Amino Acids & Proteins

27-1

Amino Acids
Amino

acid: A compound that contains both an amino group and a carboxyl group.
-Amino acid: An amino acid in which the amino group is on the carbon adjacent to the carboxyl group. although -amino acids are commonly written in the unionized form, they are more properly written in the zwitterion (internal salt) form.
O RCHCOH N H2 -Amino Acid O RCHCO N H3 + Zwitte rion form
27-2

Chirality of Amino Acids

With

the exception of glycine, all protein-derived amino acids have at least one stereocenter (the -carbon) and are chiral.
the vast majority have the L-configuration at their carbon.

COOH N H3 + CH3 D-Alanine H 3N

COOH CH3 L-Alanine

27-3

Amino Acids with Nonpolar side chains

COO- Alanine (Ala, A) N H3 + COO- Glycine (Gly, G) N H3 + COO- Isoleucine (I le, I) N H3 + COO N H3 + S
-

COO- Phenylalanine (Phe, F) + N H3 COO- Proline (Pro, P) H COO- Tryptophan (Trp, W) N H3 +

N H

Leucine (Le u, L)

N H

COO- Methion ine (Met, M) N H3 +

COO- Valine (Val, V) N H3 +

27-4

With Polar side chains

H 2N O O H 2N COON H3 + Glutamin e (Gln, Q) COON H3 + Asparagine (As n, N) HO COOSerine (S er, S)

N H3 + OH COO- Thre onin e (Thr, T) NH +

3

27-5

With Acidic & Basic Side Chains

O O O

COOAspartic acid + N H3 (As p, D) COO-Glutamic acid N H + (Glu, E)

3

N H2 + H 2N N H

COO- Arginine (Arg, R) NH +

3

N N H + H 3N

COON H3
+

HS

COO N H3 + COO

Cys teine (Cys , C) Tyrosine (Tyr, Y)

Histidine (His, H)

COON H3
+

HO

N H3 +

Lys ine (Lys, K)

27-6

Acid-Base properties, Non polar and polar side chains

N onpolar & polar side ch ains alanin e asp aragine glutamine glycine isoleucine leucine methionine ph enylalanine proline serine threonine tryp top han valine pK a of COOH 2.35 2.02 2.17 2.35 2.32 2.33 2.28 2.58 2.00 2.21 2.09 2.38 2.29 pK a of NH3 9.87 8.80 9.13 9.78 9.76 9.74 9.21 9.24 10.60 9.15 9.10 9.39 9.72

27-8

Acid-Base Properties, Acidic/Basic Side Chains

Acid ic Side Ch ains aspartic acid glu tamic acid cysteine tyros ine Basic Side Ch ains arginin e histid ine lys ine pK a of pK a of pK a of Side Ch ain 3.86 4.07 8.00 10.07 pK a of Side Ch ain 12.48 6.10 10.53 Sid e Chain Grou p carboxyl carboxyl su fh yd ryl ph enolic Sid e Chain Grou p guanid ino imidazole 1 amin o
27-9

COOH NH3 2.10 9.82 2.10 9.47 2.05 10.25 2.20 9.11 pK a of pK a of

COOH NH3 2.01 9.04 1.77 9.18 2.18 8.95

Acidity: -COOH Groups

The

average pKa of an -carboxyl group is 2.19, which makes them considerably stronger acids than acetic acid (pKa 4.76).
The greater acidity is accounted for by the electronwithdrawing inductive effect of the adjacent -NH3+ group.
RCHCOOH NH3
+
+

H2 O

RCHCOO NH3
+

+ - + H3 O

pK a = 2.19

Note that the NH2 will be protonated at these low pHs

27-10

Acidity: side chain -COOH

Due

to the electron-withdrawing inductive effect of the -NH3+ group, side chain -COOH groups are also stronger than acetic acid.
The effect decreases with distance from the -NH3+ group. Compare: -COOH group of alanine (pKa 2.35) -COOH group of aspartic acid (pKa 3.86) -COOH group of glutamic acid (pKa 4.07)

