For water to go into glass like structure (not ice)

Preserve structure of object in aqueous environment but limit radiation damage

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12
Q

In Cryo EM how is the signal to noise and resolution enhanced?

A 
EM crystallography  (3D and 2D crystals possible) 
Regular lattice of objects allows averaging over many particles as well as using the periodicity of the objects to enhance resolution (diffraction of electrons leads to diffraction pattern)

Single particle reconstruction
Accumulate many images of individual particles to improve signal to noise

EM tomography:
Rotate object and record a series of (2D images, projections)
Reconstruct 3D object from

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13
Q

What are the strengths and limitations of Negative Stain EM?

A

strengths:
small amount of sample is needed.
can image a wide range of samples.

limitations:
resolution of 20A
artefacts though staining.

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14
Q

strengths and limitations of Cyro EM?

A

Strengths:
small amount of sample
wide range of samples can be imaged
Doesnt need to be stained so less artefacts

limitations:
resolution depends on averaging techniques (10A)
object has to be greater than 200Kda

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15
Q

Strengths and limitaitons of EM diffraction

A

Strengths:
requires only 2D crystals (Good for membrane proteins)
resolution of 3-4A is possible

Limitation:
EM always struggles with radiation damage

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16
Q

What are the strengths and limitations of X ray and neutron scattering?

A

Strengths:
-Applicable to large range of macromolecules such as protiens, DNA/RNA, small to large complexes etc

  • Measurement in solution
  • aprox a mg of pure protein is required.

limitations:
resolution of 20A

only shape info

best used in combo with other techniques

neutron scattering requries more material and best suited with some level of deuteration. (I.e recombinant protien production)

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17
Q

What is the strucutre of the GAG protein of HIV?

A

MA-CA-P1-NC-P2-P6

CA (caspid domain is made up of 2 domains N-CA and C-CA)

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18
Q

how many helicies do the NCA and CCA domains have?

A

6 in NCA and 4 in CCA

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19
Q

the Zn2+ knuckle fold can be found where?

A

in the NC domain. binds to the hairpin loops SL1-4 of the viral RNA.

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20
Q

The gag protein assembles into what sort of strucuture?

A

A hexametric lattice.

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21
Q

descibe the interdomain linkers of the GAG protein?

A

MA to NCA is flexible linker
NCA to CCA is flexible linker
SP1 region is under investigation
SP2 and P6 fexible region

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22
Q

How can the direction of the polypeptide chain be reversed?

A

By turns and loops. Turns can have various types with defined structures that are typically less than 5 amino acids.

Loops are more than 5 aa and have less regular structual elements

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23
Q

what are some of the associations of beta strands?

A

assoiciate via backbone H bonds and can be arranged antiparalell or parralel. mixes of the two lead to various beta sheet configurations.

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24
Q

what is an example of a mainly beta sheet protein?

A

Fatty acid binding protein.

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25
Q

describe the characterisitics of a regular alpha helix and its bond pattern.

A 
phi: -60° psi: -45°
turn per amino acid: 100°
number of amino acids per turn: 3.6
pitch: 5.4A
H bond pattern: CO(i) NH (i+4)
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26
Q

describe the characterisitcs and H bond pattern of a 3-10 helix.

A 
phi:-49° psi: -26°
turns per aa: 120°
number of aa per turn: 3.0
pitch: 6A
H bond pattern: CO(i) NH(i+3)
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27
Q

describe the characteristics and H bond pattern of a π helix.

A 
Phi: 55° Psi: 70°
turns per aa: 87°
number of aa: 4.1
pitch: 4.7A
H bond Pattern: CO(i) NH(i+5)
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28
Q

what is an amphipathic helix?

A

a protein sequence that folds into a helical structure upon contact with polar/non polar interface. can be found in many stably folded proteins.

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29
Q

amphipathic helices have what that makes them orrientate?

A

have a hydrophillic and hydrophobic side.

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30
Q

what is the orientation of amphipathic helices in water and lipid bilayers?

A

hydrophilic sides out in water and hydrophobic sides out in lipid bilayers.

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31
Q

what are leucine zippers?

A

helices made up of leucines that form dimers.

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32
Q

protein teritary strucutres are stabalised by multiple weak inteactions such as?

A

Van der waals,
ionic interacitons
H bonds

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33
Q

what drives the structual formation of tertiary strucutres\?

A

Hydrophobic effect.

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34
Q

what amino acids are disulphide bonds formed between and what does this do?

A

Between cysteine residues and can help some protein structures be stabilised.

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35
Q

Why do protein post translational modifications occur? and how and what is charateristic about them.

A

covalently modified to alter protein function.
tends to be typically reversible and are enzymatically catalyzed.
they enhance functional diversity and have local, temporal control of function

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36
Q

what is an example of post translational modification?

