Chapter3 Spring 2013 MB

7/27/2019 Chapter3 Spring 2013 MB

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Lehninger Principles of Biochemistry

Fall 2012

Chapter 3

Amino Acids, Peptides, and Proteins


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Basic structure of amino acids (a.a.s)

All are -amino acids except for proline

The R is a side chain that distinguishes the amino acids

Aas are in the L configuration

Figure 3-2 in 6e

Figure 3-3 in 5e


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a.a.s Terminology

- Carbon

Charge states

Nomenclature

Full name, 1- Letter, 3- Letter codes

Figure 3-9 in 5e


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Aromatic Amino Acids

Figure 3-5 in 5e

Aromatic a.a.s

Abbreviations

Phe, F

Tyr, Y Trp, W

Unique Properties


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Spectroscopic Properties of

Aromatic a.a.s

Beers Law: A= abc

Box 3-1 in 5e


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Spectroscopic Properties of

Aromatic a.a.s

Spectroscopic properties of thesea.a.s allow us a way to measurethe concentrations of proteins in

solutions using at Abs at 280 nm

and Beers Law. Trp has the largest aromatic ring

structure and the strongest

absorbance, proteins high in Trp

residues have high Abs, while

those with little to no Trp/Tyr

have little to no Abs at 280 nm.

Figure 3-6 in 5e

Raise in Abs at 190

nm is due to the

peptide bond


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Polar, uncharged a.a.s

Polar, unchargeda.a.s

Abbreviations

Ser, S

Thr, T

Cys, C

Asn, N

Gln, Q Unique Properties

Figure 3-5 in 5e


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Disulfide Bonds

Figure 3-7 in 5e


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Basic a.a.s

Positively charged R

groups

Abbreviations

Lys, K Arg, R

His, H

Unique Properties

Figure 3-5 in 5e


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Acidic a.a.s

Negatively charged

R groups

Abbreviations

Asp, D Glu, E

Unique Properties

Figure 3-5 in 5e


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Uncommon Amino Acids

Figure 3-8a in 5e


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Charged states of a.a.s

Side chains also play a role in the charged states! If they havea pKa the side chain will play a role in overall charge.

Figure p79 in 5e


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a.a. pKas The proximity of the COOH group and the NH3 group

affects both of their pKa values.

Figure 3-11 in 5e


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pKas

Table 3-1 in 5e


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Expected pKa ranges for a.as in proteins

funct ional group pKa

alpha-carboxyl 3.5- 4.0

side chain carboxyl(Asp and Glu)

4.0- 4.8

imidazole (His) 6.5- 7.5

t hiol (Cys) 8.5- 9.0

phenol (Tyr) 9.5-10.5

alpha-amino 8.0- 9.0

side chain amino (Lys) 9.8-10.4

guanidinyl (Arg) ~12


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How to Draw a Titration Curve

pH vs. Equivalents of NaOH Looking at the ionization of each

ionizable group (where protons can

be removed)

Start with a completely protonatedamino acid/peptide, then

deprotonate each group based upon

the pKa value.

What happens when the pH = pKa?

0

2

4

6

8

10

12

14

0 0.5 1 1.5 2 2.5

pH

Equivalents of NaOH


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Consider the titration

1 equivalent of Gly with 0.5 equivalents of NaOH

1 equivalent of Gly with 1 equivalent of NaOH

1 equivalent of Gly with 1.5 equivalents of NaOH

1 equivalent of Gly with 2 equivalents of NaOH

pKa1= 2.34 pKa2= 9.60


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Whats the pI?

The isoelectric point, the pH when the charge is 0 Each amino acid/peptide has only ONE pI

pI = (pKa1+ pKa2)

pI

Figure 3-10 in 5e


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Consider the titration

Aspartate has 3 ionizable groups

What would a titration curve look like?

What is the pI?

pKa1= 1.88 pKa2= 9.60 pKaR= 3.65

Consider a dipeptide Ala-Cys

Ala pKa1= 2.35 pKa2= 9.87 Cys pKa1= 1.92 pKa2= 10.78 pKaR= 8.33


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Determining whether something is positively charged or

negatively charged

Using the pI

pI

pH scale

0

14 If the pH falls in avalue above the pI, the

a.a./peptide will be

negatively charged

If the pH falls in a

value below the pI, the

a.a./peptide will be

positively charged

-+


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Titration Curves, where is the pI?

Figure 3-12 in 5e


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Acid-Base Problem with a.as

Determine the %composition of Glu

(in terms of ionization

state) in a sample atpH of 8

Figure 3-12 in 5e


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Peptide Bond

Figure 3-13 in 5e


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Mechanism of Peptide Bond

Formation


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Classification of Amino Acid

polymers

Oligopeptidefew amino acids Polypeptidemany amino acids, 10,000 MW


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First look at a peptide

Written from N terminus tothe C terminus

What are the ionizable

groups?


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First look at a peptide

Identify each amino acid

What are the ionizable groups?

