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Flashcards in Ch 3 Deck (53):
1

what is the proteome

the full complement of proteins expressed from an organism's genome

2

what does the proteome vary with?

cell type, developmental conditions, environmental conditions

3

how does the size of the proteome differ compared to the genome?

much larger

4

what is the general strategy for protein purification?

Tissue --> disrupt cells --> crude fractionation --> fractionation

5

how do we recognize the protein we are looking for?

perform an assay to identify a unique identifying property of enzye.

6

what is specific activity

ratio of enzyme activity to the amount of protein in a the mixture

7

how does dialysis separate proteins

it separates on the basis of size using a semipermeable membrane with a specific molecular pore size. large molecules cannot pass and get eluted first

8

how does gel filtration work?

also separates by size: column consists of porous beads. large molecules flow faster and elute first

9

what is the advantages of salting out a protein

maintains protein in a negatively folded state, simple process, and fast process

10

ion exchange chromatography

separation is based on net charge at pH 7. if a protein has a positive charge, it will bind to the charged carboxylate groups: negatively charged proteins will not bind. protein is eluted by increasing the concentration of salt added to running buffer. positively charged ions compete with protein for binding

11

affinity chromatography

use a chemical group protein of interest has a high affinity for. Concanavalin A binds to glucose. add it to a column with glucose in it, then elute it with concentrated glucose

12

what is a his tag

a string of 6-12 histidine residues added to N or C terminus of a protein. binds nickel (II) ions.

13

how is a his tag used to purify protein?

protein with his tag is put through column containing Nickel II ions. protein is eluted with imidazole solution that binds to nickel II ions

14

gel electrophoresis

movement of charged solutes through a gel in response to an electric field

15

polyacrylamide

chemically inert; polymerized acrylamide matrix of controlled pore size; allows separation of proteins based on mass and charge

16

how does size affect migration in polyacrylamide gels?

larger proteins migrate slower
smaller proteins migrate faster

17

what does Sodium Dodecyl Sulfate do?

negatively charged detergent used to denature proteins: 1 SDS/2 amino acids. results in a denatured protein with negative charge proportional to protein mass

18

what does mercaptoethanol and dithiothreitol do?

reducing agents that break disulfide bonds and help denature tertiary structure of proteins

19

electrophoretic mobility relationship to mass?

electrophoretic mobility of many proteins is inversely proportional to the logarithm of mass

20

what is isoelectric focusing?

separating proteins by electrophoresis based on native charge

21

what is native charge dependent on?

dependent on the number of acidic and basic residues

22

how is the gel made for isoelectric focusing?

gel is made containing a gradient of pH

23

in isoelectric focusing, where does protein migrate to?

isoelectric point: pH at which the protein's net charge is 0

24

two dimensional electrophoresis

combined isoelectric focusing and SDS page electrophoresis to achieve high resolution separation of proteins.

25

what does ultracentrifugation teach us about?

mas, density, shape

26

what is the sedimentation coefficient?

a means of quantifying the movement of a particle when subjected to a centrifugal force

27

s=m(1-vp)/f what are each of these coefficients

m = mass of the particle
v = partial specific volume (the reciprocal of density)
! = density of the medium
f = frictional coefficient (a measure of the shape of the particle)
(1-vp) = buoyant force exerted by the liquid medium

28

sedimentation coefficients are expressed in what unit?

svedberg units which is s x 10^-13

29

what is the relationship between s and how fast a molecule moves in a centrifugal force?

the smaller s is, the slower a molecule moves in a centrifugal field. the larger the s value, the faster a molecule moves in a centrifugal field

30

how does a particles mass relate to how fast it sediments?

A more massive particle sediments faster than a lighter one.

31

how does shape affect how fast it sediments?

Elongated particles sediment more slowly than compact,
speherical particles

32

how does density affect how fast it sediments?

A dense particle sediments faster due to less opposing buoyant
force.

33

when do particles sink? when do they float? when do they not move? in terms of density?

Particles sink when vp < 1
float when vp > 1
and do not move when vp = 1

34

5 paramters to measure protein purification?

total protein
total activity
specific activity
yield
purification level

35

total protein:

quantity of protein present in a fraction; determined by multiplying the protein concentration by the total volume

36

total activity

determined by mutiplying the activity for a
given volume of protein used in an assay by the total
volume

37

Specific Activity

total activity divided by the total protein

38

Yield

measure of the activity retained after each step;
the activity of the initial crude step is taken to be 100%

39

purification level

measure of the increase in purity after
each step; calculated by dividing the specific acitivity,
after a purification step, by the specific activity of the
initial extract

40

ideally, how do specific activity, purification level, and yield change with each purification step?

specific activity and purification level increase while yield decreases

41

what is maldi-tof?

matrix assisted laser desorption ionization.

1. protein is ionized using laser pulse, acidic matrix ionizes and imparts positive charge to proteins
2. proteins accelerated towards detector using electric field
3. time of flight (tof) is measured
4. smaller molecules travel faster

42

how can you use malditof to identify proteins?

cleave proteins with proteolytic enzymes followed by mass spec to create a fingerprint that can be compared to other protein mass specs. also you can predict cleavage pattern from genomic DNA sequence

43

how would you use maldi-tof with proteolytic digests to identify proteins in a nuclear pore complex?

first use HPLC to fractionate nuclear pore complex and run it on a gel. isolate the protein band from the gel and digest it with trypsin and subject to mass spec.

from this 3 new proteins were identified

44

why is the amino acid sequence important?

1. similarity with a known protein could help infer function
2. comparison of the same protein in different organisms can give info about evolutionary history
3. many proteins contain signal sequences causing them to be localized to one region of the cell

45

what are 2 techniques to determine 3D structure of proteins?

x ray crystallography and nuclear magnetic resonance (nmr)

46

what is a requirement for protein crystallization:

protein must be very pure >90 percent

47

how is a protein crystallized?

purified protein is subject to conditions of high salt, variations in pH, addition of long polymers (polyethylene glycol) to form an ordered lattice of protein molecules

48

what are the 3 components of xray crystallography

protein crystal, x ray source, and an x ray detector

49

how does x-ray diffraction work?

when x-rays pass through protein crystals, they are deflected by the protein molecule's electron clouds, larger atoms (more electrons) will scatter xrays more efficiently than lighter atom

crystal's repeating symmetric formation will allow for deflected xrays to have constructive interference with one another

the intensity of each spot of the diffraction pattern is measured. the image of the protein is then reconstructed from this data using the Fourier transform

xray diffraction data is converted to an electron density map which indicates overall shape of molecule

a molecular model is then built to fit this map

50

when can you use NMR

when proteins do not readily crystallize

to see protein structures' state in a free solution

51

what is required for NMR

highly concentrated solutions of proteins (15 mg/ml)

52

what does nmr depend on

the fact that certain atomic nuclei function as small magnets

53

how does nuclear magnetic resonance work?

spinning of the proton of hydrogen generates a magnetic moment (spin) which can take 2 orientations: alpha or beta

application of radio frequency pulse can cause the spin to go from alpha lower energy to beta (higher energy) the change is called resonance

resonance spectrum is obtained by keeping magnetic field constant and changing the radio frequency

change in spin state is affected by neighboring atoms which causes a chemical shift

nuclei of perturbed samples absorbs radio frequency at a specific frequency

measured in parts per million relative to a solvent standard