文稿案例生化上-biochemistry-chapter3aa and primary structure of proteinpart p

Protein

Purification

TechniquesAmino

Acid

Composition

of

ProteinsDetermining

the

Sequence

of

AminoAcid

residuesComparisons

of

the

PrimaryStructures

of

Proteins

RevealEvolutionary

RelationshipsProteins

is

most

importantbiological

functional

molecules:Enzymes,

thebiochemicalcatalysts(photosynthesis,thermophilicbacteria,HIV-1

revers

transcriptase)Storage

andtransportof

biochemicalmolecules

(Hb,

Mb)Physical

cell

support

and

sh

(tubulin,actin,

collagen)3.1 Outline

of

ProteinsMechanicalmovement

(flagella,mitosis,muscles)Decoding

information

in

the

cell(translation,

regulation

of

geneexpression)Hormones

or

hormone

receptors(growthhormone,

insulin

receptor)Other

specialized

functions(antibodies,toxinsetc)2.

Classification

of

proteins:According

to

sh

of

proteinmolecules;Globular

proteinsUsually

water

soluble,

compact,

roughly

sphericalHydrophobic

interior,

hydrophilic

surface

globular

proteins

include

enzymes,

carrier

andregulatory

proteinsFibrous

proteinsProvide

mechanical

support

–

not

water

solubleOften

assembled

into

large

cables

orthreadsα-Keratins:

major

components

of

hair

and

nailsCollagen:

major

component

of

tendons,

skin,

bonesand

teeth(2)

According

to

polymerization

ofprotein

molecules;Monomeric

proteinsOligomeric

proteins

(multimeric

proteins)(3)

According

to

conjugation

of

proteinmolecules;Simple

proteinsConjugated

proteins3.

Molecular

size

and

Mr

ofproteinsProteins

are

homogeneous.Mr

of

proteinsvary

fromabout

6000

Da

to

1

×106Da

or

more。Usually

insulin

(5700

Da)

or

RNase

(126000Da)was

as

the

boundary

of

proteins

andpolypeptides.Mr

ofproteins

≈

Mr

of

aminoacid

×110.Primary

structure

-

amino

acid

linear

sequenceSecondarystructure-

regionsof

regularlyrepeating

conformations

of

the

peptide

chain,such

as

-helices

and

-sheetsTertiary

structure

-

describes

the

sh

of

thefully

folded

polypeptide

chainQuaternarystructure-arrangement

of

twoormore

polypeptide

chains

intomultisubunitmolecule4.

Structure

levels

and

conformation

of

proteins3.2 Structures

of

Amino

Acids1.

General

structure

of

amino

acids.More

than

200

different

AAs

arefound

in

living

anisms.2.

Structures

of

the

20

common

(standard)amino

acids.All

of

the

common

amino

acids

found

inproteins

are

α–amino

acids.CHH2NCOOHRCOO-RgroupAmino

groupCarboxylic

groupH

=

GlycineCH3

=

AlanineH3

N

+H3.

Ionization

of

amino

acidsThe

R

groups

are

differentin

20

AAs.Zwitterionic form

ofamino

acidsUnder

normal

cellular

conditions

aminoacids

are

zwitterions(dipolar

ions):Amino

group=-NH3

(Protonated)+Carboxyl

group

=-COO-

(Ionized)StereochemistryStereoisomers

-compounds

that

have

thesame

molecular

formula

but

differ

in

thearrangement

of

atoms

in

spaceEnantiomers

-

nonsuperimposable

mirrorimagesChiral

carbons

-

have

four

different

groupsattached4.