Figure 3-14 in 5e


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Aspartame- the sweetener

Figure 3 p83 in 5e


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Protein Structure


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Protein Purification

Purify proteins based upon protein properties Expression- Native vs. non-native

Problem: Have your protein, but its mixed with everything

else How do you get it out? How do you separate it?


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Break open cells, centrifuge

First you need toget the protein out

of the cell. Cells

are broken open

by sonicaters,homogenizers,

etc., then

centrifuge

Next you need toget the protein

from the other

mixture.


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Fractionation by Salting Out

Changing the amount of salt alters the solubility of a protein,

water will more favorable interact with the small salt ions

than the large protein molecule.

http://irfanchemist.wordpress.com/2009/04/19/isolation-of-protein/


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Column Chromatography

Separating proteins basedupon their different

properties, collect

fractions (fractionate) as

they elute from a columnto separate molecules.

Mobile phase: solution

(buffer/protein)

Stationary phase: beads

Figure 3-16 in 5e


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Size-exclusion chromatography

Separates proteins based onsize.

Stationary phase: beads with

small pores

Beads have small pores, the

smaller proteins will travel

through the pores traveling a

longer distance making them

travel slower. The larger

proteins elute first.

Figure 3-17 in 5e


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Y h ll th f ti


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You have all these fractions, now

what?

You have to find the sample with your protein to do yourassays.

How do you find it?

SDS-PAGE

Activity assays

SDS Page


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SDS-Page Mix protein 1:1 with a 2X Loading Dye, the run it on a gel

Loading Dye contains: SDS and a Reducing Agent (ME or

DTT), it reduces and lines the protein.

Smallest proteins run the farthest.

Figure 3-18 in 5e


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Native PAGE Versus SDS-PAGE

Native PAGE does not usethe loading dye. The

protein is not reduced or

lined with SDS. Hence it

runs differently on the gel.

http://www.ncbi.nlm.nih.gov


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Using SDS-PAGE to find MW

log(Mr) vs.Relative

Migration can

get you the

molecularweight of your

protein.

Figure 3-19 in 5e


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Based on the following information, determine the

molecular weight of the unknown peptide

Size of MW standard

(kDa)

Log MW Relative Migration

Distance of the

protein on the Gel

10 1 1

30 1.48 0.8

40 1.6 0.7

80 1.9 0.3

Unknown MW peptide 0.5

Isoelectric Focusing


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Isoelectric Focusing

Figure 3-20 in 5e

What is the charge of the following peptide at pH


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What is the charge of the following peptide at pH

2, 5, and 8? What is the isoelectric point of the

peptide?

Lys-Asp-Ala


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2-D Gels

Figure 3-21 in 5e

Run an IEF gel to

separate based onpI, then put the gel on

top of a SDS-gel and

separate based on size.


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Draw the SDS-PAGE gel, IEF Gel, and

2-D SDS Gel for the following proteins

Sample pI MW (kDa)

Protein 1 3 80

Protein 2 6 80

Protein 3 3 200


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Activity vs. Specific Activity

Activity: mM Product/s Specific Activity: mM

Product/s * mg protein

Specific activity takes into

consideration the purity of

the protein, the higher the

specific activity the more

pure the protein.

Figure 3-22 in 5e


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Activity vs. Specific Activity


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Primary sequence determination

Determine amino acid composition

Determine N and C terminus

Disulfide bond cleavage

Separation of chains (if necessary)

Cleavage into peptide fragments

Sequence determination

a a Composition


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a.a. Composition-

Acid Hydrolysis of Protein

Boil protein in 6 M HCl complete hydrolysis of protein

destroys Trp

converts amide amino acidsAsnAsp + NH3Asx=Asp +Asn

Gln Glu + NH3Glx=Glu +Gln

MDGALVWNRYKAC

a a Composition


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a.a. Composition-

Base hydrolysis

boil protein in 4 M NaOH complete hydrolysis of protein

destroys Cys, Ser, Thr, Arg

converts amide amino acidsAsnAsp + NH3Asx=Asp +Asn

Gln Glu + NH3Glx=Glu +Gln

MDGALVWNRYKAC


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Amino acid identification

After acid/base hydrolysis, you need to label amino acidswith chromophore, then use chromatography to separate and

determine amino acid composition

Ex: Label with Nihydrin or o-Phthalaldehyde then separate

by HPLCO

O

OH

OHR CH

NH3

COO

R C

O

H CO2+

O

O

O

O

2

N

R CH

NH3

COO

HSCH2

CH2

OH

-Mercaptoethanol

O

O

H

H

N CH

COO

R

S

H2

C

H2

C OH

+ 2 H2O + H+


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Amino acid Identification

AspThrSer

Glu

Pro

Gly

Ala

CysVal

Met

IleLeu

TyrPhe

His

Lys

NH3

Arg


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Cleave Disulfide bonds

Separate chains,in order to get

linear chains

and separate 2

chains linked bydisulfide bonds

Figure 3-24, 3-26 in 5e


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Determining the N-terminus

One method: Label the peptide with Dansyl chloride then acidhydroylze. The amino acid dansylated is the N-terminus.

