basic protein structure and stability i: formation of peptide bonds/ properties of amino acids...
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Basic protein structure and stability I:Formation of peptide bonds/
properties of amino acids
Biochem 565, Fall 2008
08/25/08
MQTLSERLKKRRIALKMTQTELATKAGVKQQSIQLIEAGVTKRPRFLFEIAMALNCDPVWLQYGTKRGKAA
atgcaaactctttctgaacgcctcaagaagaggcgaattgcgttaaaaatgacgcaaaccgaactggcaaccaaagccggtgttaaacagcaatcaattcaactgattgaagctggagtaaccaagcgaccgcgcttcttgtttgagattgctatggcgcttaactgtgatccggtttggttacagtacggaactaaacgcggtaaagccgcttaa
augcaaacucuuucugaacgccucaagaagaggcgaauugcguuaaaaaugacgcaaaccgaacuggcaaccaaagccgguguuaaacagcaaucaauucaacugauugaagcuggaguaaccaagcgaccgcgcuucuuguuugagauugcuauggcgcuuaacugugauccgguuugguuacaguacggaacuaaacgcgguaaagccgcuuaa
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.
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