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Tools of biochemical discovery > TOoLs > Flashcards

TOoLs Flashcards

1
Q

What wavelengths does the peptide backbone absorb light at?

A

190 and 220nm

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2
Q

What wavelengths do alpha helices have a trough in?

A

208nm and 222nm

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3
Q

What is the threshold voltage of spectroscopy at which the detector becomes unreliable?

A

650 volts

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4
Q

Define mass spectrometry:

A

The separation of gaseous ions by their mass and charge to determine their mass and compostion

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5
Q

What is the Lorentz force law?

A

F=q(E + v x B)

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6
Q

Above which FWMH is considered high resolution?

A

10000

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7
Q

What are the two ‘soft’ ionisation methods?

A

ESI and MALDI

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8
Q

What does MALDI stand for?

A

Matrix Assisted Laser Desorption Ionisation

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9
Q

What does ESI stand for?

A

Electrospray Ionisation

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10
Q

What are the four main stages of mass spectrometry?

A
  1. Sample preparation
  2. Ionisation
  3. Measure intact ions or fragment ions
  4. Detect ions and data analysis
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11
Q

What are the four main types of Mass spec?

A

Time of Flight
FT-ICR
Quadrupoles
Ion-traps

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12
Q

What can mass spectrometry determine?

A

Mass/charge of ions
Intensity of peptide and fragment ions
Liquid chromatography gives retention for the peptide

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13
Q

What are the uses of Flourescence Microscopy?

A

Localisation- flourescence microscopy
Binding interactions- Environment sensitive probes, FRET
Accessibility of molecules- use of quenching agents
Mobility of proteins- Flourescence anisotropy, polarisation
Molecular ruler- FRET

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14
Q

What are the fluorescence parameters?

A

Excitation wavelenght
Emission wavelength
Quantum efficiency (QE) -emitted photons/absorbed photons

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15
Q

What is the effect of a polar environment on flourescence?

A

Fluorescence moves to red and and becomes less intense

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16
Q

What is the effect of a non-polar environment on fluorescence?

A

Fluorescence moves to blue and and becomes more intense

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17
Q

What is photobleaching?

A

Permanent loss of fluorescence due to photo induced chemical modification of a molecule

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18
Q

Name three quenching agents:

A

Iodide
Acrylamide
O2

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19
Q

KD is used as a measure of what?

A

How well a molecule fits a receptor

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20
Q

What is the Languiir 1:1 model?

A
  1. The two binders are pure
  2. All sites are equivalent
  3. Each binding site can hold one molecule of lingand
  4. There are no interactions between adjacent sites
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21
Q

What do neonicotineoids bind to?

A

A nAChR subtype unique to insects- Alpha4 Beta2

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22
Q

Name 3 label free assays:

A

SPR, Biacore
Fluorescence
Thermal shift

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23
Q

What does SPR stand for and what does it do?

A

Surface Plasmon Resonance

It characterises proteins as they interact with other proteins

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24
Q

What are the requirements for SPR?

A

Microgram quantities
Based on 5 mins cycles per compound:
-6 concs+ 1 duplicate+ 1 zero= 8 injections/compounds
-40 mins to characterise each positive hit

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25
Q

What is IC50?

A

the concentration of an inhibitor where the response (or binding) is reduced by half
It is a frequent biological estimation of inhibitor efficacy measured by bioassay

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26
Q

What is the Van’t Hoff equation?

A

▲G=▲H-T▲S

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27
Q

What does ITC stand for and what can it determine?

A 
Isothermal Titration Colourimetry
Binding constants (KD), reaction stoicheometry (n), enthalpy(▲H), and entropy (▲S)
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28
Q

In ITC what is the equation to determine sample concentration?

A

M=cKn

Sample concentration= (5-500)(expected affinity)(expected stoichiometry)

29
Q

What is Lipinski’s rule (Ro5)?

A

A compound is more likely to exhibit poor absorbtion or permeation when two or more of the physiochemical criteria are fulfilled:

  • The Mr is greater than 500Da
  • The calculated 1-octoanol-water partition coefficient is greater than 10
  • There are more than 5 hydrogen bond donors
  • There are more than 10 hydrogen bond acceptors
30
Q

What genes did the two plasmids contain in the first yeast-2-hybrid assay?

A

Plasmid One- Gal4DBD, SNF1, His3

Plasmid Two- Gal4AD, SNF4, Leu2

31
Q

What beads are used in immunoprecipitation?

A

Protein A sepharose (binds to Fc regions of antibodies)

32
Q

What are commonly used affinity tags?

A

FLAG-DYKDDDDK
Hemaglutinin
C-myc
They are cloned on to a terminus of the protein

33
Q

What do bait and prey bind to?

A

Bait: The known protein/ligand
Prey: The interacting protein/ligand we’re looking for

34
Q

What can protein microarrays can be used to screen for?

A
  • Biochemical activity
  • Protein/protein activity
  • Protein/DNA or protein/RNA interactions
  • Protein/ligand interactions
35
Q

What was whole-genome yeast protein chips used to identify?

A

Many new calmoduling and phospholipid interacting proteins

A common binding motif for calmodulin

36
Q

What does BiFC stand for, what is it, what does it enable?

