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SURVEY OF BIOCHEMISTRY

Amino Acids and Proteins

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Amino Acids

Can you give the 1-letter and 3-letter names for all 20 amino acids within 5 minutes?

Can you draw a oligopeptide of any given sequence?

With correct stereochemistry?

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Amino Acids: Nonpolar Side Chains

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Amino Acids: Polar, Uncharged Side Chains

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Cysteine & Disulfide Bonds

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Cysteine & Disulfide Bonds

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Amino Acids: Polar, Uncharged Side Chains

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Amino Acids: Polar, Uncharged Side Chains

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Amino Acids:Polar, Charged

Be able to draw,name and give1-letter/3-letter

codes for all 20

amino acids!

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pKa and pI

• pI = Isoelectric pointthe pH at which a molecule carries no net electric charge

pKa1pKa2

pI = [pKa1 + pKa2]

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1

2

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Polypeptide SequenceNomenclature

Tetrapeptide: AYDGBe able to drawa polypeptide!

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Peptide Bond

Peptide bond links one amino acid to another.

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Torsion Angles in Proteins

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Psi(Ψ)

Phi(Φ)Peptides typically assumethe trans

conformation

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Torsion Angles Defined…

• Psi: angle made from atomsN-Calpha-Ccarbonyl-Nn+1

NH

NN

O O

OH

H

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Torsion Angles Defined…

• Phi: angle made from atomsCcarbonyl,n-1-Nn-Calpha-Ccarbonyl,n

NH

NN

O O

OH

H

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Alpha Helix

Sequence coils in a right-handedmanner.

Notice hydrogen bonding along the helical axis from carbonyl oxygen

(of residue n) to the amino hydrogenof residue (n+4).

Hydrogen bonding stabilizes the alpha helical structure!

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Beta Sheets

Notice the hydrogen bondingfrom the carbonyl

oxygen to the amino

hydrogen

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Protein Purification Overview

Courtesy of

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Salting Out: (NH4)2SO4

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Gel Filtration Chromatography

Separation based on Size

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Ion Exchange Chromatography

Separation based on Charge

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Affinity Chromatography

Affinity What could be to cause proteinsto elute off of anaffinity column?

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Ways to Assess Protein Purification

• Total Protein Concentration Assays– Beer’s Law– Colorimetric Assays

• Specific Protein Assays– Activity Assays– Immunoassays

• ELISA• RIA

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Beer’s Law

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A = εbcWhat is the molar concentration of a

solution of Bovine Serum Albumin (BSA) that exhibits

an A280 of 0.75 with a path length of 1 cm?

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Beer’s Law

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A = εbcWhat is the molar concentration of a

solution of Bovine Serum Albumin (BSA) that exhibits

an A280 of 0.75 with a path length of 1 cm? Conc. =

0.75(43,824 M-1cm-1) x (1 cm)

Conc. = 0.00001711 M = 17 µM

Must know the value of

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ε

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Bradford Protein Assay

Suppose you have a protein mixture and you need to determine the protein concentration.

You cannot use Beer’s Law.Because you would not know the extinction coefficient

for the protein mixture at 280 nm

Alternative approach: Bradford Protein Assay

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Bradford Protein Assays

How is data generated?

How is data analyzed?

Bradford Protein Assay reagent

contains Coomassie brilliant blue which reacts with basic

(esp. Arg) and aromatic amino acids to yield a blue

color with intensity proportional

to the protein concentration.

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Data Generation: Bradford Assay

Reference contains only buffer

Create a series of protein standards at known

increasing concentrations and mix with the Bradford reagent

2.5 5 10 15 20 25 µg/mL

Treat the protein mixture of unknown concentration

in the same manner as the standards and compare -

visually and spectrophotometrically!

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Bradford Assay Data Analysis

Graph the absorbance at 595 nm as a

function of [standard] in µg/mL.

Fit the line and use itto deduce the [protein]

in the mixture based on

its absorbance.