lecture 8 (9/29/17) - boston university · 9/29/17 2 the 4 s’s for the peptide bond (recap):...

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• Reading: Ch3; 97-102

• Problems: Ch3 (text); 18, 19, 20, 23Ch3 (Study guide); 9Ch4 (Study guide); 3

NEXT• Reading: Ch4; 119-122, 125-126, 131-133

Ch4; 123-124, 130-131, 133, 137-138• Problems: Ch4 (text); 2, 3, 4, 8, 13, 14

Lecture 8 (9/29/17)

OUTLINEI. Protein Structure

A. Primary1. Determination

a. Sequence determination; CHEMICALi. Cleavage of peptides bondsii. Amino acid composition and stoichiometryiii. Determine number of chains;iv. Divide & Conquer; v. Edman Degradation

b. Sequence determination; PHYSICALi. Mass Spectrometry for proteinsii. Use of tandem MS/MS for sequence determinationiii. Isolation of proteins by 2D PAGE; Isoelectric focusing x SDS-PAGE

c. Sequence determination; BIOLOGICALi. Genome sequencedii. Bioinformatics to predict protein sequences in predicted genesiii. Use of CHEMICAL and/or PHYSICAL methods to get partial sequence

Lecture 8 (9/29/17)

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The 4 S’s for the Peptide Bond (recap):

Shape –trans & double-bond, planar character due to resonanceSize –7 Å repeat length & two rotatable bonds at Ca (f & y)

Solubility –highly polar bond due to resonanceStability –high energy bond due to resonance

Peptide Bond

Peptide Bond - Stability

H2OH2OThe DG of hydrolysis is –2.4 kcal/mole (~ –10 kJ/mole)

Why doesn’t your hair fall apart into AA & water, or all other proteins?

What is the energetics of hydrolysis?

Kinetically stable, thermodynamically unstable

What does it take to hydrolyze proteins?

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The catalysis of hydrolysis of the Peptide Bond:

Acid – 6 N HCl, 16 hr at 110 C°All peptide (and amide) bonds cleaved

Base –3 N NaOH, 6 hr at 100 C°All peptide (and amide) bonds cleaved, but

hydroxyl groups and guanidino groups are destroyed

Enzymes –proteolysis; incomplete due to specificity of enzymes

Use hydrolysis to answer 2 questions:1) determine amino acid composition2) determine entire sequence by fragmenting into

small peptides

Peptide Bond

Primary structure

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Determination of primary structure

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1) Purify protein2) Determine the amino-acid composition,

including stoichiometry3) Disrupt structure (2°, 3°, 4°, and disulfides)4) Determine the number of peptide chains by

counting number of amino terminal ends5) Divide into fragments and determine sequence6) Divide into different set of fragments and

determine sequence7) Determine overlaps and piece original sequence

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How many AA are “resolved?” …....18Where are the other 2? …....Asn & Gln have already been converted to Asp & Glu, respectively

Absorbance is at 570 nm (purple). How did they make the amino acid residues colored? …......ninhydrin

Amino Acid Composition

Determination of primary structureAmino Acid

Composition

Calculate the “mole%” or “mole fraction”; in other words the stoichiometry:Example; protein MW = 11,000 Da

Approximate #AA = 11,000/110 (Ave MW for AA) = 100 AAIf 10% of A570 is 10% for Arg: 100 x 0.10 = 10 Arg in protein

Cation exchanger=Dowex-50 (sulfonic acid on polystyrene)

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Disrupt structure (2°, 3°, 4°, and disulfides)

What holds these levels of structure together?

What have you used in the lab that might disrupt non-covalent bonds? …....Urea, SDS, pH extremes, heat, etc.

…....non-covalent bonds (H-bonds, van der Waals, ionic, hydrophobic)

What about the covalent S-S bond? …....2-mercaptoethanol (b-mercaptoethanol, BME) or dithothreitol (DTT).

To keep disulfides from reforming…..1) keep BME at high concentration in buffers2) alkylate the SH groups

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Fig 3-26

Determine the number of peptide chains by counting number of amino terminal ends

Absorbs at 353 nm (yellow)

Frederick Sanger(1918-2013)

Sanger’s Reagent

Example:small tripeptide

• AA comp = A,K,L• C-term = K• DNP-A• Sequence is A-L-K

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Determine the number of peptide chains by counting number of amino terminal ends

Excitation at 330 nmEmission at 519 nm Fig 3-26


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Divide into fragments Proteolytic Cleavage

Carboxylpeptidase A Bovine pancreas Rn = C-terminal; Rn-1 ≠ Pro

“TABLE 5-5” Specificities of Exopeptidase

O–

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Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.Figure 5-15

Divide into fragments Cyanogen Bromide Cleavage

Methionine

Similar result as an endopeptidase, but leaves a homoserine lactone as the C-terminal residue

Imine intermediate (Schiff base)

Separation and isolation of peptide fragments

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Example: paper electrophoresis

Also, TLC, silica gel, etc.

to Ninhydrin

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• Determine amino-acid composition• Dansyl chloride or FDNB to determine amino-

termini and number• Proteases: Cleaves peptide bonds only after specific

residues.• Cleave protein with 2 different proteases.• Sequence fragments with Edman degradation. Piece

together sequence from overlapping fragments.

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Determination of primary structure: Divide & Conquer

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Determination of primary structure: Divide & Conquer


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Copyright © 2016 John Wiley & Sons, Inc. All rights reserved.Figure 5-16

Edman Degradation Animated Figure

(https://wileyassets.s3.amazonaws.com/Voet_Fundamentals_of_Biochemistry_5e_ISBNEPROF12533/media/animated_figures/ch05/5-15.html)

Determine the Sequence: Edman degradation

Phenylthiocarbamyl (PTC)-polypeptide

Phenylthiohydantoin (PTH)-amino acid


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Wiley Guided_explorations

Determine overlaps and piece original sequence back together

lecture 8 (9/29/17) - boston university · 9/29/17 2 the 4 s’s for the peptide bond (recap):...

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