27-11

Acidity: -NH3+ groups

The

average value of pKa for an -NH3+ group is 9.47, compared with a value of 10.76 for a 1 alkylammonium ion.
RCHCOO + H3 O NH2
+

RCHCOO + H2 O + NH3

pK a = 9.47

CH3 CHCH3 + H2 O + NH3

CH3 CHCH3 + H3 O NH2

pK a = 10.60

27-12

Details: The Guanidine Group of Arg

The basicity of the guanidine group is attributed to the large resonance stabilization of the protonated form relative to the neutral form.
NH2 NH2
+

:
RNH C NH2
+

RNH C NH2

NH2 + RNH C NH2

:
H2 O

: :
RN C

:
NH2 NH2 + H3 O
+

pK a = 12.48

27-13

Details: Imidazole Group

The imidazole group is a heterocyclic aromatic amine.
H N N+ H H NH3
+ -

+ N N H

NH3

COO

COO-

H2 O H3 O+

N ot a p art of th e aromatic sextet; the proton accep tor

N N H

NH3 +
+ + H3 O COO -

pKa 6.10

27-14

Useful Recall from buffer solutions

Ka = [H+] [A-] [HA]

If Ka = [H+] then [A-] = [HA]

27-15

Isoelectric Point
Isoelectric

point (pI): pH at which an amino acid, polypeptide, or protein has a total charge of zero.
The pI for glycine, for example, falls between the pKa values for the carboxyl and amino groups.

pI = 1 2

( pKa COOH + pKa NH3 )

= 1 (2.35 + 9.78) = 6.06 2

27-16

Isoelectric Point of glycine continued

Again
pI = 1 2 ( pKa COOH + pKa NH3 )
+

= 1 (2.35 + 9.78) = 6.06 2

pKa = pH-log([conj base]/[acid])

At pH = 6.06 For carboxyl group 2.35 = 6.06 - log ([RCO2-]/[RCO2H]) 6.06- 2.35 = 3.71 = log ([RCO2-]/[RCO2H]) ([RCO2-]/[RCO2H]) = 103.71 or 99.98% ionized as neg ion. For amino group 9.78 = 6.06 - log([RNH2]/[RNH3+]) ([RNH2]/[RNH3+]) = 10-3.72 or 99.98% protonated. On average Zero Chg. 27-17

Furthermore..

A+

C-

[A+] = [C-]
27-18

Titration of conjugate acid of glycine with NaOH.

Strongly acid -CO2H and -NH3+

Buffer solution. [-CO2H] = [-CO2-]

Isoelectric. Zero net charge. Buffer solution. [-NH3+]=[-NH2] Strongly basic -CO2- and NH2
27-19

Isoelectric Point
N onpolar & pK a of pK a of polar side + COOH NH3 ch ains alanin e 2.35 9.87 asp aragine 2.02 8.80 glutamine 2.17 9.13 glycine 2.35 9.78 isoleucine 2.32 9.76 leucine 2.33 9.74 methionine 2.28 9.21 ph enylalanine 2.58 9.24 proline 2.00 10.60 serine 2.21 9.15 threonine 2.09 9.10 tryp top han 2.38 9.39 valine 2.29 9.72 pK a of Sid e Chain ---------------------------------------pI 6.11 5.41 5.65 6.06 6.04 6.04 5.74 5.91 6.30 5.68 5.60 5.88 6.00

If pH is lower than pI then more protonated molecules. If higher then more negative charge.

