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Chapter 5: OutlineAmino Acids

Amino acid classes Stereoisomers

Bioactive AA Titration of AA

Modified AA AA reactions

Peptides

Proteins (We are here)

Protein structure

Fibrous proteins

Globular proteins

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Protein Function1. Catalysis

2. Structure

3. Movement

4. Defense

5. Regulation

6. Transport

7. Storage

8. Stress Response

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Proteins by Shape-1Fibrous proteins exist as long stranded molecules: Eg. Silk, collagen, wool. A collagen segment in space-filling mode illustrates this point.

Red spheres represent oxygen, grey carbon, and blue nitrogen

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Proteins by Shape-2Globular proteins have somewhat spherical shapes. Most enzymes are globular. Eg. myoglobin, hemoglobin. Myoglobin in space-filling mode is the chosen example.

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Proteins by CompositionSimple

Contain only amino acids

Conjugated

simple protein (apoprotein)

prostetic group (nonprotein)

glycoproteins

lipoproteins

metaloproteins

etc.

Holo-protein

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Four Levels of Protein Structure

Primary, 1o

the amino acid sequenceSecondary, 2o

3-D arrangement of backbone atoms in space

Tertiary, 3o

3-D arrangement of all the atoms in space

Quaternary, 4o

3-D arrangement of subunit chains

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Determining Primary Structure1. Hydrolyze protein with hot 6M HCl.

Identify AA and % of each.

Usually done by chromatography

2. Identify the N-term and C-term AAs

C-term via carboxypeptidase

N-term via Sanger’s Reagent, DNFB

2,4-dinitrofluorobenzene

Often step 2 can be skipped today.

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Det. Primary Structure: 23. Selectively fragment large proteins

into smaller ones.

Eg. Tripsin: cleave to leave Arg or Lys as C-term AA

Eg. Chymotrypsin: cleave to leave Tyr or Trp or Phe as C-term AA

Eg. Cyanogen bromide cleaves at internal Met leaving Met as C-term homoserine lactone

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Det. Primary Structure: 34. Determine AA sequence of peptides

with AA sequencer using Edman’s reagent:

phenyl isothiocyanate which reacts with the N-term AA

See the next slide

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Det. Primary Structure: 3bNH3

+COO

-1 2 3

N C S

NH C

S

NH COO-

1 2 3

NH3

+COO

-2 3NH C

S

NCH

CO

R1

aqueous acid

+

N

CS

NH

CHCO

R1

RAR

protein

Edman’s reagent

Phenylthiohydantoin (PTH)derivative of N-term AA

Thiazoline derivative

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Det. Primary Structure: 45. Reassemble peptide fragments from

step 3 to give protein.

An example follows on the next slide.

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Det. Primary Structure: 4b

A twelve AA peptide was hydrolyzed.

Trypsin hydrolysis:

Leu-Ser-Tyr-Gly-Ile-Arg

Thr-Ala-Met-Phe-Val-Lys

Chymotrypsin hydrolysis

Val-Lys-Leu-Ser-Tyr

Gly-Ile-Arg

Thr-Ala-Met-Phe

Deduce the AA sequence

One isC-term

Lys is internal!

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Det. Primary Structure: 4c

Tr Leu-Ser-Tyr-Gly-Ile-Arg

Ct Gly-Ile-Arg

Ct Val-Lys-Leu-Ser-Tyr

Tr Thr-Ala-Met-Phe-Val-Lys

Ct Thr-Ala-Met-Phe

The complete sequence is:

Thr-Ala-Met-Phe-Val-Lys-Leu-Ser-Tyr-Gly-Ile-Arg

Keeping in mind the N-term AA and overlaping the sequences properly gives:

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Secondary Structure

The two very important secondary structures of proteins are:

-helix

-pleated sheet

Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain.

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Helix: Peptide w Hbonds

First six C=O to N hydrogen bonds shown

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Sheet: stick form Protein G

Chain 1

H bonds in dotted red-blue

Chainsegment 1

Seg 2

Seg 3

Seg 4

H bonds shown in dotted red-blue

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B Sheet: Lewis Structure

CH

C

N

CH

C

N

CH

C

N

O

H

H

O

H

O

N-term

C-term C-term

N-term

CH

C

N

CH

C

N

CH

C

N

O

H

H

O

H

O

Parallel sheet

CH

C

N

CH

C

N

CH

C

N

O

H

H

O

H

O

N-term

C-term

C-term

N-term

CH

C

N

CH

C

N

CH

C

N

O

H

H

O

H

O

Antiparallel sheet

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Supersecondary StructureReverse turns in a protein chain allow

helices and sheets to align side-by-side

Common AA found at turns are:

glycine: small size allows a turn

proline: geometry favors a turn

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Supersecondary Structure: 2

Combinations of helix and sheet.

