Chapter 3: Amino Acids, Peptides, and Proteins

Dr. ClowerChem 4202

Outline (part I)

Sections 3.1 and 3.2

Amino Acids

Chemical structure

Acid-base properties

Stereochemistry

Non-standard amino acids

Formation of Peptide Bonds

The building blocks of proteins Also used as single molecules in biochemical

pathways 20 standard amino acids (-amino acids) Two functional groups:

carboxylic acid group amino group on the alpha () carbon

Have different side groups (R) Properties dictate behavior of AAs

Amino Acids

R side chain

| H2N— C —COOH

|H

Both the –NH2 and the –COOH groups in an amino acid

undergo ionization in water.

At physiological pH (7.4), a zwitterion forms Both + and – charges

Overall neutral

Amphoteric Amino group is protonated

Carboxyl group is deprotonated

Soluble in polar solvents due to ionic character

Structure of R also influence solubility

Zwitterions

Classification of Amino Acids

Classify by structure of R Nonpolar

Polar

Aromatic

Acidic

Basic

Nonpolar Amino Acids

Hydrophobic, neutral, aliphatic

Polar Amino Acids

Hydrophilic, neutral, typically H-bond

Disulfide Bonds

Formed from oxidation of cysteine residues

Aromatic Amino Acids

Bulky, neutral, polarity depend on R

Acidic and Basic Amino Acids

Acidic R group = carboxylic

acid Donates H+ Negatively charged

Basic R group = amine Accepts H+

Positively charged His ionizes at pH 6.0

Remember H3PO4 (multiple pKa’s)

AAs also have multiple pKa’s due to multiple ionizable

groups

Acid-base Properties

pK1 ~ 2.2(protonated below 2.2)

pK2 ~ 9.4(NH3

+ below 9.4)

pKR

(when applicable)

Table 3-1

Note 3-letter and 1-letter

abbreviations

Amino acid organization chart

pH and Ionization

Consider glycine:

Note that the uncharged species never forms

H3N CH C

H

OH

O

H3N CH C

H

O

O

H2N CH C

H

O

O

OH-

H3O+

OH-

H3O+

Glycine ion at acidic pH

(charge = 1+)

Zwitterion of glycine (charge = 0)

Glycine ion at basic pH

(charge = 1-)

Titration of Glycine

pK1

[cation] = [zwitterion]

pK2

[zwitterion] = [anion]

First equivalence point Zwitterion Molecule has no net charge pH = pI (Isoelectric point)

pI = average of pKa’s = ½ (pK1 + pK2)

pIglycine = ½ (2.34 + 9.60) = 5.97

Animation

pI of Lysine

For AAs with 3 pKa’s, pI = average of two relevant pKa values

Consider lysine (pK1 = 2.18, pK2 = 8.95, pKR = 10.53):

Which species is the isoelectric form?

So, pI = ½ (pK2 + pKR)

= ½ (8.95 + 10.53) = 9.74

Note: pKR is not always higher than pK2 (see Table 3-1 and Fig. 3-12)

H3N CH C

CH2CH2CH2CH2NH3+

OH

O

H3N CH C

CH2CH2CH2CH2NH3+

O

O

H2N CH C

CH2CH2CH2CH2NH3+

O

O

H2N CH C

CH2CH2CH2CH2NH2

O

O

pK1 pK2 pKR

Learning Check

Would the following ions of serine exist at a pH above, below, or at pI?

H3N CH C

CH2

O

O

OH

H3N CH C

CH2

OH

O

OH

H2N CH C

CH2

O

O

OH

Stereochemistry of AAs

All amino acids (except glycine) are optically active

Fischer projections:

D and L Configurations

d = dextrorotatory l = levorotatory D, L = relative to glyceraldehyde

Importance of Stereochemistry

All AA’s found in proteins are L geometry

S enantiomer for all except cysteine

D-AA’s are found in bacteria

Geometry of proteins affects reactivity (e.g

binding of substrates in enzymes)

Thalidomide

Non-standard Amino Acids

AA derivatives Modification of AA after

protein synthesized

Terminal residues or R

groups

Addition of small alkyl

group, hydroxyl, etc.

D-AAs Bacteria

CHEM 2412 Review

Carboxylic acid + amine = ?