27-20

Isoelectric Point
Acidic Sid e Ch ains asp artic acid glutamic acid cystein e tyrosin e pK a of pK a of + Sid e COOH NH3 Chain 2.10 9.82 3.86 2.10 9.47 4.07 2.05 10.25 8.00 2.20 9.11 10.07 pK a of pK a of + Sid e COOH NH3 Chain 2.01 9.04 12.48 1.77 9.18 6.10 2.18 8.95 10.53 pK a of pI 2.98 3.08 5.02 5.63

Basic Sid e Ch ains argin ine his tidine lysine

pK a of

pI 10.76 7.64 9.74

Three buffered pHs

27-21

Aspartic acid
A+ B CD2-

pKa 2.10 pH = pKa [A+] = [B]

3.86
[B] = [C-]

9.82

pI = (2.10 + 3.86)/2 [A+] = [C-] [D2-] approx 0

Note species B has zero net charge. pKa1 and pKa2 control [A+] and [C-] which should be equal.
27-22

Arginine
A2+ B+ C D-

pI = (9.04+12.48)/2 =10.76 [B+] = [D-]; [A2+] about 0

27-23

Electrophoresis
Electrophoresis:

The process of separating compounds on the basis of their electric charge.

electrophoresis of amino acids can be carried out using paper, starch, polyacrylamide and agarose gels, and cellulose acetate as solid supports.

27-24

Electrophoresis
A sample of amino acids is applied as a spot on the paper strip. An electric potential is applied to the electrode vessels and amino acids migrate toward the electrode with charge opposite their own. Molecules with a high charge density move faster than those with low charge density. Molecules at isoelectric point remain at the origin. After separation is complete, the strip is dried and developed to make the separated amino acids visible. After derivitization with ninhydrin, 19 of the 20 amino acids give the same purple-colored anion; proline gives an orange-colored compound.
27-25

Electrophoresis
The reagent commonly used to detect amino acid is ninhydrin.
O RCHCO + 2 NH3
+ -

O OH OH O N inh yd rin O N O O Pu rp le-colored anion O

-

An -amino acid

O + RCH + CO2 + H3 O+

27-26

Polypeptides & Proteins

In

1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds to which he gave the name peptide bonds. Peptide bond: The special name given to the amide bond between the -carboxyl group of one amino acid and the -amino group of another.

27-27

Serinylalanine (Ser-Ala)
HOCH2 H H 2N O H + H 2N O Serine (S er, S) O O H H CH3 Alanine (Ala, A) peptide bond HOCH2 H H O N H H 2N O O H CH 3 Serinylalanine (S er-Ala, (S-A)

27-28

Peptides
Peptide: The name given to a short polymer of amino acids joined by peptide bonds; they are classified by the number of amino acids in the chain. Dipeptide: A molecule containing two amino acids joined by a peptide bond. Tripeptide: A molecule containing three amino acids joined by peptide bonds. Polypeptide: A macromolecule containing many amino acids joined by peptide bonds. Protein: A biological macromolecule of molecular weight 5000 g/mol or greater, consisting of one or more polypeptide chains.
27-29

Writing Peptides
By convention, peptides are written from the left, beginning with the free -NH3+ group and ending with the free -COO- group on the right.
+

H 3N N-terminal amino acid

N OH O OH COOSer-Phe-Asn

C6 H5 H N

C-termin al amino acid

27-30

Writing Peptides
The tetrapeptide Cys-Arg-Met-As At pH 8 it would be At pH 6.0, its net charge is +1. half ionized.
p Ka 8.00 N-termin al amin o acid SH H N O O N H NH H2 N NH2 + O SCH3 H N O C-terminal amino acid ONH2 O pK a 12.48

H3 N

27-31

Primary Structure
Primary

structure: The sequence of amino acids in a polypeptide chain; read from the N-terminal amino acid to the C-terminal amino acid: Amino acid analysis:
Hydrolysis of the polypeptide, most commonly carried out using 6M HCl at elevated temperature. Quantitative analysis of the hydrolysate by ionexchange chromatography.

