Basic protein structure and stability I:Formation of peptide bonds/

properties of amino acids

Biochem 565, Fall 2008

08/25/08

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Proteins are the primary functionalmanifestation of the information in genomes

DNA sequence

RNA sequence

proteinsequence

proteinstructure

proteinfunction

transcription

translation

-amino acids--the building blocks

of proteins

H2N CH C

R

OH

O

H3N CH C

R

O

O

The zwitterionic form isthe predominant form atneutral pH

amino group carboxylic acidgroup

side chain

alpha carbon

H3N CC

R

O

O

H

The alpha carbon is a chiral center--naturalproteins are made ofL amino acids (shownabove) as opposed to D

The protein alphabet--the 20 amino acid R groups

CH2 β

N

HN

CH2 β

OH γ

CH β

γ1 HO CH3 γ2

CH2 β

CH2 γ

C δ

NH2 ε2O

CH2 β

C γ

NH2 δ2O

CH2 β

SH γ

HCH3 β

CH β

γ1 H3C CH3 γ2

CH β

γ2 H3C CH2 γ1

CH3 δ

CH2 β

CH γ

δ1 H3C CH3δ2

H2C

H2C

HC N

CH2

A C

CH2 β

CH2 γ

C δ

OO

CH2 β

C γ

OO

CH2 β

NH

CH2 β CH2 β

CH2 γ

CH2 δ

CH2 ε

NH3 ζ

CH2 β

CH2 γ

S δ

CH3 ε

CH2 β

CH2 γ

CH2 δ

NH ε

C

NH2H2N

D E F G

β

γ

δ

δ1

ε1ε2

δ2δ1δ2

ε1ε2

ζ

CH2 β

Y

δ1δ2

ε1ε2ζ

OHη

δ1

ε1

γ γ

γ

γ

δ2

ε2

ε3

ζ2η2

ζ3ζ

η2η1

H I

WVTSRQPNM

K L

δ1ε1

Aromatic ring numbering/naming (IUPAC)

NH2

CH

C

H2C

OH

O

HN12

3

4

7

56

β

3a7a

NH2

CH

C

H2C

OH

O

N

HN

12

3 4

5

(π)

(τ)

β

NH2

CH

C

H2C

OH

O

HO1

23

4

5 6β

IUPAC nomenclature:http://www.chem.qmw.ac.uk/iupac/AminoAcid/index.html

Proteins are made by controlled polymerization of amino acids

H2N CH C

R1

OH

O

H2N CH C

R2

OH

O

H2N CH C

R1

NH

O

CH C

R2

OH

O

peptide bond is formed

+ HOH

residue 1 residue 2

two amino acidscondense to form...

...a dipeptide. Ifthere are more itbecomes a polypeptide.Short polypeptide chainsare usually called peptideswhile longer ones are calledproteins.

water is eliminated

N or aminoterminus

C or carboxyterminus

Solid phase peptide synthesis (SPPS)

Resin

O

AA1HNFmoc

Fmoc

P1

Resin

O

AA1H2N

P1

O

AA2HNFmoc

P2

OH

O

AA2HNFmoc

P2

A

A

Resin

O

AA1NH

P1

O

AA2HNFmoc

P2

A

activation deblocking

coupling repeat stepsfor each aminoacid in peptide,then deblock, deprotect, cleave off resin

Resin

Fmoc

P2P1

AA2AA1

A

solid support

fmoc protecting group

protecting groupsfor side chains

1st and 2ndamino acids

carbonyl activatinggroup

adapted from Sigma-Aldrich website

Solid phase peptide synthesis (SPPS)

Resin

O

AA1NH

P1

O

AA2HNFmoc

P2

Fmocfinaldeblocking

Resin

O

AA1NH

P1

O

AA2H2N

P2

deprotection andcleavageP1P2Resin

O

AA1NH

O

AA2H2N

OH

at the end a final deblocking is done followed by removal of the side-chain protecting groups and cleavage from the resin to recover the peptide

SPPS using Fmoc canbe used to make peptides up to 70-100residues in length(chemical ligation can be used to make longer ones)