Structure of amino acid

R C OH

O

+ H2N R R C NH

O

+ H2Oheat

R

H2N C CO2H

H

R

The Peptide Bond

Chain of amino acids = peptide or protein Amino acid residues connected by peptide bonds Residue = AA – H2O

The Peptide Bond

Non-basic and non-acidic in pH 2-12 range due to delocalization of N lone pair

Amide linkage is planar, NH and CO are anti

C

O

N

H

O

N

HRigid

restricted rotation

Polypeptides

Linear polymers (no branches) AA monomers linked head to tail Terminal residues:

Free amino group (N-terminus) Draw on left

Free carboxylate group (C-terminus) Draw on right

pKa values of AAs in polypeptides differ slightly from pKa values of free AAs

Naming Peptides

Name from the free amine (NH3+)

Use -yl endings for the names of the amino acids The last amino acid with the free carboxyl group (COO-)

uses its amino acid name

(GA)

Amino Acid Ambiguity

Glutamate (Glu/E) vs. Glutamine (Gln/Q) Aspartate (Asp/D) vs. Asparagine (Asn/N) Converted via hydrolysis Use generic abbreviations for either

Glx/Z Asx/B

X = undetermined or nonstandard AA

Write the name of the following tetrapeptide using amino acid names and three-letter abbreviations.

Learning Check

CH CH3

CH3

H3N CH C

O

N

H

CH C

O

N

H

CH C

O

N

H

CH C O-OCH CH2

CH2

S

CH3

CH2

SH

CH3

Learning Check

Draw the structural formula of each of the following peptides.A. Methionylaspartic acid

B. Alanyltryptophan

C.Methionylglutaminyllysine

D.Histidylglycylglutamylalanine

Outline (part II)

Sections 3.3 and 3.4 Separation and purification Protein sequencing

Analysis of primary structure

Protein size

In general, proteins contain > 40 residues Minimum needed to fold into tertiary structure

Usually 100-1000 residues Percent of each AA varies Proteins separated based on differences in

size and composition Proteins must be pure to analyze, determine

structure/function

Factors to control

pH Keep pH stable to avoid denaturation or chemical degradation

Presence of enzymes May affect structure (e.g. proteases/peptidase)

Temperature Control denaturation (0-4°C) Control activity of enzymes

Thiol groups Reactive Add protecting group to prevent formation of new disulfide bonds

Exposure to air, water Denature or oxidize Store under N2 or Ar Keep concentration high

General Separation Procedure

Detect/quantitate protein (assay) Determine a source (tissue) Extract protein

Suspend cell source in buffer Homogenize

Break into fine pieces Cells disrupted Soluble contents mix with buffer Centrifuge to separate soluble and insoluble

Separate protein of interest Based on solubility, size, charge, or binding ability

Solubility

Selectively precipitate protein Manipulate

Concentration of salts Solvent pH Temperature

Concentration of salts

Adding small amount of salt increases [Protein]

Salt shields proteins from each other, less

precipitation from aggregation Salting-in

Salting out Continue to increase [salt] decreases [protein]

Different proteins salt out at different [salt]

Other Solubility Methods

Solvent Similar theory to salting-out Add organic solvent (acetone, ethanol) to interact with

water Decrease solvating power

pH Proteins are least soluble at pI Isoelectric precipitation

Temperature Solubility is temperature dependent

Chromatography

Mobile phase Mixture dissolved in liquid or

solid

Stationary phase Porous solid matrix

Components of mixture

pass through the column

at different rates based on

properties

Types of Chromatography

Paper Stationary phase = filter paper

Same theory as thin layer chromatography (TLC)

Components separate based on polarity

High-performance liquid (HPLC) Stationary phase = small uniform particles, large surface area

Adapt to separate based on polarity, size, etc.

Hydrophobic Interaction Hydrophobic groups on matrix

Attract hydrophobic portions of protein

Types of Chromatography

Ion-exchange Stationary phase =

chemically modified to

include charged groups

Separate based on net

charge of proteins

Anion exchangers Cation groups (protonated

amines) bind anions

Cation exchangers Anion groups (carboxylates)

bind cations

Types of Chromatography

Gel-filtration Size/molecular exclusion

chromatography Stationary phase = gels

with pores of particular size

Molecules separate based on size

Small molecules caught in pores

Large molecules pass through

Types of Chromatography

Affinity Matrix chemically

altered to include a molecule designed to bind a particular protein

Other proteins pass through

UV-Vis Spectroscopy

Absorbance used to

monitor protein

concentrations of each

fraction

= 280 nm Absorbance of aromatic

side groups

Electrophoresis

Migration of ions in an electric field

Electrophoretic mobility (rate of movement) function of

charge, size, voltage, pH

The positively charged proteins move towards the negative

electrode (cathode)