Configuration

of

amino

acids.Stereochemistry

of

amino

acids19

of

the

20

common

amino

acids

have

achiral

-carbon

atom

(Gly

does

not)Threonine

andisoleucine

have

2chiralcarbons

each

(4possible

stereoisomerseach)Mirror

imagepairs

ofamino

acids

aredesignated

L

(levo)

and

D

(dextro)Proteins

are

assembled

from

L-amino

acids(few

D-amino

acids

occur

innature)Mirror

Images

of

AminoAcid

Mirror

image

of

StereoisomersThe

19

chiral

amino

acids

used

in

the

assemblyofproteins

are

all

of

the

L

configuration,although

afew

D-aminoacids

occur

innature.Why

?Ball-and-stick

modelprojectiosprojections

-

horizontal

bonds

froma

chiralcenter

extend

toward

the

viewer,

vertical

bondsextend

away

from

the

viewer(1).The

three-letter

and

one-letter

abbreviations.5.Classification

of

the

20

common

amino

acids(2).

Classification

of

amino

acids

bychemical

construction.A.Aliphatic

R

groups:B.Aromatic

R

groups:C.Sulfur-containing

R

groups:D.Side

chains

with

alcohol

groups:E.Basic

R

groups:F.Acidic

R

groups

and

theiramidederivatives:A. Aliphatic

(hydrophobic)

R

Glycine

(Gly,

G)

-Gthreou-cpasrbon

isnot

chiralsince

there

are

two

H’s

attached

(R=H)

=smallest

and

fits

easiest

into

small

nitchesFour

amino

acids

have

saturated

side

chains:Alanine

(Ala,

A)

Valine

(Val,

V)Leucine

(Leu,

L,

The

occurrence

inproteinsisthe

highest.)Isoleucine

(Ile,

I)Proline

(Pro,P)

3-carbon

chainconnects-C

and

N

=ring

structureFour

aliphatic amino

acidstructuresImportant

in

protein

structure

and

foldingsincetheir

R

groups

cluster

away

from

waterProline

has

a

nitrogen

in

thealiphatic

ring

systemProline(Pro,

P)-

has

a

threecarbon

side

chain

bonded

tothe

-aminonitrogenThe

heterocyclic

pyrrolidinering

restricts

the

geometry

ofpolypeptides

=

causes

abruptchanges

in

the

direction

of

thepolypeptide

chainB.

Aromatic

R

Groups(benzene

ring

properties)Side

chains

have

aromatic

groupsPhenylalanine

(Phe,

F)

- benzene

ringTyrosine

(Tyr,

Y)

- phenol

ringTryptophan

(Trp,

W)

-

bicyclic

indole

groupThe

occurrence

in

proteins

is

the

lowest.Aromatic

amino acid

structuresthey

absorb

UV

light

at

280

nmC.

Sulfur-Containing

R

GroupsMethionine

(Met,

M)

- (-CH2CH2SCH3)Cysteine

(Cys,

C)

-Methionine

usually(-CH2SH)amino

acid

in

a

proteinTwo

cysteine

side

chains

can

be

cross-linked

byforming

a

disulfide

bridge

(-CH2-S-S-CH2-)Disulfide

bridges

may

stabilize

thethree-dimensional

structures

of

proteinsMethionine

and

cysteineMany

extracellular

proteins

contain

disulfidebridges

that

stabilize

the

3D

structure

ofproteins

by

joining

adjacent

peptide

chains.D. Side

Chains

with

AlcoholGroupsSerine

(Ser,

S)

and

Threonine

(Thr,

T)haveuncharged

polar

side

chains

=

hydrophilicE.

Basic

RGroupsHydrophilic

with

nitrogenouechainsHistidine

(His,

R)

-

imidazoleLysine

(Lys,

K)

- alkylamino

groupArginine

(Arg,

R)

