1. 2 ester (functional group, acid + alcohol) handout 2-9 top a trigyceride (fat)

1

2

Ester (functional group, acid + alcohol)}

Handout 2-9 top A trigyceride (fat)

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cis

C C| |

HH

HH| |

| || C C| |

HH|| ||

- 2H

X

Free rotation about single bonds

No free rotationabout double bonds

cis

Solid fats

Oils

Effect of fatty acid structure on physical properties

trans

cis

Free rotation about single bonds

No free rotation about double bonds

C C|

|H

H

||

|

|

X

trans

Unsaturated fatty acid

Saturated fatty acid

Hydrogenation of oil to solidify it

4

Fatglobule

Nuc.

Adipocyte (fat storage cell)

Adipose tissue

Fat is a good compact source of energy, about twice the calories as starch, pound for pound.

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6

[HO]

[HO]

Handout 2-9

Phospholipids:

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O ||

HO-P-O-

|

O-

Phosphoric acid(phosphate ion)

+ R-OH

an alcohol(hydroxyl)

O ||R-O-P-OH |

O-

a phosphoester

8

If R=another alcohol:a phospho-diester

If R=H, phosphatydic acid

}HO

HO

Handout 2-9

HO –CH2CH2N+H3

(alcohol = ethanolamine) phophatydyl ethanolamine

(2 FAs implied)

x y

9

HOHHOH

Phosphate head

2 fatty acid tails each

Biological membranes are phospholipid bilayers

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Incidentally, note the functional groups we have met so far:

HydroxylAmineAmideCarboxylCarbonylAldehydeKetoneEster: Carboxylic acid ester

PhosphoesterPhosphodiester

And:

Glycosidic bondsC=C double bonds (cis and trans)

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Amino acids (the monomer of proteins)

PROTEINS

R = ONE of 20 CHEMICAL GROUPS

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At pH 7, ,most amino acids are zwitterions (charged, but electrically neutral)

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Equilibrium state of the carboxyl group lies far towards the ionized molecule at pH7

+H+

pH7

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+OH- ( = -H+)

+H+

pH:Net charge:

50-50 charged-uncharged at ~ pH9 (=the pK)

50-50 charged-uncharged at ~ pH2.5 (=the pK)

R OH | /+H3N - C – C=O

| H

R O-

| /

H2N - C – C=O

| H

R O-

| /+H3N - C – C=O

| H

1+1

11-1

70

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Numbering (lettering) amino acids

alpha-carbon

alpha-carboxyl (attached to the α-carbon)alpha-amino

β

γδ

ε

ε-amino group

The 2 amino groups and the carboxyl are assumed to be charged (understood)even if unwritten.

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Sho

wn

unch

arge

d (a

s on

exa

ms)

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18

19

H+ H+

~10% charged at pH7 guanido +1

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Ball and stick physical model of an amino acid

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Amino acids in 3 dimensions

• Asymmetric carbon (4 different groups attached)

• Stereoisomers• Rotate polarized light• Optical isomers • Non-superimposable• Mirror images

• L and D forms

From Sadava text

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Mannose

A

B

C

D

Any compound

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Condensation of amino acids to form a polypeptide(must be catalyzed)

dehydration again

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Parts of a polypeptide chain

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Handout 3-3

The backbone is monotonous It is the side chains that provide the variety

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“Polypeptides” vs. “proteins”

• Polypeptide = amino acids connected in a linear chain (polymer)

• Protein = a polypeptide or several associated polypeptides (discussed later)

• Often used synonymously

• Peptide (as opposed to polypeptide) is smaller, even 2 AAs (dipeptide)

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(Without showing the R-groups)

The backbone is monotonous

It is the side chains that provide the variety

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Proteins do most of the jobs in the cell

E.g., egg albumin, hemoglobin, keratin, estrogen receptor,immunoglobulins (antibodies), enzymes (e.g., beta-galactosidase)

Each is a polymer or assemblage of polymers made up of amino acidsEach particular protein polymer (polypeptide) has a unique sequence of amino acids . . . . and an English name.Each molecule of a particular protein has the same sequence of amino acids.

E.g., met-ala-leu-leu-arg-glu-leu-val- . . . .

How is this sequence determined?

