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Biochem > Exam 1: M3 > Flashcards

Exam 1: M3 Flashcards

1
Q

protein ___ is the most fundamental relationship btwn proteins

A

homology

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2
Q

2 categories of protein homologs

A

paralogs and orthologs

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3
Q

paralogs

A

homologs w/in same species; very similar sequences like Mb and Hb

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4
Q

orthologs

A

homologs w/in diff species; related protein in 2 diff species

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5
Q

evolutionary relationships can be detected through ____ matrices

A

substitution; ex. BLOSUM-62 Matrix

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6
Q

conservative

A

similar; ex. aspartic acid and glutamic acid are both -

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7
Q

nonconservative

A

dissimilar; aspartic acid and tryptophan- one is polar and charged other is bulky and hydrophobic

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8
Q

conservative can also be…

A

– and + charge; just look for presence of charge

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9
Q

if score matrix is >0

A

conservative

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10
Q

if score matrix is <0

A

nonconservative

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11
Q

how to obtain protein of interest

A
  1. DNA sequence amplified by PCR
  2. Amplified DNA& circular plasmic treated w specific restriction enzymes
  3. Digested DNA placed into plasmid (ligation)
  4. Transofmred bacteria grown to certain density
  5. Add small molecule to ‘turn on’ expression machinery
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12
Q

types of centrifugation

A
  1. Differential
  2. Density gradients
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12
Q

how to separate cell debris from soluble proteins or separate proteins according to mass and shape

A

centifugation

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12
Q

differential centrifugation

A

applying low G forces to separate soluble proteins from other larger debris. expose to higher G force to separate out soluble proteins from aggregated proteins…using differential amounts of F force to separate diff molecules we care abt

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13
Q

density gradients

A

prepare sucrose and apply the sample to top and spin it at very high G forces and our proteins migrate through that density gradient till they reach the same density of the protein in which they would stop

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14
Q

how to separate protein from other macromolecules like other proteins

A

column chromatography

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15
Q

what is column chromatography

A

separation of particles based on chemical and physical properties; use a solid matrix (natural or synthetic polymers) w/ specific chemical properties (stationary phase) and pass a soln of protein and other components (mobile phase) through column/matrix (mobile)

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16
Q

in column chromatography, what is connected to the polymer

A

a specific property (charge for ex) that would help fish out protein of interest

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17
Q

what are the 4 types of columns

A
  1. affinity
  2. gel filtration (size exclusion)
  3. Ion exchange
  4. Hydrophobic
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18
Q

what is an affinity column

A

using specialized tags or antibodies to fish out protein of interest

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19
Q

what is gel filtration/size exclusion

A

using size or shape of a protein to separate proteins in a complex mixture

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20
Q

what is an ion exchange column

A

cationic and anionic; separate molecules based on charge

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21
Q

hydrophobic column

A

can separate based on degree of hydrophobicity–> nonpolar side chains

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22
Q

the matrix has a special functional group that allows for the protein of interest to bind to in what type of chromatography

A

affinity

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23
Q

affinity chromatography: what can u add to protein of interest on either N or C terminus

A

6 His tag

24
Q

Affinity chromatography: function of 6 His tag

A

binds to Zn or Ni linked to agarose so the protein of interest is attached to matrix which will be later washed and eluted by imidazole

25
Q

Affinity chromatography: why can imidazole be used to release/elute protein of interest

A

it acts as a salt and looks like His so it competes for Ni and bump 6His tag off column

26
Q

describe the gel filtration/size exclusion

A

series of beads w tiny pores on them
when soln runs through column, larger ones go thru faster and smaller ones travel slower; can run standards to predict when protein will elute

27
Q

anion exchanger

A

attracts anions to a cation linked to the matrix

28
Q

what is important to consider for ion exchange

A

pk, pi, solvent ph

29
Q

how to elute proteins of interst from cation or anion exchnage columns

A

se high salt conc or change the ph

30
Q

describe hydrophobic chromatgraphy

A

hydrophobic interactions drive exclusion of nonpolar residues; use high salt buffer; substitute matrix w/ hydrophobic groups

31
Q

why is a high salt buffer used for hydrophobic chromatography

A

more ionic soln makes hydrophobics want to interact even more strongly

32
Q

how to elute sample from hydrophobic chromatography

A

dec salt conc or add detergetns or change ph

33
Q

4 protein identifying assays

A

SDS PAGE
Western blotting
absorbance spectroscopy
mass spec

34
Q

SDS-PAGE involves proteins by ____

A

mass

35
Q

every molecule of SDS binds ___ aa

A

2

36
Q

how does SDS PAGE work

A

in prescence of SDS, proteins carry overall net negative charge and migrate towards anode; heat denatures protein of interest and SDS binds to protein of interest stoichiometrically so every protein will have same mass:charge ratio–> apply sample to gel matrix which pulls proteins thru matrix by applying electrophoresis so they move to positive end of gel

37
Q

T/F: on SDS page, smallest proteins are on top

A

false

38
Q

what is a blue dye we use to stain gels

A

coomassie blue

39
Q

western blotting combines SDS-page electrophoresis w

A

immunoblotting

40
Q

T/F: WB gives us info abt purity

A

F

41
Q

explain absorbance spectroscopy

A

estimates protein conc using Beer lambert law

42
Q

which aa absorb light in absorbance spec

A

Tyr, Phe, Trp @ 280nm

43
Q

absorbance spec is highly dependent on

A

protein seq

44
Q

define circular dichroism

A

form of absorption spec

45
Q

explain. circular dichroism

A

diff secondary structures absorb polarized light diff

46
Q

circular dichroism: alpha helix

A

-208 min and -222 min (starts high)

47
Q

circular dichroism: beta sheet

A

195 max and -218 min

48
Q

circular dichroism: random coil

A

-196 min and >210 max

49
Q

4 diff 3D structural informations

A
  1. X Ray crystallography
  2. NMR
  3. Electron microscopy
  4. Protein Data Bank
50
Q

explain x ray crystallography

A

expose protein crystals to x-rays to obtain diffraction patterns; need info from diffraction patterns (wave intensities) & phase to calc e- density map

51
Q

technical considerations to x ray crystallography

A

need to grow crystals
no limits on protein size
up to atomic resolution (1.5-3A)
snapshot of structure only

52
Q

define resolution

A

distance btwn distinguishable factors
higher resolution = lower distance
lower resolution - blurry image, higher number

53
Q

what does NMR tell you

A

inter-atomic distance btwn 2 nearby nuclei that are covalently attached or close in 3D space

54
Q

how does NMR work

A

aligns nuclear spins in constant magenetic field and pulse w radio waves

55
Q

technical considerations of NMR

A

don’t need crystals
smaller proteins (30 kDa)
need nuclei w spin
ensemble of structures/messy

56
Q

what is cryo-electron microscopy

A

pass e- beam through sample frozen in vitreous ice and get direct image

57
Q

technical considerations for cryo-electron microscopy

A

no crystals required
molecules and assemblies–> can be very large and heterogenous
not good for small proteins
resolution limits traditionally low but ever inc
low contrast

58
Q
A

Biochem (6 decks)

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