27-32

Ion Exchange Chromatography

Analysis

of a mixture of amino acids by ion exchange chromatography

27-33

Cyanogen Bromide, BrCN

BrCN Selectively cleaves of peptide bonds formed by the carboxyl group of methionine.
cyan ogen b romide is specific for the cleavage of this p eptide bond from the N-termin al end O O P N -C-NH CH C NH-PC CH 2 CH 2 -S-CH3 from the C-termin al end

27-34

Cyanogen Bromide, BrCN

O HN pe pt id e COOH3 N pe pt id e C-N H O + Br C N side chain of meth ionine S- CH3 O O A s ubstitute d -lactone of th e amino acid homoserine H 3 N pe pt id e COOCH3 S- C N Methyl th iocyanate 0.1 M HCl H2 O

O H3 N pe pt id e C-N H

Mechanism to follow
27-35

Cyanogen Bromide, BrCN

Step 1: Nucleophilic displacement of bromine.
O H3 N C-NH O :S-CH3 HN COOH3 N O C-NH O Br Br C N a sulfon ium ion; a good leaving group :S-CH3 C N HN COO
-

Cyanogen bromide

27-36

Cyanogen Bromide, BrCN

Step 2: Internal nucleophilic displacement of methyl thiocyanate.
O H3N C-NH HN O : S-CH3 C N H3N O C-NH HN O An iminolactone hydrobromide

COO-

COO+ CH3-S-C N Methyl th iocyanate

: :

27-37

Cyanogen Bromide, BrCN

Step 3: Hydrolysis of the imino group.
O H3 N C-N H HN O O H3 N C-N H O O H 3N COOCOOH2 O

A s ubstituted -lactone of th e amino acid homos erine

27-38

Enzyme Catalysis
A

group of protein-cleaving enzymes called proteases can be used to catalyze the hydrolysis of specific peptide bonds.
Enzyme Tryps in Catalyzes Hydrolysis of Pep tide Bond Formed b y Carboxyl Group of Argin ine, lysine

Ch ymotrypsin Phen ylalan ine, tyrosin e, tryptoph an

27-39

Edman Degradation
Edman

degradation: Cleaves the N-terminal amino acid of a polypeptide chain.

R + H3 N O N-terminal amino acid NH COO+ S=C=N-Ph

Phenyl isothiocyan ate R HN S N Ph A p henylthiohydantoin

27-40

O + H2 N

COO

Edman Degradation
Step 1: Nucleophilic addition to the C=N group of phenylisothiocyanate and proton tautomerization
R H2 N NH O Ph N C S COOHN R O S NH COO
-

Ph N H

A d erivative of N-phen ylth iourea

27-41

Edman Degradation
Step 2: Nucleophilic addition of sulfur to the C=O of the adjacent amide group and acid catalysis.
R HN Ph N H R HN O Ph-N S + + H3 N COOH S H+ O HN NH COOH + Ph-N H S R OH NH H+ H+ COOH

A thiazolinone

27-42

Edman Degradation
Step 3: Isomerization of the thiazolinone ring.
R HN Ph-N S O + H-Nu HN Ph N R HN S NH Ph O Nu - H-Nu HN S N Ph SH R O Nu R O keto-en ol tautomerism

substitution

A th iazolin on e

A p henylthiohydantoin

27-43

DNFB tagging of N terminal AA

27-44

Carboxypeptidase cleavage of C terminal AA

Treatment

of peptide with carboxypeptidase cleaves the peptide linkage adjacent to the free alpha carboxyl group. It may then be identified.

27-45

Primary Structure, example

Deduce the 1 structure of this pentapeptide
Expe rimental Procedure pentape ptide Edman Degradation Hydrolysis - Chymotryps in Fragme nt A Fragme nt B Hydrolysis - Trypsin Fragme nt C Fragme nt D Amino Acid Compos ition Arg, Glu, His , Phe, Se r Glu Glu, His, Ph e Arg, S er Arg, Glu, His , Phe Ser

GluGlu-His-Phe

Glu-His-Phe-Arg-Ser

using
Enzyme Tryps in Catalyzes Hydrolysis of Pep tide Bond Formed b y Carboxyl Group of Argin ine, lysine

