Chapter 3 Amino Acids, Peptides, and Proteins

Lehninger Principles of Biochemistry Fall 2012

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

a.a.s Terminology
- Carbon

Charge states

Nomenclature
Full name, 1- Letter, 3- Letter codes
Figure 3-9 in 5e

Non-polar Sidechains
Nonpolar a.a.s Abbreviations
Gly, G Ala, A Pro, P Val, V Leu, L Iso, I Met, M

Unique Properties
Figure 3-5 in 5e

Aromatic Amino Acids

Aromatic a.a.s Abbreviations
Phe, F Tyr,Y Trp, W

Unique Properties

Figure 3-5 in 5e

Spectroscopic Properties of Aromatic a.a.s

Beers Law: A= abc

Box 3-1 in 5e

Spectroscopic Properties of Raise in Abs at 190 Aromatic a.a.s nm is due to the

Spectroscopic properties of these
peptide bond

a.a.s allow us a way to measure the 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

Polar, uncharged a.a.s

Polar, uncharged

a.a.s Abbreviations
Ser, S Thr, T

Cys, C
Asn, N Gln, Q

Unique Properties

Figure 3-5 in 5e

Disulfide Bonds

Figure 3-7 in 5e

Basic a.a.s
Positively charged R

groups Abbreviations
Lys, K Arg, R

His, H

Unique Properties

Figure 3-5 in 5e

Acidic a.a.s
Negatively charged

R groups Abbreviations
Asp, D Glu, E

Unique Properties

Figure 3-5 in 5e

Uncommon Amino Acids

Figure 3-8a in 5e

Charged states of a.a.s

Side chains also play a role in the charged states! If they have

a pKa the side chain will play a role in overall charge.

Figure p79 in 5e

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

affects both of their pKa values.

Figure 3-11 in 5e

pKas

Table 3-1 in 5e

Expected pKa ranges for a.as in proteins

f unctional group alpha-carboxyl side chain carboxyl (Asp and Glu) imidazole (His) t hiol (Cys) phenol (Tyr) alpha-amino side chain amino (Lys) guanidinyl (Arg) pKa 3.5- 4.0 4.0- 4.8 6.5- 7.5 8.5- 9.0 9.5-10.5 8.0- 9.0 9.8-10.4 ~12

How to Draw a Titration Curve

14 12 10 pH 8 6 4 2 0 0 0.5 1 1.5 2 2.5

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

Equivalents of NaOH

ionizable group (where protons can be removed) Start with a completely protonated amino acid/peptide, then deprotonate each group based upon the pKa value. What happens when the pH = pKa?

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

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

pI = (pKa1+ pKa2)

Figure 3-10 in 5e

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

Using the pI
Determining whether something is positively charged or

negatively charged
pH scale
14
If the pH falls in a value 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

pI

Titration Curves, where is the pI?

Figure 3-12 in 5e

Acid-Base Problem with a.as

Determine the %

composition of Glu (in terms of ionization state) in a sample at pH of 8

Figure 3-12 in 5e

Peptide Bond

Figure 3-13 in 5e

Mechanism of Peptide Bond Formation

Classification of Amino Acid polymers

Oligopeptide few amino acids Polypeptide many amino acids, <10,000 MW Proteins- larger polypeptides, >10,000 MW

First look at a peptide

Written from N terminus to

the C terminus What are the ionizable groups?

First look at a peptide

Identify each amino acid What are the ionizable groups?

Figure 3-14 in 5e

Aspartame- the sweetener

Figure 3 p83 in 5e

Protein Structure

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?

Break open cells, centrifuge

First you need to

get the protein out of the cell. Cells are broken open by sonicaters, homogenizers, etc., then centrifuge Next you need to get the protein from the other mixture.

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/

Column Chromatography
Separating proteins based

upon their different properties, collect fractions (fractionate) as they elute from a column to separate molecules. Mobile phase: solution (buffer/protein) Stationary phase: beads

Figure 3-16 in 5e

Ion exchange chromatography

DEAE: + Charged stationary phase SP or CM: - Charged Stationary

phase Charges of a peptide:

At pH > pI (meaning the pH is

more basic), a net negative charge exists. At pH < pI (meaning the pH is more acidic), a net positive charge exists. When pH=pI no net charge
How do you elute bound protein?
Figure 3-17 in 5e

Size-exclusion chromatography
Separates proteins based on

size. 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

Affinity chromatography
This system takes advantage a

proteins natural affinity for a ligand. Stationary phase: Beads with a ligand covalently bound Proteins that bind that ligand bind to the column. How do you elute bound protein?

Figure 3-17 in 5e

Predict Separations of these mixtures

Mixture 1 and 2 on an DEAE Column at pH 7
Mixture 1: Ala-Glu / Lys-Asp / Arg-Gly / Ala-Val Mixture 2: Peptides with pIs of 4.5, 7.2, 8.6

Mixture 3:

Protein
A B C D

pI
3.5 8 8 8

MW
50, 000 50,000 20,000 100,000

You have been given a mixture of 4 proteins, how would you

separate them? Using Buffers at pH 7

You have all these fractions, now what?

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

assays. How do you find it?

SDS-PAGE Activity assays

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

Native PAGE Versus SDS-PAGE

Native PAGE does not use

the loading dye. The protein is not reduced or lined with SDS. Hence it runs differently on the gel.