A

phosphorylation. tends to be a naturally very slow reaction so gets catalysed with kinases and then the removal is done by phosphatase.

37
Q

what is the strucuture of the kinase domain used for the catalysis of phosphoryaltion.

A

has an N lobe thats predominantly Beta sheets, an ATP binding cleft and then a C lobe thats predominantly Alpha helix.

38
Q

what residues get phosphorylated? and how

A

Tyrosine, Serine, Threonine. A phosphate from ATP gets transfered to them via the kinase domain.

39
Q

What state of a protein does the unfolded protein want to get to?

A

The Native 3D structure

40
Q

how are unfolded proteins degraded?

A

proteases

41
Q

unfolded proteins may get to the intermediate state and still get degraded? T/F

A

True

42
Q

How does the protein form its 3D structure? how is this known.

A

its encoded in the amino acid sequence.

when there is oxidation under denatured conditions it leads to the formation of the wrong disulphide bonds. tho renaturation first then oxidation leads to the correct bond formation showing how its encoded.

43
Q

what is the folding process of single protein domains?

A

typically undergo an all or nothing 2 state folding process.

44
Q

What factors effect folding state in the protein folding funnel graph?

A

number of native interactions and the number of residue contacts.

the native state has high levels of both.

45
Q

what is the unfolded state characterised by?

A

there are many configurations. can have many interactions between residues but there are few native like interactions.

46
Q

what is the transition state?

A

is where there are many contacts and key residues have native contacts.

47
Q

what is the native state?

A

this is the folded state where there are many interactions that are all native interactions.

48
Q

Why is misfolding dangerous?

A

it can lead to disease.

49
Q

What is Prion?

A

an infectious agent entirely composed of protein Prp. it causes BSE (mad cows disease), Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep.

50
Q

what is the definition of an infections agent?

A

requires DNA or RNA to allow replication in host.

51
Q

Prp comes as the normal PrpC and the infectious agent PrpSc. whats the difference structually between them?

A

PrpSc is richer in beta strands.

52
Q

How are domains identified?

A

by aa sequence profiles. they also have disulphide patterns of 1-3, 2-4, 5-6.

53
Q

What is the C1 variant of the IG domain?

A

4 stranded beta sheet DEBA and 3 stranded beta sheet GFC

54
Q

what is the C2 variant of the IG domain?

A

3 stranded beta sheet EBA and 4 stranded beta sheet GFCC’

55
Q

What are the IG domains found in a MHC class I?

A

α3 and β2m

56
Q

What is titin?

A

a giant muscle protein that is 90% Ig or IG like domains. tehre are 166 IG domains and 132 FN3 domains.

no complete strucuture is available but the domains give insight into the protein dimensions.

57
Q

what is an intrinsically disordered protein?

A

parts of protein sequences or entire proteins that lack a defined 3D structure.
they can be entirely random coil or partially structured and carry out their function as unstructured proteins.

58
Q

approximately what percentage of the eukaryotic genome are IDPs (intrinsically disordered proteins)

A

30%

59
Q

what are some examples of IDPs?

A

alpha-synuclein which is a human protein of 140aa and has function in synaptic transmission.
has different strucutal features depending on enviroment.
lipid bound tetramer that has a beta rich fibrilar state.

60
Q

how are the primary strucutres of proteins studied?

A

chemically with Edman degradation which gives us the sequence of aa.

or by mass spec which is the identification of the protein fragments.

61
Q

what is the equation used in spectroscopic methods? linking absorption and concentration?

A

A=ε l c

Beer Lambert Law

ε: molar extinction coefficient

l: path length in cuvette
c: concentration of protein (DNA,…)

62
Q

How can the secondary protein structure be identifed?

A

Circular dichroism which uses the polarisation of light and its orientation alteration by molecules with chiral centres.

63
Q

what is the CD spectrum?

A

the measurement of the differences between left and right polarised light as the function of wavelength of light.

64
Q

describe the graphical characteristics of differnt secondary structures in a CD spectrum.

A

alpha helices start of with high CD and then have 2 minimums at 210 and 220 forming a sort of w shape

beta strands start lower CD adn go below zero adn then back up to zero. shallow wave.

unfolded protiens are negative and then become positive and then back to zero.

65
Q

how does the matrix of the GAG protein inetract with the membrane?

A

via myristol modificaton

66
Q

what resolution does Transmission EM image at?

A

20A

67
Q

To increase contrast of the samples what is done to them before analysis? what alternative can be done to reduce the number of artefacts?

A

metal staining. can also do sample freezing to reduce the number of artefacts.

68
Q

What happens to the sample in cryo EM and why?

A

samples are flash frozen. the water turns into a glass like structure not ice. it preserves the structure of sample in aqueous environment but limits the radiation damage.

69
Q

what are the 3 ways to enhance signal to noise in Cryo EM?