The negatively charged proteins move toward the positive

electrode (anode)

A protein at its pI (neutral) will not migrate in either direction

Variety of supports (gel, paper, starch, solutions)

Protein Sequencing

Determination of primary structure Need to know to determine 3D structure Gives insight into protein function Approach:

Denature protein Break protein into small segments Determine sequences of segments

Animation

End group analysis

Identify number of terminal AAs Number of chains/subunits

Identify specific AA

Dansyl chloride/dabsyl chloride Sanger method (FDNB) Edman degradation (PITC)

Bovine insulin

Dansyl chloride

Reacts with primary amines N-terminus

Yields dansylated polypeptides Dansylated polypeptides

hydrolyzed to liberate the modified dansyl AA

Dansyl AA can be identified by chromatography or spectroscopy (yellow fluorescence)

Useful method when protein fragmented into shorter polypeptides

N

SO2

Cl

+

HN CH

R

C

O

N

SO2

H2N CH

R

C

O

HCl +H3O+

HN CH

R

C

O

OH

N

SO2

+ other free AAs

Dabsyl chloride and FDNB

Same result as dansyl chloride

Dabsyl chloride

1-Fluoro-2,4-dinitrobenzene (FDNB) Sanger method

SN

NN O

O

Cl

Edman degradation

Phenylisothiocyanate (PITC) Reacts with N-terminal AA to produce a phenylthiocarbamyl (PTC) Treat with TFAA (solvent/catalyst) to cleave N-terminal residue Does not hydrolyze other AAs Treatment with dilute acid makes more stable organic compound

Identify using NMR, HPLC, etc. Sequenator (entire process for proteins < 100 residues)

Fragmenting Proteins

Formation of smaller segments to assist with

sequencing

Process: Cleave protein into specific fragments

Chemically or enzymatically

Break disulfide bonds

Purify fragments

Sequence fragments

Determine order of fragments and disulfide bonds

Cleaving Disulfide Bonds

Oxidize with performic acid

Cys residues form cysteic acid

Acid can oxidize other

residues, so not ideal

H C

O

O OH

Cleaving Disulfide Bonds

Reduce by mercaptans (-SH) 2-Mercaptoethanol

HSCH2CH2OH

Dithiothreitol (DTT)

HSCH2CH(OH)CH(OH)CH2SH

Reform cysteine residues

Oxidize thiol groups with

iodoacetete (ICH2CO2-) to

prevent reformation of disulfide

bonds

Hydrolysis

Cleaves all peptide bonds Achieved by

Enzyme Acid Base

After cleavage: Identify using chromatography Quantify using absorbance or fluorescence

Disadvantages Doesn’t give exact sequence, only AAs present Acid and base can degrade/modify other residues Enzymes (which are proteins) can also cleave and affect results

Enzymatic and Chemical Cleavage

Enzymatic Enzymes used to break

protein into smaller peptides

Endopeptidases Catalyze hydrolysis of

internal peptide bonds

Chemical Chemical reagents used to

break up polypeptides Cyanogen bromide (BrCN)

An example

Fundamentals of Protein Fundamentals of Protein StructureStructure

Our life is maintained by Our life is maintained by molecular network systemsmolecular network systems

Molecular network system in a cell

(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)

Proteins play key roles in a Proteins play key roles in a living systemliving system

Three examples of protein functions

Catalysis:Almost all chemical reactions in a living cell are catalyzed by protein enzymes.

Transport:Some proteins transports various substances, such as oxygen, ions, and so on.

Information transfer:For example, hormones.

Alcohol dehydrogenas

e oxidizes alcohols to

aldehydes or ketones

Haemoglobin carries oxygen

Insulin controls the

amount of sugar in the

blood

Amino acid: Basic unit of Amino acid: Basic unit of proteinprotein

COO-NH3+ C

R

HAn amino

acid

Different side chains, R,

determin the properties of 20

amino acids.