Peptide bond formation in vivo

N

NN

N

NH2

O

OHO

HH

HH

O

O

NH

H

R1

P

O

O O

N

NN

N

NH2

O

OHO

HH

HH

O

O

H2N

HR2

P

O

O O

peptide

P-site A-site

aminoacylt-RNA esteractivatescarbonyl, makingpeptide bond formation favorable

adenine 2451of 23S ribosomalRNA abstracts protonfrom amino group,catalyzing nucleophilic attack

chemical protecting groupsare not necessary becausethe ribosomal machineryensures selective positioningand activation of the reactants

t-RNA

Peptide bond formation in vivo

N

NN

N

NH2

O

OHOH

HH

HH

OP

O

O O

N

NN

N

NH2

O

OHO

HH

HH

O

OHN

HR2

P

O

O O

P-site A-site

O

HN HR1peptide

peptidyl t-RNA shifts to P-sitenew aminoacyl t-RNA comes into A-site

deacylated t-RNAleaves P-site

Properties of the amino acid side chains

• size• acid-base equilibria• hydrophobicity/polarity• tautomerism• oxidation/reduction of cysteine • chemical reactivity (next lecture)

Sizes of amino acids

a.a vol (Å3) surface area(Å2)A 88.6 115R 173.4 225D 111.1 150N 114.1 160C 108.5 135E 138.4 190Q 143.8 180G 60.1 75H 153.2 195I 166.7 175L 166.7 170 K 168.6 200

a.a vol (Å3) surface area(Å2)M 162.9 185 F 189.9 210P 112.7 145S 89.0 115 T 116.1 140 W 227.8 255Y 193.6 230V 140.0 155

volume: Zamyatin A Prog Biophys Mol Biol 24, 107 (1972)surface area: Chothia C J Mol Biol 105, 1 (1975)

Acid-base titration curves of ionizable side chains

3 4 5 6 7 8 9 10 11 12 13 14

Arg+

Lys+Tyr

Cys

His+

AspandGlu

pH

eq.OH-

added

1

0

pKa

physiological pH

acid

base

The basic side chains

CH2

NH

HN

histidineHis H2.3%

pKa ~ 6means thatoften it isnot charged

arginineArgR5.1%

pKa ~ 12almost alwayspositively chargedin proteins

lysineLysK5.9%

pKa ~ 10almost alwayspositively chargedin proteins

NH2CHC

CH2

HO

O

CH2

CH2

CH2

NH3

CH2

CH2

CH2

NH

C

NH2

H2N

pct occurrencein proteins

The acidic side chains

aspartateAspD5.3%

glutamateGluE6.3%

CH2

CH2

C

O

O

NH2CHC

CH2

HO

O

C

O

O

asparagineAsnN4.3%

glutamineGlnQ4.3%

CH2

CH2

C

NH2

O

NH2CHC

CH2

HO

O

C

NH2

O

and thesecarboxylicacid side chainsare closely related to theiramide versions...

generally negative charged in prote insbecause conjugate carboxylic acids have pKa of about 4

Shifting of side chain titration curves

3 4 5 6 7 8 9 10 11 12 13 14

His+

pH

eq.OH-

added

1

0

pKa

physiological pH

acid

baseH2C

N

NH

H2C

NH

NH

Poorly populated but highly reactive forms of amino acids

NHCHC

CH2

HN

O

CH2

CH2

CH2

NH2

NHCHC

CH2

HN

O

CH2

CH2

CH2

NH3

NH2CHC

CH2

HO

O

CH2

CH2

CH2

N

RH

R

O

H

H2O

pH 7

– H+

base form of lysine not highly populated in general at physiological pH, but is a reactive nucleophile, and if present even in minuscule amounts may do chemistry

Kyte-Doolittle hydropathy of amino-acid residues

side chain hydropathy indexIle 4.5Val 4.2Leu 3.8Phe 2.8Cys 2.5Met 1.9Ala 1.8Gly -0.4Thr -0.7Trp -0.9

side chain hydropathy indexSer -0.8Tyr -1.3Pro -1.6His -3.2Glu -3.5Gln -3.5Asp -3.5Asn -3.5Lys -3.9Arg -4.5