- guanidino

groupSide

chains

are

nitrogenous

bases

which

aresubstantially

positively

charged

(protonacceptors)

at

pH

7

=

gives

a

positive

chargetoproteinsStructures

of

histidine,

lysine

andarginineF. Acidic

R

Groups

and

AmideDerivativesConfer

negativechares

to

proteins

becausetheir

side

chains

are

ionized

at

neutral

pHAspartate

(Asp,

D)

and

Glutamate

(Glu,

E)are

dicarboxylic

acids,

and

are

negativelycharged

at

pH

7Asparagine

(Asn,

N)

and

Glutamine

(Gln,

Q)are

uncharged

but

highly

polar

and

are

amidesof

Asp

and

Glu

whose

R

groups

can

form

HbondsStructures

of

aspartate,

glutamate,asparagine

and

glutamineuLe

L-C-C-CONH2-C-CONH2-C-COOH-C-C-COOH-H-CH3-C-OH-C-C-S-CPPro-C-C

CNN+3-C-C-C-C-NH

+-C--C- -OH-C-NAliphaticAmideAcidicImino,CircularSulfurAlcoholAromaticBasic

-C-C-C-N-C-NN+=C-C-C-CC-C-C-CC

C-CCC

CHN

C-COOH-C-COHAsnNGlnQAspDGluEPhe

FArgRLysKHisHGlyGAlaAVal

VIle

ITyr

YSerSThrTMet

MCys

C

-C-SHTrp

W(3).

Classification

of

amino

acids

by

polarity.Highly

hydrophobic:Highly

hydrophilic:IleVal

LeuMetPheHisAspGluGlnAsnLysArgLess

hydrophobic:TyrCysSerThrTrp

Ala

GlyProThe

Hydrophobicity

ofAmino

Acid

Side

ChainsHydropathy: the

relative

hydrophobicity

ofeachamino

acid

(5

are

highly

hydrophobic

and

7arehighly

hydrophylic)The

larger

the

hydropathy,

the

greater

thetendency

of

an

amino

acid

to

prefer

ahydrophobic

environmentHydropathy

affects

protein

folding:hydrophobic

side

chains

tend

to

be

in

the

interior

hydrophilic

residues

tend

to

be

on

the

surfaceHydropathy

scale

for

aminoacid

residues(Free-energy

change

fortransfer

of

an

amino

acidfrom

interior

of

alipidbilayer

to

water)Free-energy

changefor

transfer

(kjmol-1)AminoacidThe

relative

hydrophobicityorhydrophilicityof

eachaminoacid

is

called

its

hydropathy.-0.29-0.75-1.1-1.7-2.6-2.7-2.9-3.0-4.6-7.5Nutritional

requirement

of

human

onamino

acids:Nonessentialamino

acidEssentialamino

acidGly

Ala

Ser

Tyr

Cys

ProAsn Asp

Glu

Gln

ArgMet

Trp

Lys

Val

IleLeu

Phe Thr

(His)3.3 Other

Amino

Acids

andAmino

Acid

DerivativesMore

than

200

different

AAs

arefound

in

living

anisms.Most

species

contain

a

variety

ofL-AAs

that

are

either

precursorsof

the

common

AAs

orintermediates

in

other

biochemicalpathways.For

examples:Homocysteine,

homoserine,

ornithine,

citrulline(AAs

precursors

or

intermediates);SAM

(CH3

donor),γ-aminobutyrate

(neurotransmitter),

histamine (blood

pressure,HCl

secretion),epinephrine,

Thyroxine

(metablism

regulation),N-formylmethionine

(bacteria),selenocystein

(enzyme).Precursor

AA:(a)glutamate (b)

histidine

(c)tyrosine (d)

