Chapter 4 Proteins: Determination Of Primary Structure

Question 1

Proteins

Answer 1

Structural support, storage, movement, signaling, defense, enzymes/regulation

Question 2

Protein classification

Answer 2

Classified based on non-aa components
-simple: protein alone

-Conjugated: non-aa components
Nucleoproteins
Lipoproteins
Glycoproteins
Chromoproteins
Mealloproteins

Question 3

Life cycle of a protein

Answer 3

Synthesis —–> Folding——->Processing——–> covalent modification——-> translocation——->activation——>catalysis———-> Aging———> ubiquitination——–> Degradation

Question 4

Factors controlling protein activity

Answer 4

pH - keep pH stable to avoid denaturation or chemical degradation
Enzymes - May affect structure
Temperature- control denaturation, and controls enzyme activist
Thiol Groups - Reactive, add protecting group to prevent formation of new disulfide bonds
Exposure to air or water - denature or oxidize, Store under N2 or Ar, keep concentration high

Question 5

Proteins expressed at high levels

Answer 5

Collagen and hemoglobin

Question 6

Proteins expressed at low levels

Answer 6

Repressors and signaling

Question 7

To Study proteins

Answer 7

Have to purify proteins

• Choose source
• Separate proteins using fractionation based of physical characteristics
• Solubility
• electrical charge or polarity
• size and shape
• affinity for other molecules

Question 8

Purification based on charge

Answer 8

1 Ion exchange chromatography
2 Electrophoresis
3 Isoelectric Focusing

Question 9

Protein Purification on Size

Answer 9

1 Dialysis and ultracentrifugation
2 Gel electrophoresis
3 Gel filtration chromatography

Question 10

Protein purifying on specificity

Answer 10

Affinity chromatography

Question 11

Protein purification based on polarity

Answer 11

1 Adsorption chromatography
2 paper Chromatography
3 Reverse-phase chromatography
4 Hydrophobic chromatography

Question 12

Primary Structure

Answer 12

Particular sequences of amino acids in a protein, backbone of a peptide chain or protein.

• Sequence determines structure and function of a protein.
• Conversion of Glu to Val in hemoglobin which leads to sickle cell anemia

Question 13

Insulin

Answer 13

First protein to have its primary structure determined

• Starts as a single peptide chain (preproinsulin)
• Mature form has 2 chains connected by disulfide bonds

Question 14

Protein identification by Mass Spectrometry

Answer 14

Proteins need to be ionized by

• Electrospray Ionization
• Matrix-AssistedLaser Desorption

Question 15

Post- Translational Modifications by MS

Answer 15

• Almost all proteins are modified during or after synthesis to create a much larger repertoire from limited genes
• Side chain modification
• Cleavage

Question 16

Post Translational Modifications

Answer 16

• Used for regulation of protein activity, transport, and secretion
• Cannot be definitively predicted from DNA sequence
• Can involve complex systems and enzymes
• Dynamic since some are reversible (phosphorylation and acetylation)
• Can cause some diseases

Question 17

Advantages of MS

Answer 17

High specificity, high sensitivity, high coverage, multiple proteins can be identified in a single analysis, type of PTM and exact location of modified residues can be determined.

Question 18

Enzyme

Answer 18

Catalyze chemical reactions

Question 19

Hormones

Answer 19

Messengers that regulate bodily functions

Question 20

Storage proteins and transport proteins

Answer 20

• Storage proteins make essential substances readily available
• Transport proteins carry substances through body fluids

Question 21

Structural, protective and contractile proteins

Answer 21

• Structural proteins support and maintain cell shape
• Protective proteins provide defense
• Contractile proteins do mechanical work

Question 22

2D-Gel Electrophoresis

Answer 22

1. Subject the protein sample to isoelectric focusing in a pl 3-10 gradient
2. Place IEF gel horizontally on top SDS-PAGE gel further resolving the proteins.
3. Stain gel with Coomassie blue

Question 23

Western lot

Answer 23

1. Separate proteins
2. Transfer separated proteins onto nitrocellulose or PVDF membranes
3. Blocking
4. Incubation of member pane with primary and secondary antibodies
5. Detect using chemiluminescence or colorimetric methods

Question 24

Using Edman Degradation to determine primary sequence

Answer 24

1. Introduce Edman’s reagent to label the amino-terminal residue.
2. PTH derivatives can be removed generating a new amino-terminal residue
3. Use successive rounds of derivatization with Edman’s reagent to sequence many residues of one peptide
4. Identify PTH derivatives by RP-HPLC or CE retention times determining the primary sequence

Question 25

Tandem MS

Answer 25

1. Proteases break proteins into peptides 2. A Tandem MS breaks the peptides down into fragmented ions measuring the mass of each. 3. MS accelerates the fragmented ions. (The heavier the slower) 4. MS measures mass/charge ratio of the ion.. 5. These fragmented ions are used to determine primary sequences using protein databases.