Kyte J & Doolittle RF J Mol Biol 157, 105-32 (1982)

Many attempts have been made to quantify polarity, nonpolarity (hydrophobicity) of amino-acid residues in terms of scales. Kyte-Doolittle is a classic one. It is based on transfer free energies from nonpolar solvents to water combined with measurements of the tendency of residues to be buried in proteins. nonpolar--blue; polar--red; ambiguous--purple

The aliphatic amino acids (plus methionine)

prolineProP5.2%

only amino acid withside-chain fused to backbone in two places to make a ring

leucineLeuL9.1%

alanineAlaA7.8%

the amino-acid equivalentof vanillaice cream

glycineGlyG7.2%

sometimesconsidered a "polar"amino acid

isoleucineIleI5.3%

the mostcommontype in proteins

valineValV6.6%

these are branchedat the beta-carbon

NH2CHC

H

HO

O

CH3 CHH3C

CH3 H3C CH2

CH3

H CH2

CHH3C

CH3

NHC

HO

O

CH2

CH2

S

CH3

methionineMetM2.2%

not aliphaticbecause ofsulfurbut is similarin characterin many ways(nonaromatic,nonpolarresidue)

Aromatic side chains

CH2

N

HN

CH2 CH2

OH

NH3+CHC

CH2

-O

O

NH

histidineHis H2.3%

often not groupedwith otheraromatics and also can becharged/polar

tyrosineTyr Y3.2%

also sometimesconsidered anuncharged polarresidue

phenylalaninePheF3.9%

tryptophanTrp W1.4%

both these usually alsoconsidered hydrophobic amino acids

The polar uncharged side chains

CH2

N

HN

histidineHis H2.3%

pKa ~ 6for conjugateacid meansthat some-times it'scharged inproteins

glutamineGlnQ4.3%

threonineThrT5.9%

also hassomehydrophobiccharacterdue to methyl

NH2CHC

CH2

HO

O

OHHO

CH3H CH2

CH2

C

NH2

O

CH2

C

NH2

O

serineSerS6.8%

asparagineAsnN4.3%

these two havea bifunctional character in thesense of having both hydrogen bonddonor and acceptorgroups

CH2

OH

tyrosineTyrY3.2 %

not really very polar

pKa ~ 10means that it can bedeprotonated

CH2

SH

cysteineCysC6.8%

not as polaras its sisterserine but much easierto ionize toanion

Histidine--the “ambidextrous”

side chain acid

base

pKa ~ 7

Histidine is just barely acidic enough to populate base forms at neutral pH therefore, its base form is about the strongest base that can exist under physiological conditions the base form has two tautomers: one nitrogen can act as a base/ nucleophile, while the other can act as a hydrogen donor--”ambidextrous”

H2C

NH

N

NH

CH2

N

HN

NH

O O

H2C

NH

HN

NH

O

1 (δ1)

3 (ε2) 2 (ε1)

4 (δ2)

5 (γ)

β

predominant form in model peptides

Cysteine and cystine

CH2SH2 1/2O2 CH2S SCH2 H2O

cys tine2 cys te ines

R1 S R2S SR2 R2 S R1S SR2

disulfideformation

disulfideexchange

disulfide exchange occurs through the thiolate anion at neutral to basic pH

Pairs of cysteines frequently undergo oxidation to a disulfide bonded form called “cystine”

more hydrophobic than cysteine

• amino acids don’t fall neatly into classes--they are different combinations of small/large, charged/uncharged, polar/nonpolar properties

• how we casually speak of them can affect the way we think about their behavior. For example, if you think of Cys as a polar residue, you might be surprised to find it in the hydrophobic core of a protein unpaired to any other polar group. But this does happen.

• the properties of a residue type can also vary with conditions/environment

Key points about the character of amino acid side chains

Grouping the amino acids by properties

from http://www.russell.embl-heidelberg.de/aas/

which adapted it from Livingstone & Barton, CABIOS, 9, 745-756, 1993.