Ch ymotrypsin Phen ylalan ine, tyrosin e, tryptoph an

27-46

Polypeptide Synthesis
The

problem in protein synthesis is how to join the -carboxyl group of aa-1 by an amide bond to the -amino group of aa-2, and not vice versa.
O + O + H3 NCHCO + H3 NCHCO aa2 aa1 ? O + O H3 NCHCNHCHCO + H2 O aa1 aa2

27-47

Polypeptide Synthesis Strategy

Protect the -amino group of aa-1. Activate the -carboxyl group of aa-1. Protect the -carboxyl group of aa-2.
protecting group O activating group O protecting group form pe ptide bond

Z-N HCHC- Y + H2 N CH C-X a a2 a a1

O O Z-N HCHCN HCHC-X + H- Y a a1 a a2

27-48

Amino-Protection
convert them to amides.

O PhCH2 OCCl Benzyloxycarb on yl ch loride O O ( CH3 ) 3 COCOCOC(CH3 ) 3 D i-t ert-b utyl dicarbonate

O PhCH2 OCBenzyloxycarb on yl (Z-) group O (CH3 ) 3 COCt ert -Bu toxycarbonyl (BOC-) grou p

27-49

Amino-Protection
Treatment of an amino group with either of these reagents gives a carbamate (an ester of the monoamide of carbonic acid).
O O O 1 . NaOH + PhCH2 OCNHCHCOH + H3 NCHCO 2 . HCl, H2 O CH3 CH3 Benzyloxycarbonyl Alanin e N -Benzyloxycarbonylalanin e chloride (Z-Cl) (Z-A la) O PhCH2 OCCl

A carbamate is stable to dilute base but can be removed by treatment with HBr in acetic acid.
O PhCH2 OCNH-p eptide HBr CH3 COOH PhCH2 Br + CO2 + H3 N-pep tide Benzyl bromide U nprotected p eptide

A Z-pro tected peptid e

27-50

Amino-Protecting Groups
The

benzyloxycarbonyl group is also removed by hydrogenolysis.

The intermediate carbamic acid loses carbon dioxide to give the unprotected amino group.
O PhCH2 OCNH-p eptide + H2 Pd PhCH3 + CO2 + H2 N-pept ide Toluen e U nprotected p eptide

A Z-pro tected peptid e

27-51

Carboxyl-Protecting Groups. Esters

Carboxyl

groups are most often protected as methyl, ethyl, or benzyl esters.

Methyl and ethyl esters are prepared by Fischer esterification, and removed by hydrolysis in aqueous base under mild conditions. Benzyl esters are removed by hydrogenolysis; they are also removed by treatment with HBr in acetic acid

27-52

Peptide Bond Formation

The

reagent most commonly used to bring about peptide bond formation is DCC.
DCC is the anhydride of a disubstituted urea and, when treated with water, is converted to DCU.
N C N
+ H2 O

1,3-D icy clo hexyl carbod ii mide (D CC)

H

O N C N

In bringing about formation of a peptide bond, DCC acts a dehydrating agent.

N,N' -d icy clo hexy lu rea (D CU)

27-53

Peptide Bond Formation

O O

Z-N HCHC- OH + H2 NCHCOCH3 + DCC R1 R2 Amino-protected Carboxyl-prote cte d aa1 aa2 O O Z-N HCHC- NHCH COCH3 + DCU R1 R2 Amino and carboxyl protected dipeptide

CHCl 3

27-54

Solid-Phase Synthesis
Bruce

Merrifield, 1984 Nobel Prize for Chemistry

Solid support: a type of polystyrene in which about 5% of the phenyl groups carry a -CH2Cl group. The amino-protected C-terminal amino acid is bonded as a benzyl ester to the support beads. The polypeptide chain is then extended one amino acid at a time from the N-terminal end. When synthesis is completed, the polypeptide is released from the support beads by cleavage of the benzyl ester.

27-55

Solid-Phase Synthesis
The

solid support used in the Merrifield solid phase synthesis.

