Ch. 5

Question 1

Fibrous Protein

Answer 1

Simple, linear structure
Insoluble in water

Question 2

Ex. of Fibrous Protein

Answer 2

Collagen, Keratin

Question 3

Globular Protein

Answer 3

Spherical structure
nonpolar inside and polar outside
soluble in water

Question 4

Ex. of Globular Protein

Answer 4

Enzymes

Question 5

Membrane Protein

Answer 5

Embedded in cell membrane
polar inside and nonpolar outside
not soluble in water

Question 6

Crude Isolation/Purification Techniques

Answer 6

Centrifugation, Salting out, and Dialysis

Question 7

Centrifugation

Answer 7

Separates based on density

Question 8

Salting out

Answer 8

Increase salt concentration until proteins precipitate out

Question 9

Dialysis

Answer 9

Separate Proteins from small molecules/ions by diffusion across semipermeable membrane

Question 10

Chromatography Techniques

Answer 10

Ion Exchange, Size Exclusion, Hydrophobic Interaction, Affinity Chrom., and HPLC

Question 11

Ion Exchange

Answer 11

Separates based on charge
charged matrix
like charges move faster through matrix

Question 12

Size Exclusion

Answer 12

Matrix made of porous beads
Larger proteins move faster bc small proteins get trapped in the beads

Question 13

Hydrophobic Interaction

Answer 13

Matrix of covalently linked hydrophobic groups
Hydrophobic sticks, hydrophilic pass through quickly

Question 14

Affinity Chromatography

Answer 14

Proteins bind certain functional groups or ligand
Matrix has ligand or functional group attached
Binding interaction traps proteins

Question 15

HPLC (High-performance liquid chromatography)

Answer 15

Limited to small scale due to tightly packed matrix and high pressure
Works well with other techniques

Question 16

Electrophoresis Techniques

Answer 16

SDS-PAGE, Isoelectric Focusing, and 2D Electrophoresis

Question 17

SDS-PAGE (Sodium dodecylsulfate)

Answer 17

Disrupts tertiary structure and gives everything a (-) charge
Proteins separated based on MW (Smaller=Faster)
Makes protein soluble

Question 18

Isoelectric Focusing

Answer 18

Based on relative amts. of acidic and basic AA
Matrix is a pH gradient from low to high, protein stops at pI

Question 19

Isoelectric Point

Answer 19

pH at which protein is neutral
Low pI = more acidic groups
High pI = more basic groups

Question 20

2D Electrophoresis

Answer 20

Mixture separated by pI then SDS-PAGE

Question 21

How to break a protein into individual AA’s

Answer 21

6M HCl and 110C

Question 22

Structure Determination Steps

Answer 22

1. Separate and purify polypeptide chains
2. Cleave S-S bonds
3. Determine N-terminal and C-terminal AA
4. Cleave polypeptide chain into shorter fragments
5. Repeat step 4. with diff cleavage method
6. Reconstruct AA sequence through overlaps

Question 23

1. Separate and purify polypeptide chains

Answer 23

Disrupt noncovalent forces by
changing pH, high salt conc., treat with urea or guanidinium chloride (breaks up noncovalent interactions)

Question 24

2. Cleave S-S bonds

Answer 24

Oxidation-performic acid
Reduction- 2-mercaptoethanol/dithiothreitol
Cap thiols to prevent disulfide bond from refroming

Question 25

3. Determine N-terminal and C-termial

Answer 25

N-terminal - Edmans reagent C-terminal - carboxypeptidase Y

Question 26

4. Cleave Polypeptide Chain

Answer 26

Enzymatic and Chemical Cleavage

Question 27

Enzymatic Cleavage Methods

Answer 27

Trypsin, Chymotrypsin, Staphylcoccal

Question 28

Trypsin

Answer 28

Cuts peptide bond on C-terminal side of Lysine (Lys) and Arginine (Agn)

Question 29

Chymotrypsin

Answer 29

Cuts peptide bond on C-terminal side of Phenylalanine (Phe), Tyrosine (Tyr), and Tryptophan (Trp)

Question 30

Staphylcoccal

Answer 30

Cuts peptide bond on C-terminal side of Aspartic Acid (Asp) and Glutamic Acid (Glu)

Question 31

Chemical Cleavage Methods

Answer 31

Cyanagen bromide (CNBr) Cuts peptide bond on C-terminal side of Methionine

Question 32

Hydrophobic Proteins are most commonly found where?

Answer 32

Globular Proteins

Question 33

Sequence Similarity implies evolutionary relatedness (Name?)

Answer 33

Homologous Proteins

Question 34

Conjugated Protein

Answer 34

Protein containing a non-protein part

Question 35

NH2 Protecting Groups

Answer 35

FMOC and BOC

Question 36

Reagent used to make OH a better leaving group in protein synthesis

Answer 36

DIPCDI

Question 37

Purification and Protection in protein synthesis

Answer 37

Attach C-terminal to insoluble resin (Solid phase peptide synthesis) Purification by filter

Question 38

Direction of synthesis in nature and chemically

Answer 38

Nature: Starts at N-Terminal Chemically: Starts at C-Terminal

Question 39

Examples of Ligand Bound Proteins

Answer 39

Transport proteins, enzymes, regulatory proteins, and scaffold proteins