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Primary (1o) Structure = the sequence of the amino acids in the polypeptide chain

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Determining the sequence

One way: use an enzyme: (an old method, but useful for teaching)

identify,

e.g., …. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp-ser

…. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu-asp +

…. arg-leu-leu-val-gly-ala-gly-phe-trp-lys-glu +

Carboxypeptidase: hydrolyzes the peptide bond

ser

asp

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(-) (+)

AA mixture (ala, glu, lys

METHODS . . .

Anode Cathode

Note: The cathode is negative in an electrophoresis apparatus even though it is positive in a battery (voltaic cell)

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A paper electrophoresis apparatus

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AAs applied at lower end

Side view

Handout 3-4

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“Rf”

0.82

0.69

0.45

0.27

0.11

After stopping the paper chromatography and staining for the amino acids:

1.00“front” =

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Paper chromatography apparatus

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N C

Trypsin (lys, arg)

Chymotrypsin (trp, tyr, phe)

The order of the subpeptides is unknown.The sequence is reconstructed by noting the overlap between differently produced subpeptides

(1)

(2)

Sequence overlap

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N C

Trypsin (lys, arg)

Chymotrypsin (trp, tyr, phe)

The order of the subpeptides is unknown.The sequence is reconstructed by noting the overlap between differently produced subpeptides

(1)

(2)

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Sub-peptides

Fingerprinting a protein: analysis of the sub-peptides

(without breaking them down to their constituent amino acids)

Application to sickle cell disease(Vernon Ingram, 1960’s)

Hemoglobin protein

No further digestion to amino acids; left as sub-peptides

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Oligopeptides behave as a composite of their constituent amino acids

Net charge = -1: moves toward the anode in paper electrophoresesFairly hydrophobic (~5/6): expected to move moderately well in paper chromatography

Nomenclature: ala-tyr-glu-pro-val-trp or AYEPVW or alanyl-tyrosyl-glutamyl-prolyl-valyl-tryptophan

+

-

-

E.g.:

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In fingerprinting, these spots containpeptides, not amino acidsThe mixture of

all sub-peptides formed

------glutamate----- (normal)

------valine------(sickle)

More hydrophobic

More hydrophilic

Negativelycharged

Positivelycharged

Negativelycharged

Positivelycharged

Negativelycharged

Positivelycharged

Less negatively charged,More hydrophobic

Hb

trypsin

Protein fingerprinting

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Every different polypeptide has a different primary structure (sequence).Every polypeptide will have different arrangement of spots after fingerprinting.

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• Molecule #1: N-met-leu-ala-asp-val-val-lys-....

• Molecule #2: N-met-leu-ala-asp-val-val-lys-...

• Molecule #3: N-met-leu-ala-asp-val-val-lys-...

• Molecule #4: N-met-leu-ala-asp-val-val-lys-... etc.

3-dimensional structure of proteins

One given purified polypeptide

clothesline . . .

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Information for proper exact folding(How does a polypeptide fold correctly?)

Predicting protein 3-dimensional structure

Determining protein 3-dimensional structure

Where is the information for choosing the correct folded structure?

Is it being provided by another source (e.g, a scaffold) or does it reside in the primary structure itself?

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Denature by heat

Cool, renature?

Tangle, gel.Probably due to non-productivehydrophobic interactions

XToo long to sort out

“Renaturation” of a hard-boiled egg

ovalbuminCool, entangled

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urea

chaotropic agent

used at very high concentrations (e.g., 7 M)

gentler, gradual denaturation, renaturation

O||

N-C-N

H

H

H

H

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+ urea, denature

-urea, renature

??

“Renaturation” of pure ribonuclease after urea

“native” ribonucleaseactive enzyme

compact

denatured ribonucleaseinactive enzyme

random coil

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Now dialyze out the urea

Slow denaturation of ribonuclease by urea

O ||Urea = H2N-C—NH2

Macromolecules (protein here) cannot permeate bag material

Small molecules (H20, urea) can.Urea will move from areas of high concentrationto areas of low concentration

Ribonuclease in the bag is denatured

RENATURESRibonuclease

in the absence of any other material

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PRIMARY STRUCTURE DETERMINES TERTIARY STRUCTURE. 

Christian Anfinsen:

+ urea, denatures

- urea, renatures

“The Anfinsen Experiment”