tyrosine23

of

AAs

have

2

ionizable

groups;7

of

AAs

have

ionizable

side

chains

withadditional,measurable

pKa

values——aspartate,

glutamate,

cysteine,

tyrosine,lysine,

histidine

and

arginine3.4 Ionization

of

Amino

AcidsEach

ionizable

group

is

associated

with

aspecificpKa

value.When

the

pH

of

the

solution

is

below

thepKa,

the

protonated

form

predominates;When

the

pH

ofthe

solutionis

above

thepKa,

the

unprotonated

form

predominates;HighLowH

H+HlonepairelectronsHHH+NNAminoH+Ampholyte

contains

both

positive

and

negative

groups

on

its

moleculeCarboxylicCOOHCOOProton

Is

Adsorbed

or

DesorbedpKaLowHighpKaCOOHN

H3+R-C-HCOO-N

H3+R-C-HNH2R-C-HCOO-Acidic

environmentNeutral

environmentAlkaline

environmentA+A-AopK1

~

2pK2

~

9pH

=

pKa

+

log[proton

acceptor]/[protondonor]Henderson-Hasselbalch

equationThe

pKa

values

of

AAs

are

determinedfrom

titration

curves.(Ala)pK1

=

2.4pK2

=

9.9The

net

charge

on

Ala

at

pH2.4

averages+0.5

and

the

net

charge

at

pH9.9

averages-0.5.At

pH6.15

,

the

avergae

net

charge

ofAla

is

zero.

The

pH

is

referred

to

as

theisoelectric

point

(pI).Ala

would

not

migrate

in

a

electric

fieldat

its

pI.pH

>

pI

，

toward

anodepH

<

pI

，

toward

cathodepH

=

pI

，

no

migrateIonization

andthe

titrationcurve

of

Glu.Ionization

andthe

titrationcurve

of

His.At

pH7.0,

the

ratio

of

imidazole

toimidazolium

ion

is

10:1.

Thus,

the

2formsof

His

are

both

present

in

significantconcentrations

near

physiological

pH.This

property

makes

the

side

chain

ofHisideal

for

the

transfer

of

protonswithin

thecatalytic

sites

of

enzymes.2Amino

acids-COOH-NH-RGlyGAVLISTMFWNQPDEHCYKR2.349.60Ala2.349.69Val2.329.62Leu2.369.68Ile2.369.68Ser2.219.15Thr2.6310.4Met2.289.21Phe1.839.13Trp2.389.39Asn2.028.80Gln2.179.13Pro1.9910.6Asp2.099.823.86Glu2.199.674.25His1.829.176.0Cys1.7110.88.33Tyr2.209.1110.07Lys2.188.9510.53Arg2.179.0412.48pK1pK1pHpK2pK2pK3[OH-]pI

?pK1

+

pK22two

pKapIthreepKa??Howto

calculate

pI

of3NH

+HHOOC-CH2-C-COOHHOOC-CH2-C-COO-3NH

+H-OOC-CH2-C-COO-3NH

+H-OOC-CH2-C-COO-NH2HA+AoA-A2-pK1

=

2.1pK2

=

3.9pK3

=

9.8

2.1

+

3.92=3.0secondthirdIsoelectric

point

is

the

averageofthetwopKa

flankingthezeronet-chargedformpK1pK2pK3Aspartic

acid-2-10+1[OH]The

pI

of

20commonAAsAApIAApIAApIAApIAla6.02Pro6.30Thr6.53Asp2.77Val5.97Phe5.48Cys5.02Glu3.22Leu5.98Trp5.89Tyr5.66Lys9.74Ile6.02Gly5.97Asn5.41Arg10.76Met5.74Ser5.68Gln5.65His7.59The

pKa

of

α–COOH

of

free

AAs

are

lowerthanthose

of

typical

carboxylicacids.

Because

of

theeffect

of

α–NH2

and

R

group.According

to

Henderson-Hasselbalchequation,free

AAs

exist

predominantly

as

zwitterions

atneutral

pH.γ-COOH

of

Glu

is

further

removed

from

theinfluence

of

the

α–NH2

,

andbehaves

as

aweakacidwith

apKa

of

4.1

similar

to

acetic

acid.The

pKa

of

ionizableside

chainsin

proteins

candiffer

from

those

ofthe

free

AAs.Because:

(1)

α–NH2

and

α–COOH lose

theircharges

in

peptide

chain;(2)

the

position ofan ionizable

sidechain

within

the

3D

structure

of

aprotein

canaffectitspKa.The

measurement

of

AAsconcentrationWhether

can

AAs

concentration

beenmeasured

by

acidic

or

basic

titration

?pH

=

pKa2

+

log[H2NCH2COO-]/[H3N+CH2COO-]Reactions

of

α-NH2：Reactions

of

α-COOH：Reactions

of

α-NH2

and

α-COOH：Reactions

of

R

groups：3.5 Chemical

reactions

of

AAs:1.Reactions

of

α－NH2：(1)