27-56

27-57

Solid-Phase Synthesis
Merrifield synthesized the enzyme ribonuclease, a protein containing 124 amino acids

27-58

Peptide Bond Geometry

The four atoms of a peptide bond and the two alpha carbons joined to it lie in a plane with bond angles of 120 about C and N. Model of the zwitterion form of Gly-Gly viewed from two perspectives to show the planarity of the six atoms of the peptide bond and the preferred s-trans geometry.

27-59

Peptide Bond Geometry

To account for this geometry, Linus Pauling proposed that a peptide bond is most accurately represented as a hybrid of two contributing structures. The hybrid has considerable C-N double bond character and rotation about the peptide bond is restricted.
:O C :O : C H C
-

: :
C C N

C H

27-60

Peptide Bond Geometry

Two conformations are possible for a planar peptide bond. Virtually all peptide bonds in naturally occurring proteins studied to date have the s-trans conformation.

C C N H s-t rans

H C N C s-cis

27-61

Secondary Structure
Secondary

structure: The ordered arrangements (conformations) of amino acids in localized regions of a polypeptide or protein. To determine from model building which conformations would be of greatest stability, Pauling and Corey assumed that:
1. All six atoms of each peptide bond lie in the same plane and in the s-trans conformation. 2. There is hydrogen bonding between the N-H group of one peptide bond and a C=O group of another peptide bond as shown in the next screen.

27-62

Secondary Structure
Hydrogen bonding between amide groups.

27-63

Secondary Structure
On

the basis of model building, Pauling and Corey proposed that two types of secondary structure should be particularly stable:

-Helix. Antiparallel -pleated sheet.

-Helix:

A type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a right-handed spiral.

27-64

The -Helix
The polypeptide chain is repeating units of L-alanine.

H bonds (C=OH) parallel to axis of helix

27-65

The -Helix
In

a section of -helix:

There are 3.6 amino acids per turn of the helix. Each peptide bond is s-trans and planar. N-H groups of all peptide bonds point in the same direction, which is roughly parallel to the axis of the helix. C=O groups of all peptide bonds point in the opposite direction, and also parallel to the axis of the helix. The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino acid units away from it. All R- groups point outward from the helix.
27-66

The -Helix
An

-helix of repeating units of L-alanine

A ball-and-stick model viewed looking down the axis of the helix. A space-filling model viewed from the side.

27-67

-Pleated Sheet
The

antiparallel -pleated sheet consists of adjacent polypeptide chains running in opposite directions:
Each peptide bond is planar and has the s-trans conformation. The C=O and N-H groups of peptide bonds from adjacent chains point toward each other and are in the same plane so that hydrogen bonding is possible between them. All R- groups on any one chain alternate, first above, then below the plane of the sheet, etc.

27-68

-Pleated Sheet
-pleated sheet with three polypeptide chains running in opposite directions (antiparallel).

27-69

Tertiary Structure
Tertiary

structure: The three-dimensional arrangement in space of all atoms in a single polypeptide chain.
Disulfide bonds between the side chains of cysteine play an important role in maintaining 3 structure.

27-70

Tertiary Structure
A ribbon model of myoglobin.

27-71

Quaternary Structure
Quaternary

structure: The arrangement of polypeptide chains into a noncovalently bonded aggregation.

The major factor stabilizing quaternary structure is the hydrophobic effect.

Hydrophobic

effect: The tendency of nonpolar groups to cluster together in such a way as to be shielded from contact with an aqueous environment.

27-72

Quaternary Structure
The quaternary structure of hemoglobin. The -chains in yellow, the heme ligands in red, and the Fe atoms as white spheres.

27-73

Quaternary Structure
If two polypeptide chains, for example, each have one hydrophobic patch, each patch can be shielded from contact with water if the chains form a dimer.
Pro tein N umber o f Subun its

Al coho l dehyd rog enase 2 Al dol ase 4 Hemog lo bin 4 Lactate d eh ydrog enase 4 Insul in 6 Glu tamine sy nthetase 12 Tobacco mosai c vi rus 17 protei n d isc
27-74