cellular peptides

8/12/2019 Cellular peptides

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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, Charged

Be able to draw,

name and give1-letter/3-letter

codes for

all 20

amino acids!


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pKaand pI

pI = Isoelectric pointthe pH at which a molecule carries nonet electric charge

pKa1pKa2

pI = [pKa1+ pKa2]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

Psi()

Phi()Peptides

typically

assume

the transconformation


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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

N

NO O

OH

H


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Alpha Helix

Sequence coils in a right-handed

manner.

Notice hydrogen bonding along thehelical axisfrom carbonyl oxygen

(of residue n) to the amino hydrogen

of residue (n+4).

Hydrogen bonding stabilizes the

alpha helical structure!


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Beta Sheets

Notice the

hydrogen

bonding

from thecarbonyl

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

AffinityWhat could beto cause proteins

to elute off of an

affinity column?

W t A P t i


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Ways to Assess ProteinPurification

Total Protein Concentration AssaysBeers Law

Colorimetric Assays

Specific Protein Assays

Activity Assays

Immunoassays ELISA

RIA


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Beers Law

A bcWhat is the molar concentrat ion o f a solut ion of

Bovine Serum Album in (BSA) that exhibi ts anA 280of 0.75 w ith a path leng th o f 1 cm?


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Beers Law

A bcWhat is the molar concentrat ion o f a solut ion of

Bovine Serum Album in (BSA) that exhibi ts anA 280of 0.75 w ith a path leng th o f 1 cm?

Conc. =0.75

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

Conc. = 0.00001711 M = 17 M

Must know

the value of


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Bradford Protein Assay

Suppose you have a protein mixture and you need to

determine the protein concentration.

You cannot use Beers 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 increasingconcentrations and mix with

the

Bradford reagent2.5 5 10 15 20 25 g/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 g/mL.

Fit the line and use it

to deduce the [protein]

in the mixture based on

its absorbance.

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