Reaction

with

HNO2（deamination）:Van

Slyke

methods

to

measure

content

of

AAs(2)

Reaction

with

acylating

agent（for

protection

of

－NH2

inpeptides

chemical

synthesis):NH2R-CH-COOH

+

HNO2OHR-CH-COOH

+

N2

+

H2OAcylating

reactionOR1

C

X+

H2N

CHCOO-R2X=-Cl,

OH,-OCOROH-COO-R2OR1

C

HN

CHN(CH3)2R

OSO2

HN

CH

CN(CH3)2R

O+

H2N

CH

C水解N(CH3)2R

OHN

CH

C

OH+氨基酸SO2Cl丹磺酰氯多肽N-端丹磺酰N-端氨基酸Reaction

with

DNS-Cl:DNS-氨基酸SO2For

the

measurement

of

N-terminus

ofproteins.DNFBF

＋

H2NNO2O2NRCH

COOHDNP

-

AAO2NNO2HNRCH COOH＋

HF(3)

Hydrocarbylation

Reaction:For

the

measurement

of

N-terminus

of

proteins

by

Sanger.Edman

reaction＋NCNH

RCHS40℃，H+、硝基甲烷C

ORCH

COOHH2NPITC异硫

酸苯酯PTC-

AA苯氨基硫甲酰氨基酸PTH-氨基酸苯乙内酰硫脲氨基酸HNCNHC

OCHRN

C

S40℃，弱碱OHS可用层析法鉴定出aa的种类(4)

Forming

Shifebase:(5)

Deamination

reaction

by

enzymecatalyse：RCH

COO-3NH

+R4OCH

COO-

+

NH

+氨基酸氧化酶(6)

Reaction

with

fluorescent

agents:For

the

labelling

and

modification

ofproteins;

measurement

of

free

amine

of

Lys.λEX=390nm，λEM=475nmFluorescentammoniaFluorescentExcessive

fluorescent

ammonia

will

been

hydrolyzed

within

1min.+RNH2PH9，RT+

RNH2

+

SH-R’o-Phthalaldehyde(邻苯二)：λEX=340nm，λEM=455nmFluorescent2.

Reaction

ofα－COOH：(1)

Reactions

to

form

salt

andester（for

protection

of

－COOH

in

peptides

chemical

synthesis):+

R2OHR3CH

COO-NH

+NH2.HClR

CH

COOR2

+

H2O(2)

Reactionstoformacyl

chloride:（for

activating

of

－COOH）PCl3,

PCl5

or

SOCl2RCHCOO-NHPGNHPGR

CH

COCl(3)Decarboxylation:In

vivo:

decarboxylase

catalyse.In

vitro:Ba(OH)2

，ΔRCH2NH2

+CO2NH2—CH—COOHR(4)

Reactions

to

form

azide（

for

activating

of

－COOH）酰化氨基酸甲酯酰化氨基酸酰肼酰化氨基酸叠氮还原性茚三酮OOH3

2OH＋

NH

＋

CO

＋

RCHO水合茚三酮＋RCH－COOHNH2OOO＋32NH

＋OOOHHOOO＋

3H2O蓝紫色化合物O＋NH4ONO

O茚三酮反应常用于aa的定性和定量分析3. Reactions

of

α－NH2

and

α－COOH：(1)

Reaction

with

ninhydrin

reagent：Pro

的茚三酮反应呈黄色,λ=440nmλ=570nm(2)

Reaction

to

form

peptides：4.

Reactions

of

R

groups:Tyr：iodonation

and

nitration:（For

isotope

labelling

）Pauly

reaction：（C）Millon

reaction（phenolic

group）：Tyr

+

Hg(NO3)2

(含HNO2的HNO3溶液)

△

redcompounds（D