Module 1 Section 4

Question 1

Nested Protein levels

Answer 1

the four protein levels exist in a manner that one level higher is completely comprised of level below

Question 2

Primary structure

Answer 2

-linear sequence of AA, read from N-term to C-term

Question 3

Peptide backbone constraints

Answer 3

• alpha carbons separated by 3 covalent bonds
• C-N is the peptide bond
• the resonance in the backbone results in planar structure, partial double bond character
• no rotation around peptide bond
• N-C(alpha) angle is phi
• C(alpha)-C angle is psi

Question 4

Ramachandran Plot

Answer 4

represents the allowed phi and psi for each AA

• glycine has larger range of allowed values (small R)
• proline is greatly restricted
• easily allowed conformations are darker on the plot

Question 5

Secondary structure

Answer 5

• H-bonds form between polar atoms in backbone chain
• alpha helix=10-15 residues long
• beta sheet =3-10 residues

Question 6

Alpha-helix

Answer 6

• most stable arrangement of backbone
• 3.6 AA per turn
• one turn is 5.4 angstroms long
• R groups protrude from helix

Question 7

B-sheet

Answer 7

• second most stable backbone arrangement
• H-bonds between backbone amide and carbonyl groups
• can accommodate large aromatic residues

Question 8

B-sheet orientation

Answer 8

• can run parallel or antiparallel orientations

- opposite N-term to C-term directions strands are antiparallel

Question 9

B-turns

Answer 9

• reversal in direction with 4 AA residues
  1. backbone carbonyl O of 1st bonds to amide H of 4th
• no inter residue bonds between 2nd and 3rd residues
• usually Pro position 2, Gly position 3

Question 10

Gamma turns

Answer 10

• less common than B-turn
• only 3 AA
• 1st+3rd residues form H-bond
• 2nd not involved

Question 11

Tertiary structure

Answer 11

3-D orientation of all secondary structures

-main overall shapes: fibrous, globular

Question 12

Globular protein

Answer 12

-water soluble, spherical

Question 13

Fibrous proteins

Answer 13

• elongated shape, play structural role

- ie. keratin, collagen

Question 14

quaternary structure

Answer 14

• the connection between 2+ polypeptide chains

- protein of 2 subunits called dimer

Question 15

Multisubunit composition

Answer 15

• avoids problem of folding one large polypeptide (ie. one domain not folding properly renders entire protein useless)
• if one subunit misfolds, is excluded from oligomer
• if one subunit denatures, is replaced by another subunit

Question 16

Denaturation

Answer 16

loss of biological activity

Question 17

Oligomer/multimer

Answer 17

protein with multiple polypeptide chains

Question 18

subunits/protomers

Answer 18

individual polypeptide chains

Question 19

homooligomer

Answer 19

oligomer with identical subunits

Question 20

heterooligomer

Answer 20

oligomer with non-identical subunits

Question 21

Protein folding

Answer 21

• some proteins require assistance to fold

- structure always in flux, vibrating rapidly

Question 22

Regular + Misfolded proteins

Answer 22

• nature state of protein selected at AA level, which is the lowest energy state
• if states achieve lower free energy than functional protein conformation, will lead to irreversible aggregations

Question 23

Steps of protein purification

Answer 23

1. Cell lysis
2. Centrifugation
3. Fractionation
4. Protein detection

Question 24

Cell lysis methods

Answer 24

• Detergent (compromise membrane integrity)
• Shear force (apply high freq sonic waves [sonification])
• low ionic salt (cells osmotically absorb water + burst)
• Pressure changes (high pressure breaks cells)

Question 25

About centrifugation of cell lysate

Answer 25

• uses sedimentation principle (denser particles sediment at bottom)
• pellet at bottom, supernatant at top
• differential centrifugation is at different speeds

Question 26

Fractionation by Column Chromatography

Answer 26

• mixture dissolved in mobile phase
• mobile phase passes through stationary phase
• stationary phase causes components of mobile phase to move at different speeds (ie. size, charge)

Question 27

Steps of column chromatography

Answer 27

1. protein mixture applied to column with resin or matrix
2. buffer passed through column to wash away proteins that don’t bind with matrix
3. another buffer applied that causes bound proteins to dissociate from matrix (elution)
4. eluted proteins collected in fraction collector

Question 28

Types of column chromatography

Answer 28

1. Ion exchange
2. Size-exclusion
3. Affinity

Question 29

About Ion exchange chromatography

Answer 29

• resin contains anion/cation exchange resin bonds anions/cations respectively
• elution due to increasing salt solution concentration
• pI used for each protein, character depends on the pH of solution dissolving proteins

Question 30

About size exclusion chromatography

Answer 30

• matrix has pores of specific size, proteins smaller than pore can enter
• proteins that enter pores take longer to elute
• large proteins elute fastest

Question 31

About affinity chromatography

Answer 31

• uses fact that many proteins specifically bind other mlc/ligands as part of their function
• column can have ligand covalently attached to matrix

Question 32

SDS-PAGE steps

Answer 32

• used for protein detection
  1. treated samples loaded into wells @ top of gel, electric field applied to gel (SDS highly -ve so proteins move towards +ve cathode @ bottom)
  2. proteins migrate at diff rates accoording to relative molecular mass (smaller=faster)
  3. Gel removed from between glass, soaked in acidic buffer to ‘fix’ proteins (gel treated w dye that selectively binds to proteins)