A

Bimolecular Fluorescence Complementation
The formation of a fluorescent complex by fragments of an xFP brought together by the association of two interacting partners
It enables visualisation of the subcellular sites of protein interactions

37
Q

What is the mechanism of BiFC?

A
  1. Divide YFP into two fragments (half barrels)
  2. Fuse these fragments at the C-terminal ends of the bZIP domains of Fos and Jun
  3. Express in E.Coli, alone or together
38
Q

Define the interactome:

A

A map of all protein interactions in a given cell. Proteins of known structure, cellular location and function tend to cluster together

39
Q

What wavelengths do X-ray diffraction experiments generally use?

A

0.5 to 2 Amstroms

40
Q

What are Millers indices?

A

Sets of planes diffract only if they cut each of the unit cell edges an integral number of times

41
Q

What does the convolution theory state?

A

The Fourier transform of the convolution of two products is equal to the product of the Fourier transforms of the two separate functions

42
Q

What does the Inversion theorum state?

A

If function f is the Fourier transform of function g then function g is the Fourier transform of function f

43
Q

What are the characteristics of purification?

A

Protein should be chemically and conformationally homogenous
Proteins should be soluble at concentrations around 5-10 mg/ml
Proteins should generally not be aggregated

44
Q

How are X-rays generated?

A

Electrons boil off the cathode and are accelerated by the high potential towards the anode. Interaction with the anode generates X-rays

45
Q

What does the process of cyrocrystallography entail?

A

Diffraction experiments are conducted at liquid N2 temperatures by to reduce crystal destruction by x-rays
The protein lattice is protected from damage due to ice crystals by briefly soaking it in a cryproprotectant
Freeze trapping catalytic intermediates

46
Q

What are the characteristics of syncotrons?

A

More intense highly collimated x rays
Use smaller crystals
More information from weakly diffracting crystals
You can change the wavelengths of the X-rays

47
Q

What are four methods of determining phase?

A

Direct methods
Isomorphous replacement
Multiwave anomalous dispersion (MAD)
Molecular replacement

48
Q

What are common heavy metal reagents and what do they bind to?

A

Mercury-Exposed thiol groups
Gold- Histidine
Platinum- methionine

49
Q

What is the Patterson Function and how can it be calculated?

A

Fourier transform of the structure factor intensities

Can be calculated directly without knowing phases

50
Q

What is anomalous dispersion?

A

Generally, an atom scatters X-rays without changing their phase: The atomic scattering factor can be drawn as a real number

51
Q

What are the two stages of molecular replacement?

A

A translation function

A rotation function

52
Q

What is NMR?

A

Nuclear magnetic resonance is the physical phenomenon that certain nuclei in a magnetic field can absorb and re-emit electromagnetic radiation at specific frequency

53
Q

What is scalar coupling?

A

Interaction through chemical bonds (

54
Q

What does NOE stand for and what is it?

A

The Nuclear Overhauser Effect is the transfer of nuclear spin polarisation from one spin system to another through cross relaxation

55
Q

What does COSY detect?

A

Coupling between directly connected neighbouring protons

56
Q

What does TOCSY stand for and what it detect?

A

Total Correlation Spectroscopy shows all the coupling if there is a complet chain of coupled protons

57
Q

What does NOESY stand for and what does it detect?

A

Nuclear Overhauser Effect Spectroscopy detects all continuous proton-proton coupling through space

58
Q

What does HSQC stand for and what does it detect?

A

Heteronuclear Single-Quantum Correlation spectroscopy detects direct proton-carbon connection

59
Q

What does HMBC stand for and what does it detect?

A

Heteronuclear Multiple-Bond Correlation spectroscopy detects direct H-C coupling through multiple bonds, the carbon which hydrogen is directly connected to is not observed

60
Q

What are the two main types of electron microscope?

A

Transmission Electron Microscope (TEM) - electrons go through the sample
Scanning Electron Microscope (SEM) - electrons are reflected back off the sample

61
Q

What is vitreous ice?

A

Ice lacking hexagonal or cubic crystal order, generated by shock freezing

62
Q

What is the theory of common lines?

A

Fourier transforms of two projections originating from the same object can intersect at a common line (Used in angular reconstitution)

63
Q

Name three methods to determine orientations in Cryo-EM:

A

Angular reconstitution
Projection matching
Analyse FT of ordered sample

64
Q

What are the two types of Cryo-EM contrast?

A

Amplitude contrast gives clear images but information is lost
Phase contrast provides noisy images containing 3D information on the original object

65
Q

What does Cryo-electron Tomography allow?

A

3D images of complex structures can be obtained

No need for averaging procedures so you can image a unique sample

66
Q

What are the limitations of Cryo-electron Tomography?

A

The sample has to be thin
(less than 500nm thick, thinner than most eukaryotic cells)
The electron dose should not be greater than 3000-5000 electrons nm-2

67
Q

What is the process involved in a CCD detector?

A

Electrons stimulate photon release from scintillator
Photons transferred by fibre optic layer
Photons converted to electronic signal which is then transmitted and stored

68
Q

Who developed the Ballcock?

A

Thomas Crapper

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