1 Determining the sequence One way: use an enzyme: (an old method, but useful for teaching)...

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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

ser asp

2

(-) (+)

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

2000 to 4000 volts DC, dangerous

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AAs applied at lower end

Side view

Handout 3-4

Isopropanol

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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” =

Most hydrophobic = furthest

Most hydrophilic = least far

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Paper chromatography apparatus

(felt tip black marker ink demonstration)

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“Sub-peptides”

Polypeptide chain

Ordering the sub-peptides within the polypeptifde:

• Treatment of a polypeptide with trypsin• Trypsin is a proteolytic enzyme.• It catalyzes cleavage (hydrolysis) after lysine and arginine residues

Determine sequence of eachsubpeptide using the carboxypeptidase technique

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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

Done!

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Quick way to compare two proteins without sequencing:Fingerprinting a protein: analysis of the sub-peptides themselves.(Without sequencing, i.e., without breaking them down to their constituent amino acids)

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

Sub-peptides

Hemoglobin protein

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

trypsin

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Oligopeptides behave as a composite of their constituent amino acids

Net charge = -2+1= -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

+

-

-

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In fingerprinting, these spots containpeptides, not amino acids

The mixture of all sub-peptides formed

More hydrophobic

More hydrophilic

Negativelycharged

Positivelycharged

negatively charged

positivelycharged

Less negatively charged,more hydrophobic

Hb protein

trypsin

---glutamate--- (normal)

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

Sequence just this peptide:

Single AA substitution

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Every different polypeptide has a different primary structure (sequence). By definiton.

The migration behavior of each sub-peptide depends on its composite properties.

The properties are suffiently complex such that most subpeptides in a given polypeptide will behave differently.

Every polypeptide will have different arrangement of spots after fingerprinting.

Four polypeptide fingerprints

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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 template)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 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 permeate.

Urea will move from areas of high concentration to 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”

20Julio Fernandez lab, CU: a modern version of the Anfinson experiment

For

ce n

eede

d to

pul

lRelax force, re-contracts, renatures Pull

Length

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Denaturation/renaturation of domains ofa protein (titin) using the atomic force microscope.

Julio Fernandez and colleagues, Columbia Univ.

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BUT:Chaperonins

(made of proteins themselves)

• Help fold proteins during synthesis

• Perhaps by preventing illegitimate interactions, like intermolecular contacts via exposed hydrophobic groups of partially folded proteins

• Also help re-fold proteins that have denatured after passing through a membrane’s P-lipid bilayer, e.g., during transport into a mitochondrion (organelle).

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Zsolt Török, Laszlo Vigh and Pierre Goloubinoff, 1996 The Journal of Biological Chemistry, 271, 16180-16186.

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See bottom of handout 3-3

Primary structure itself results in some folding constraints:

Protein folding

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These 4 red atoms are in one plane(C of C=O central)

so 6 atoms in one plane

And these 4 atoms are in one plane (N central)

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28

29

30

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There’s still plenty of flexibility

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This graphic intentionally left blank.

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Amino acids shown simplified, without side chains and H’s.

Secondary structure: the alpha helix

Almost every N-H and C=O group can participate

H

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Poly alanineSide chains = -CH3 (lighter gray)H’s not shown

C = graysN = blueO = red

Alpha helix depictions

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Linus Pauling and a model of the alpha helix.1963

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AA residue

H-bond

Secondary structure: beta pleated sheet

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Beta sheet (i.e., beta pleated sheet)

antiparallel

parallel

antiparallel

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Anti-parallel Parallel

Beta-sheets

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secondary structure (my definition):

structure produced by regular repeated interactions between atoms of the backbone.

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Tertiary structure: The overall 3-D structure of a polypeptide.

These “ribbon” depictions do not show the side chains, only the backbone

3 alpha helices

This is a popular “ribbon” modelof protein structure. Get familiar with it. The ribbons are stretchesof single polypeptide chains. A single ribbon is NOT a sheet.

A beta sheet

Neither

41Tertiary structure

(overall 3-D)

ionic

hydrophobic

H-bond

Ion - dipoleinteraction

Van der Waals

cys

covalent

In loop regions and in regions of secondary structure

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R-CH2-SH HS-CH2-R+ R-CH2-S-S-CH2-R½ O2

+ HOH

cysteine cysteine cystine

Disulfide bond formation

An oxidation-reduction reaction: Cysteines are getting oxidized (losing H atoms, with electron; NOT losing a proton, not like acids.)Oxygen is getting reduced, gaining H-atoms and electronsActually it’s the loss and gain of the electrons that constitutes oxidation and reduction, respectively. No catalyst is usually needed here.

Sulfhydryl groupDisulfide bond

(covalent, strong)

Two sulfhydryls have been oxidized (lost H’s)Oxygen has been reduced (gained H’s).Oxygen was the oxidizing agent (acceptor of the H’s).

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Stays intact in the jacuzzi at 37 deg C

Usually does not require the strong covalent disulfide bond to maintain its 3-D structure

Overall 3-D structure of a polypeptide is tertiary structure

[Tuber mode]l

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Space-filing,with surface charge

Backbone only

Protein structures are depicted in a variety of ways

Ribbon

Space-filling

Small molecule bound

blue = +

red = -

Continuous lines, ribbons=backbone (not sheets)

Drawing attention to a few side groups

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Most proteins are organized into

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474o, QUATERNARY STRUCTURE

Monomeric protein (no quaternary structure)

Dimeric protein (a homodimer)

Dimeric protein (a heterodimer)

A heterotetramer

A heteropolymeric protein (large one)

Also called: multimeric proteins

The usual weak bonds

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Hemoglobin

Molecular weight

16,000

16,000

64,000

Subunit molecular weight

Subunit molecular weight

Protein molecular weight

One protein

Four polypeptide chains, 2 identical alphas and 2 identical betasFour “subunits”

64,000, even though the 4 chains are not covalently bonded to each other

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Two heavy chains (H),Two light chains (L)

Interchain disulfide bonds

Tetramer

The 4 weak bond types

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Normal Sickle cell

glu glugluglu valval valval

Sickle cell disease

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Pyridoxal phosphate

Some small molecules can by added to a protein via covalent bonds.One form of a “prosthetic group”.

= Vitamin B6

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Riboflavin~ vitamin B2

Heme

Tetrahydrofolic acid~ vitamin B9

Most prosthetic groups are bound tightly via weak bonds.

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Membrane proteins

54Membrane proteins

Hydrophobic side chains on the protein exterior for the portion in contact with the interior of the phospholipid bilayer.

Anions are negatively charged.

Cations are positively charged

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Too far

Small molecules bind with great specificity to pockets on protein surfaces

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57Estrogen receptor binding estrogen, a steroid hormone

estrogen estrogen

detail

Estrogen receptor is specific, does not bind testosterone

58Protein binding can be very specific

Testosterone Estrogen