meander

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Tertiary StructureThe configuration of all the atoms in the

protein chain:

side chains

prosthetic groups

helical and pleated sheet regions

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Tertiary Structure: 2

Protein folding attractions:

1. Noncovalent forces

a. Inter and intrachain H bonding

b. Hydrophobic interactions

c. Electrostatic attractions

+ to - ionic attraction

d. Complexation with metal ions

e. Ion-dipole

2. Covalent disulfide bridges

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Tertiary interactions: diag.

NH3

+

Polypeptide Chain

COO CH2 S

CH2S

O HC

O O

CH3CH3

CH2OHCH2 OH

CHCH3 CH3CHCH3 CH3 CO

ONH3

+

Mg2+

ionic

disulfide

hydrophobicH bondsor dipoleIon-dipole

metal coord’n

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DomainsDomains are common structural units

within the protein that bind an ion or small molecule.

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Quaternary Structure-1Quaternary structure is the result of

noncovalent interactions between two or more protein chains.

Oligomers are multisubunit proteins with all or some identical subunits.

The subunits are called protomers.

two subunits are called dimers

four subunits are called tetramers

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Quaternary Structure-2If a change in structure on one chain

causes changes in structure at another site, the protein is said to be allosteric.

Many enzymes exhibit allosteric control features.

Hemoglobin is a classic example of an allosteric protein.

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Denaturation-loss of protein structure, 2o 4o, but not

1o.1. Strong acid or base2. Organic solvents3. Detergents4. Reducing agents5. Salt concentrations6. Heavy metal ions7. Temperature changes8. Mechanical stress

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Denaturation-2

Denaturing destroys the physiological function of the protein.

Function may be restored if the correct conditions for the protein function are restored.

But! Cooling a hardboiled egg does not restore protein function!!

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Fibrous ProteinsFibrous proteins have a high

concentration of -helix or -sheet. Most are structural proteins.

Examples include:

a-keratin

collagen

silk fibroin

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Globular ProteinsUsually bind substrates within a

hydrophobic cleft in the structure.

Myoglobin and hemoglobin are typical examples of globular proteins.

Both are hemoproteins and each is involved in oxygen metabolism.

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Myoglobin: 2o and 3o aspectsGlobular myoglobin has 153 AA arranged in eight -helical regions labeled A-H.

The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue.

His E7 and F8 are important for locating the heme group within the protein and for binding oxygen.

A representation of myoglobin follows with the helical regions shown as ribbons.

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Some helical regions

Myoglobin: 2o and 3o aspects

Heme group with iron (orange)at the center

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The Heme Group

NN

NN

CH2CH2COO-

CH2CH2-OOC

CH3 CH3

CH

CH2

CH3

CH3 CH CH2

Fe(II)Pyrrole ring

N of HisF8 bindstofifth site onthe iron.

His E7 actsas a ”gate” for oxygen.

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Binding Site for Heme

Lower His bonds covalently to iron(II)

Oxygen coordinates to sixth site on iron and the upper His acts as a “gate” for the oxygen.

NN

H

O

O

N

N

N

N N

N

Fe

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HemoglobinA tetrameric protein

two -chains (141 AA)

two -chains (146 AA)

four heme units, one in each chain

Oxygen binds to heme in hemoglobin cooperatively: as one O2 is bound, it

becomes easier for the next to bind.

Lengthy segments of the and chains homologous to myoglobin.

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Hemoglobin: ribbons + hemesEach chain is in ribbon form and color coded.

The heme groups are in space filling form

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Oxygen Binding CurvesOxygen bonds differently to hemoglobin

and myoglobin.

Myoglobin shows normal behavior while hemoglobinn shows cooperative behavior. Each oxygen added to a heme makes additon of the next one easier.

The myoglobin curve is hyperbolic.

The hemoglobin curve is sigmoidal.

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Oxygen Binding Curves-2

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The Bohr Effect (H+ and Hb)

Lungs:

pH higher than in actively metabolizing tissue. (Low H+). Hb binds oxygen and releases H+.

Muscle at Work:

pH lower (H+ product of metabolism). Hb releases oxygen and binds H+.

HbO2 + H+ + CO2

metabolism O2 + H+-Hb-CO2

in lungs