Chapter 4 Proteins: Determination Of Primary Structure Flashcards Preview

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Flashcards in Chapter 4 Proteins: Determination Of Primary Structure Deck (25):
1

Proteins

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

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Protein classification

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

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

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Life cycle of a protein

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

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Factors controlling protein activity

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

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Proteins expressed at high levels

Collagen and hemoglobin

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Proteins expressed at low levels

Repressors and signaling

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To Study proteins

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

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Purification based on charge

1 Ion exchange chromatography
2 Electrophoresis
3 Isoelectric Focusing

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Protein Purification on Size

1 Dialysis and ultracentrifugation
2 Gel electrophoresis
3 Gel filtration chromatography

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Protein purifying on specificity

Affinity chromatography

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Protein purification based on polarity

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

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Primary Structure

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

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Insulin

First protein to have its primary structure determined
-Starts as a single peptide chain (preproinsulin)
-Mature form has 2 chains connected by disulfide bonds

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Protein identification by Mass Spectrometry

Proteins need to be ionized by
-Electrospray Ionization
- Matrix-AssistedLaser Desorption

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Post- Translational Modifications by MS

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

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Post Translational Modifications

-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

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Advantages of MS

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.

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Enzyme

Catalyze chemical reactions

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Hormones

Messengers that regulate bodily functions

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Storage proteins and transport proteins

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

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Structural, protective and contractile proteins

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

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2D-Gel Electrophoresis

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

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Western lot

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

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Using Edman Degradation to determine primary sequence

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

25

Tandem MS

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.