Amino group Carboxylic acid group

20 20 Amino acidsAmino acids

Glycine (G)

Glutamic acid (E)Asparatic acid (D)

Methionine (M)

Threonine (T)

Serine (S)

Glutamine (Q)

Asparagine (N)

Tryptophan (W)Phenylalanine (F)

Cysteine (C)

Proline (P)

Leucine (L)Isoleucine (I)Valine (V)

Alanine (A)

Histidine (H)Lysine (K)

Tyrosine (Y)

Arginine (R)

White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic

Proteins are linear polymers of Proteins are linear polymers of amino acidsamino acids

R1

NH3＋

C CO

H

R2

NH C CO

H

R3

NH C CO

H

R2

NH3＋

C COOー

H＋

R1

NH3＋

C COOー

H＋

H2OH2O

Peptide bond

Peptide bond

The amino acid sequence is called

as primary structure A AF

NGG

S TS

DK

A carboxylic acid condenses with an

amino group with the release of a water

Amino acid sequence is encoded Amino acid sequence is encoded by DNA base sequence in a geneby DNA base sequence in a gene

・CGCGAATTCGCG・

・GCGCTTAAGCGC・

DNA molecule

＝

DNA base sequence

Amino acid sequence is encoded Amino acid sequence is encoded by DNA base sequence in a geneby DNA base sequence in a gene

Second letterT C A G

Firs

t lette

r

T

TTTPhe

TCT

Ser

TATTyr

TGTCys

T

Th

ird le

tter

TTC TCC TAC TGC CTTA

LeuTCA TAA

StopTGA Stop A

TTG TCG TAG TGG Trp G

C

CTT

Leu

CCT

Pro

CATHis

CGT

Arg

TCTC CCC CAC CGC CCTA CCA CAA

GlnCGA A

CTG CCG CAG CGG G

A

ATTIle

ACT

Thr

AATAsn

AGTSer

TATC ACC AAC AGC CATA ACA AAA

LysAGA

ArgA

ATG Met ACG AAG AGG G

G

GTT

Val

GCT

Ala

GATAsp

GGT

Gly

TGTC GCC GAC GGC CGTA GCA GAA

GluGGA A

GTG GCG GAG GGG G

Gene is protein’s blueprint, Gene is protein’s blueprint, genome is life’s blueprint genome is life’s blueprint

Gene

GenomeDNA

Protein

Gene GeneGene

Gene

GeneGeneGeneGene

GeneGeneGeneGene

GeneGene

Protein Protein

ProteinProtein

Protein

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Protein

Protein

ProteinProtein

Protein

Gene is protein’s blueprint, Gene is protein’s blueprint, genome is life’s blueprint genome is life’s blueprint

Genome

Gene GeneGene

Gene

GeneGeneGeneGene

GeneGeneGeneGene

GeneGene

Protein Protein

ProteinProtein

Protein

ProteinProtein

Protein

Protein

Protein

Protein

ProteinProtein

Protein

Glycolysis network

3 billion base pair => 6 G letters &

1 letter => 1 byte

The whole genome can be recorded in just 10 CD-ROMs!

In 2003, Human genome In 2003, Human genome sequence was deciphered!sequence was deciphered! Genome is the complete set of genes of a living thing.

In 2003, the human genome sequencing was completed. The human genome contains about 3 billion base pairs. The number of genes is estimated to be between 20,000 to

25,000. The difference between the genome of human and that of

chimpanzee is only 1.23%!

Each Protein has a unique Each Protein has a unique structurestructure

Amino acid sequence

NLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAE

VPRVGFolding!

Basic structural units of proteins: Basic structural units of proteins: Secondary structureSecondary structure

α-helix β-sheet

Secondary structures, α-helix and β-sheet, have

regular hydrogen-bonding patterns.

Three-dimensional structure of Three-dimensional structure of proteinsproteins

Tertiary structure

Quaternary structure

Hierarchical nature of protein Hierarchical nature of protein structurestructure

Primary structure (Amino acid sequence)↓

Secondary structure （ α-helix, β-sheet ）↓

Tertiary structure （ Three-dimensional structure formed by assembly of secondary structures ）

↓Quaternary structure （ Structure formed by more

than one polypeptide chains ）

Close relationship between Close relationship between protein structure and its functionprotein structure and its function

enzyme

A

B

A

Binding to A

Digestion of A!

enzyme

Matching the shape

to A

Hormone receptor AntibodyExample of enzyme reaction

enzyme

substrates

Protein structure prediction has Protein structure prediction has remained elusive over half a remained elusive over half a centurycentury“Can we predict a protein structure from

its amino acid sequence?”

Now, impossible!

SummarySummary Proteins are key players in our living systems. Proteins are polymers consisting of 20 kinds of amino

acids. Each protein folds into a unique three-dimensional structure

defined by its amino acid sequence. Protein structure has a hierarchical nature. Protein structure is closely related to its function. Protein structure prediction is a grand challenge of

computational biology.