H2NC N

C NC

R1

O

H

R2

O

H

R3

S OO

Cl

NH3C CH3

SO2

NH3C CH3

HN CH

R1

C

O

HN C

R2

C

O

H+

HCl

OH-

HH

H

SO2

NH

3

C CH3

HN CH

R1

COOH

6 M HCl

+ amino acids


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Determining the N-terminus

Second Method: Label thepeptide with Sangers Reagent(1-flouro-2,4-dinitrobenzene,

FDNB) then acid hydroylze.

The amino acid labeled withFDNB is the N-terminus

F

NO2

O2N

H2NC N

C NC

R1

O

H

R2

O

H

R3

NO2

O2NHN

C NC N

C

R1

O

H

R2

O

H

R3

HH H

+ H++F

-HHH

NO2

O2NHN

C OH

R1

O

6 M HCl

H + amino acids

D t i i th N t i


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Determining the N-terminus Third Method: Using Edmond Degradation to label the first

amino acid.

N C S H2NHC

R1

C

OHN

HC

R2

C

O

NHHC

R1

C

O

NHHC

R2

C

O

C

S

HN

CH

HNC

N

CR1

O

S

H2NHC

R2

C

OHN

HC

R3

NHHC

R1

C

O

NHHC

R2

C

O

C

S

HN


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C-terminus Identification

Carboxypeptidase treatment exopeptidase that hydrolyzes the C-terminal residue

usually can use to determine 3-4 amino acids from the C-

terminus

HC

Rn-2

C

OHN

HC

Rn-1

C

OHN

HC COO

Rn

HC

Rn-2

C

OHN

HC

Rn-1

C

O

O H3NHC COO

Rn

carboxypeptidase

+

H2O

Sanger method and Edmond


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Sanger method and Edmond

Degradation

These methods canalso be used to

determine the

sequence of a small

peptide

Figure 3-25 in 5e

HR1

HR2


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Cleaving the peptide

These sequencing methods are useful, however can onlysequence small peptides, therefore we need to cut up large

peptides in order to sequence the overall protein.

Can cleave with endopeptidase or a chemical agent.

Endopeptidases:

Trypsin: Cleaves as Arg and Lys Carboxyl groups

Chymotrypsin: Cleaves at bulky aromatic amino acids Tyr, Phe,

Trp Carboxyl groups

HN CH

1

C

O

HN CH

2

C

O

HR1

HR2


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Cleaving the peptide

Chemical cleavage: Cyanogen Bromide- Reacts with thioether group of

methinonine, cleaves the petpide on the C-terminal side of

methionine and converts methionine to homoserine lactone.

HN CH C

O

HN CH C

O


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Figure 3-27 in 5e


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El t M S t t f P t i


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Electrospray Mass Spectrometry of a Protein

Determine the primary sequence of the peptide


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based on the following experimental data. Treatment of a sample of peptide (containing 31 amino acid

residues) with carboxypeptidaseA liberates Gln.

Treatment of another sample of peptide with FDNB liberates DNB-

Tyr

Treatment of a third aliquot of peptide with trypsin produces the

following peptides:

Lys Gly-Gln Asn-Ala-Ile-Val-Lys

Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys Asn-Ala-His-Lys

Ser-Gln-Thr-Pro-LeuVal-Thr-Leu-Phe-Lys



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based on the following experimental data. Treatment of another sample of peptide with chymotrypsin produces

the following peptides:

Lys-Asn-Ala-Ile-Val-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln

Gly-Gly-Phe Tyr

Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe

Whats the sequence of this


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What s the sequence of this

peptide?

Valine was released by carboxypeptidase A Acid hydrolysis of the dansylated-polypeptide yielded

dansyl-Pro

Trypsin treatment released the following peptides:

Met-Lys

Phe-Ile-Val

Pro-Gly-Ala-Arg

Ser-Arg CNBr-cleavage yielded:

Pro-Gly-Ala-Arg-Homoserine lactone and

Lys-Ser-Arg-Phe-Ile-Val


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Major Concepts of Chapter 3

properties of amino acids know all common 20 amino acids

structure

three letter abbreviations

one letter abbreviations

pKa values will be provided onexam (however should have anidea of approximate pKa values,and know how to use them)

titration curves

interpretation creation

peptides/proteins structure

nomenclature

properties

separation techniques

basic principles

be able to make predictions

about separations

primary sequence determinations

reagents and products of thereactions

exopeptidases and specificities

endopeptidases and specificities

determine the primary sequence

of a protein


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Recommended Optional Problems/Review

Study Guide #s from 5e- Topics for Discussion 1-

5, 7-12, 15-27, 29, Do you know the facts?, Applyingwhat you know

Textbook #s from 6e- 1, 4-8, 10-18, 20, 21, 23

Textbook #s from 5e- 1, 4-8, 10-18, 20-23 Website for textbook

(http://bcs.whfreeman.com/lehninger5e) online chapter 2

interactive quiz

Documents

Chapter3 Spring 2013 MB