Exam 1: M3

Question 1

protein ___ is the most fundamental relationship btwn proteins

Answer 1

homology

Question 2

2 categories of protein homologs

Answer 2

paralogs and orthologs

Question 3

paralogs

Answer 3

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

Question 4

orthologs

Answer 4

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

Question 5

evolutionary relationships can be detected through ____ matrices

Answer 5

substitution; ex. BLOSUM-62 Matrix

Question 6

conservative

Answer 6

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

Question 7

nonconservative

Answer 7

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

Question 8

conservative can also be…

Answer 8

– and + charge; just look for presence of charge

Question 9

if score matrix is >0

Answer 9

conservative

Question 10

if score matrix is <0

Answer 10

nonconservative

Question 11

how to obtain protein of interest

Answer 11

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

Question 12

types of centrifugation

Answer 12

1. Differential
2. Density gradients

Question 12

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

Answer 12

centifugation

Question 12

differential centrifugation

Answer 12

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

Question 13

density gradients

Answer 13

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

Question 14

how to separate protein from other macromolecules like other proteins

Answer 14

column chromatography

Question 15

what is column chromatography

Answer 15

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)

Question 16

in column chromatography, what is connected to the polymer

Answer 16

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

Question 17

what are the 4 types of columns

Answer 17

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

Question 18

what is an affinity column

Answer 18

using specialized tags or antibodies to fish out protein of interest

Question 19

what is gel filtration/size exclusion

Answer 19

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

Question 20

what is an ion exchange column

Answer 20

cationic and anionic; separate molecules based on charge

Question 21

hydrophobic column

Answer 21

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

Question 22

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

Answer 22

affinity

Question 23

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

Answer 23

6 His tag

Question 24

Affinity chromatography: function of 6 His tag

Answer 24

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

Question 25

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

Answer 25

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

Question 26

describe the gel filtration/size exclusion

Answer 26

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

Question 27

anion exchanger

Answer 27

attracts anions to a cation linked to the matrix

Question 28

what is important to consider for ion exchange

Answer 28

pk, pi, solvent ph

Question 29

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

Answer 29

se high salt conc or change the ph

Question 30

describe hydrophobic chromatgraphy

Answer 30

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

Question 31

why is a high salt buffer used for hydrophobic chromatography

Answer 31

more ionic soln makes hydrophobics want to interact even more strongly

Question 32

how to elute sample from hydrophobic chromatography

Answer 32

dec salt conc or add detergetns or change ph

Question 33

4 protein identifying assays

Answer 33

SDS PAGE Western blotting absorbance spectroscopy mass spec

Question 34

SDS-PAGE involves proteins by ____

Answer 34

mass

Question 35

every molecule of SDS binds ___ aa

Answer 35

2

Question 36

how does SDS PAGE work

Answer 36

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

Question 37

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

Answer 37

false

Question 38

what is a blue dye we use to stain gels

Answer 38

coomassie blue

Question 39

western blotting combines SDS-page electrophoresis w

Answer 39

immunoblotting

Question 40

T/F: WB gives us info abt purity

Answer 40

F

Question 41

explain absorbance spectroscopy

Answer 41

estimates protein conc using Beer lambert law

Question 42

which aa absorb light in absorbance spec

Answer 42

Tyr, Phe, Trp @ 280nm

Question 43

absorbance spec is highly dependent on

Answer 43

protein seq

Question 44

define circular dichroism

Answer 44

form of absorption spec

Question 45

explain. circular dichroism

Answer 45

diff secondary structures absorb polarized light diff

Question 46

circular dichroism: alpha helix

Answer 46

-208 min and -222 min (starts high)

Question 47

circular dichroism: beta sheet

Answer 47

195 max and -218 min

Question 48

circular dichroism: random coil

Answer 48

-196 min and >210 max

Question 49

4 diff 3D structural informations

Answer 49

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

Question 50

explain x ray crystallography

Answer 50

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

Question 51

technical considerations to x ray crystallography

Answer 51

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

Question 52

define resolution

Answer 52

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

Question 53

what does NMR tell you

Answer 53

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

Question 54

how does NMR work

Answer 54

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

Question 55

technical considerations of NMR

Answer 55

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

Question 56

what is cryo-electron microscopy

Answer 56

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

Question 57

technical considerations for cryo-electron microscopy

Answer 57

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