A

EM crystallography: Regular lattice of objects allows averaging over many particles as well as using the periodicity of the objects to enhance resolution (diffraction of electrons leads to diffraction pattern)

single partical reconstruction: Accumulate many images of individual particles to improve the signal to noise.

EM tomogrpahy: Rotate the object and record a series of 2D images. that can then be reconstructed into a 3D object form.

70
Q

What is X ray scattering?

A

x rays are radiated at the sample. causes scattering radiation that is detected and outputs the intensity of the radiation as a function of the angle. I(q)

I(q) is then interpreted and if one knows the individual domains they can then be arranged to give the right shape.

71
Q

What is the first step in the structural determination by X ray crystallography?

A

Making of the protein crystals. sample in a dilute buffer has ammonium sulphate added that is then left for a few days where it becomes crystals. buffer conditions will be trial and error to get condition that promotes crystal growth not aggregation.

72
Q

When x rays at radiated at the crystals in x ray diffraction there are many diffracted beams that get detected on a film. what does the film get converted into and what additional information is needed to get over the ‘Phase problem’?

A

Film gets converted into an electron density map.

additional information needed is from:

1) make additional crystals with heavy atom derivatives. 2) incorporate seleno-methionione protein using its anomalous diffraction to determine the phases
3) if a similar protein structure is known back calculate the electron density map.

73
Q

In NMR what gives the NMR line?

A

the transition state between spin states.

74
Q

Why is there spin in NMR

A

when a nucleus is put into a strong magnetic field it causes the nucleus to spin as the nucleus spin likes to align itself with the magnetic field. if there is a change in the alignment the energy level of the spin will change. different molecules will thus have different spins.

75
Q

regarding 3D structure what does chemical shift dispersion in NMR indicate?

A

indicates the presence of absence of 3D structure. more unfolded means more chemical shift dispersion.

76
Q

What are correlation peaks in NMR? what can they be used for?

A

when two protons are close in space they show correlation peaks on the NMR proton chemical shift spectrum.

can convert proximity information of proton pairs into a 3D structure via triangulation.

77
Q

What does Ligand binding with 1H-15N do to NMR?

A

causes shift of selected peaks.

since chemical shift is sensitive to enviroment by doing ligand binding can get more information about the enviroment by creating the footprint of the protein. can infer where specific ligand binding sites are where there is a movement of particulate NH.

78
Q

What are the strengths and limitations of x ray crystallography.

A 
Strengths:  
highest resolution (less then 1Å)

enormous information content: do chemistry by structure

applicable to large range of macromolecules

Proteins, DNA/RNA, small to large complexes, and assemblies

Limitations:
Molecule needs to form crystals

may prove impossible

Crystal environment may influence the result of dynamic parts of a protein or weakly interacting systems

Static structure

Several mg of pure protein required

79
Q

strengths and limitations of NMR

A

Strengths:
Versatile tool to investigate any macromolecule

Each atom a potential “spy” to investigate a protein

Experiments in solution or in solids (membranes, aggregates)

Structure determination folding,

Protein ligand interactions with local resolution

dynamics (site specific with out perturbation of protein)

highest resolution (less then 1Å)

enormous information content: do chemistry by structure

applicable to large range of macromolecules

Limitations:
Several mg of pure protein required (isotope labelling , recombinant protein production

Solutions structures limited to small (<50Kda) proteins

80
Q

What enzyme cleaves the HIV GAG protein?

A

HIV-1 protease.

81
Q

at what points in GAG does the protease cleave?

A

first clevage is just before the NC domain. the second is between the CA and MA. 3rd is just before the p6. 4 is before SP2 seperating it from the NC. 5th which is the critical one is between the CA and SP1.

82
Q

In the comparison of the XRAY and NMR structures of the MATRIX in HIV waht are the biggest differneces shown in two loop regions.

A

Ile19-Gln28 NMR multiple strucutres but differnent from XRAY.

Leu68-Ser72 NMR well defined stuctures but different from XRAY.

83
Q

Why in the matrix can there be differences found around Ser72?

A

the crystal MA forms trimers and the region around Ser72 is a trimer interface so can show differences.

84
Q

what domains are the caspid in the HIV gag protein made of?

A

N-CA and C-CA

85
Q

where is the zinc knucle fold found?

A

in the nucleocaspid domian

86
Q

what does the zinc knuckle fold bind to?

A

the hairpin loops SL1-4 of the viral RNA.

87
Q

which interdomain linkers of the gag protien are known to be flexible?

A

MA to NCA
NCA to CCA
SP2 to P6

88
Q

In a peptide bond what configuration is preffered?

A

Trans configuration as tends to be energetically much lower for all amino acids

89
Q

What amino acid can also take upon the cis form?

A

Proline. no COOH group so can go in a free peptide form in cis form.

Protein structure and function (7 decks)

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