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

Using SDS-PAGE to find MW

log(Mr) vs.

Relative Migration can get you the molecular weight of your protein.

Figure 3-19 in 5e

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
30 40 80 Unknown MW peptide

1
1.48 1.6 1.9

1
0.8 0.7 0.3 0.5

Isoelectric Focusing

Figure 3-20 in 5e

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

2-D Gels
Run an IEF gel to separate based on pI, then put the gel on top of a SDS-gel and separate based on size.

Figure 3-21 in 5e

Draw the SDS-PAGE gel, IEF Gel, and 2-D SDS Gel for the following proteins
Sample Protein 1 Protein 2 Protein 3 pI 3 6 3 MW (kDa) 80 80 200

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

Activity vs. Specific Activity

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. CompositionAcid Hydrolysis of Protein

Boil protein in 6 M HCl complete hydrolysis of protein destroys Trp converts amide amino acids Asn Asp + NH3 Asx=Asp +Asn Gln Glu + NH3 Glx=Glu +Gln MDGALVWNRYKAC

a.a. CompositionBase hydrolysis

boil protein in 4 M NaOH complete hydrolysis of protein destroys Cys, Ser, Thr, Arg converts amide amino acids Asn Asp + NH3 Asx=Asp +Asn Gln Glu + NH3 Glx=Glu +Gln MDGALVWNRYKAC

Amino acid identification

After acid/base hydrolysis, you need to label amino acids

with chromophore, then use chromatography to separate and determine amino acid composition Ex: Label with Nihydrin or o-Phthalaldehyde then separate

by HPLC
O

O
NH3 R CH COO

2
O

OH OH

H H O

NH3 R CH COO

O R C H

HSCH2CH2OH
+ CO2

-Mercaptoethanol

O N O

O
S

H2 H2 C C OH R

+ 2 H2O + H+

N CH COO

Amino acid Identification

His

Met
Gly ThrSer Asp

Glu
Ala CysVal Pro

Leu Ile Tyr

Phe

Lys

Arg
NH3

Cleave Disulfide bonds

Separate chains,

in order to get linear chains and separate 2 chains linked by disulfide bonds

Figure 3-24, 3-26 in 5e

Determining the N-terminus

One method: Label the peptide with Dansyl chloride then acid

hydroylze. The amino acid dansylated is the N-terminus.

Cl O S O O + H3C N CH3 H3C N CH3 H2N H C R1 R2 H C N H N H C H O R3 HCl R1 O H H HN CH C N C C SO2 O R2

OH-

6 M HCl R1 HN CH COOH SO2 + amino acids N

H3C

CH3

Determining the N-terminus

Second Method: Label the

peptide with Sangers Reagent (1-flouro-2,4-dinitrobenzene, FDNB) then acid hydroylze. The amino acid labeled with FDNB is the N-terminus

O2N

F NO2 H2N H C R1

R2 C N H H O

H N H C R3

O2N NO2

H N H C R1

R2 N H H O C

H N H C R3

+ +H + F

6 M HCl

O2N NO2

H N H C R1

O OH

+ amino acids

Determining the N-terminus

Third Method: Using Edmond Degradation to label the first

amino acid.
N C S O O H H H H2N C C N C C R1 OO
O O

R2

H HH HH H N C NH C NH C C C N C NH CC C NH C

SS

RR 11

RR 22

O R1 C CH N HN C S O H H H H2N C C N C R2 R3

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
O O H H H H H C C N C C N C COO Rn-2 Rn-1 Rn O O H H H C C N C C O Rn-2 Rn-1 carboxypeptidase

H2O + H H3N C COO Rn

Sanger method and Edmond Degradation

These methods can

also be used to determine the sequence of a small peptide

Figure 3-25 in 5e

Cleaving the peptide

H H N CH C N CH C O O

R1

R2

These sequencing methods are useful, however can only

sequence 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

Cleaving the peptide

Chemical cleavage:

H H N CH C N CH C O O

R1

R2

Cyanogen Bromide- Reacts with thioether group of

methinonine, cleaves the petpide on the C-terminal side of methionine and converts methionine to homoserine lactone.

Figure 3-27 in 5e

Analyze by Mass Spec

Can use mass spectrometry to determine the sequence of a

peptide or total peptide mass.

Box3-2 Figure 1 in 5e

Electrospray Mass Spectrometry of a Protein

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

residues) with carboxypeptidase A liberates Gln. Treatment of another sample of peptide with FDNB liberates DNBTyr 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 Ser-Gln-Thr-Pro-LeuVal-Thr-Leu-Phe-Lys Asn-Ala-His-Lys

Determine the primary sequence of the peptide 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 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 Lys-Ser-Arg-Phe-Ile-Val

and

Major Concepts of Chapter 3

properties of amino acids know all common 20 amino acids structure peptides/proteins structure

exam (however should have an idea of approximate pKa values, and know how to use them) titration curves interpretation creation

three letter abbreviations one letter abbreviations pKa values will be provided on

nomenclature
properties separation techniques basic principles

be able to make predictions

about separations primary sequence determinations reagents and products of the reactions exopeptidases and specificities endopeptidases and specificities determine the primary sequence of a protein

Recommended Optional Problems/Review

Study Guide #s from 5e- Topics for Discussion 15, 7-12, 15-27, 29, Do you know the facts?, Applying what 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