Lesson 5: Protein Folding and Methods in Protein Biochemistry

Question 1

Heat

Answer 1

tends to be irreversible (think fired egg)

Question 2

pH

Answer 2

tends to be reversible (but not advisable) too high/too low pH can damage amide backbone (i.e. hydorlysis)

Question 3

chaeotropic salts

Answer 3

reversible precipitation and/or unfolding

Question 4

urea

Answer 4

interacts with amide backbone (and/or solvation shell of protein); denature proteins

Question 5

ammonium sulfate (Na4)2SO4

Answer 5

changes the activity of water in molar excess; precipitates folded proteins

Question 6

Tm

Answer 6

melting temperature - temperature where 50% of protein is denatured

ex: if you “remove” one s-s bond, then less heat is required to reach Tm

Question 7

% protein folded in native state vs. [urea]

Answer 7

urea interacts with the peptide backbone - as [urea] increases, there is a linear decrease in the % of proteins in native state

Question 8

% protein folded in native state vs. [ammonium sulfate]

Answer 8

added in molar excess - changes the activity of water. Non-linear because all proteins are differential
1st (on top) - more hydrophobic, less solvation, ppt first
2nd (on bottom) - more hydrophilic, more solvation, ppt first

Question 9

how to determine % folded correctly if there are 8 -HS

Answer 9

1/7 X 1/5 X 1/3 X 1/1 = 1/105 = 0.95%
*** take away 2 each time

Question 10

result of native molecule –> reduce (BME) –> denature (urea) –> denatured molecule –> remove urea –> oxidize

Answer 10

nearly 100% of protein folded correctly
- with urea removed first, the only thing then that is missing is to “turn on” disulfides

Question 11

result of native molecule –> reduce (BME) –> denature (urea) –> denatured molecule –> oxidize –> remove urea

Answer 11

leads to completely random disulfide formation b/c there no tertiary structures yet –>

Question 12

results of Anfinsen experiment

Answer 12

• nearly 100% original enzyme activity and structure was restored (when Urea was removed first)
• protein folding is not random
• primary sequence dictates higher order structures (as long as primary structure is not disturbed)

Question 13

characteristics of protein

Answer 13

• every protein has a pI where the net charge = 0
• solubility of protein tends to be minimized when pH approaches pI
• acidic proteins have pI < 7 and basic proteins have pI > 7

Question 14

at lower valus of pH, proteins will carry a more () charge

Answer 14

positive

Question 15

at higher values of pH, proteins will carry a more () charge

Answer 15

negative

Question 16

sonification

Answer 16

• a way to prepare a protein extract from prokaryotic and eukaryotic cells *
• high frequency sound

Question 17

enzymatic and physical

Answer 17

• a way to prepare a protein extract from prokaryotic and eukaryotic cells *
• lysozyme and freeze/thaw

Question 18

column chromatography types

Answer 18

• gel filtration
• ion exchange
• affinity

Question 19

gel filtration

Answer 19

• size exclusion
• larger molecules elute first

Question 20

ion exchange

Answer 20

• charged matrix
• exploits charge difference between proteins
• uses 2 matrices: DEAE (positively charged) –> anion exchange, CMC (negatively charged) –> cation exchange
  ** remove bound proteins w/ elution buffer
  ** ions that have the same charge (either positive or negative) as the matrix elute first

Question 21

affinity

Answer 21

• highly specific, binds to ligand then elutes from column
  – to deptrotonate from bound matrix, add free ligand (DNA) or something that mimics DNA or urea

Question 22

visualizing proteins

Answer 22

proteins are charged, therefore they will migrate in an electric field
» acidic and basic proteins do not migrate in the same manner

Question 23

SDS-PAGE

Answer 23

• sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• confers the same shape and charge so proteins can be separated by their size

Question 24

how does SDS work

Answer 24

• SDS binds to proteins w/ a specific stoichiometry (one SDS molecule for every 2 amino acids)
• binds to peptide backbone and disrupts all of non-covalent forces that hold the peptide together

Question 25

SDS breaks up

Answer 25

noncovalent bonds

Question 26

BME breaks up

Answer 26

disulfide bonds

Question 27

gel electrophoresis

Answer 27

• largest molecules have least electrophoretic mobility (more friction, slower movement)
• add BME before running gel to ensure protein is completely linear (also boil it), and has no disulfide bonds

Question 28

order of flow for electrophoresis

Answer 28

(-) cathode to (+) anode