CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Peptides - General structure and properties

CHMI 2227 - E.R. Gauthier, Ph.D. 1

CHMI 2227EBiochemistry I

Peptides-General structure and properties


Peptides Peptides are polymers of amino acids;

Amino acids building blocks (residues) are linked to each other through a covalent bond: the peptide bond.

1 2

1 2

A dipeptide


Peptides

Polypeptides and proteins are simply chains of amino acids linked together through peptides bonds:

If less than 20 residues: oligopeptide; If more than 20 residues but molecular mass (Mr) < 10,000 Da: polypeptides. If Mr > 10 kDa: protein.

Particular terminology: Dipeptide (2 residues) / Tripeptide (3 residues) / Tetrapeptide (4 residues) / Pentapeptide (5

residues) / Ect, ect, ect.

Note: 1 Da (dalton) = 1 g /mol.

Little trick: Mr of a polypeptide/protein ~ number of amino acids x 110 Da.


Peptides - polarity

Each peptide has a polarity: One extremity with the NH2

(the one bonded to the C) which is not part of a peptide bond: N-terminal end;

One extremity with the COOH (the one bonded to the C) which is not part of a peptide bond: C-terminal end;

By convention: the N-terminal is always placed on the left, and the C-terminal on the right.


Peptides - nomenclature

Different ways to write this peptide (hyphen = peptide bond) : Tyrosyl-glycyl-glycyl-phenylalanyl-leucine Tyr-Gly-Gly-Phe-Leu Y-G-G-F-L YGGFL

Note that the peptide is always written with the N-ter to the left and the C-ter to the right (NH2COOH).


Peptide: hydrolysis

The composition (NOT the sequence) of a peptide in its amino acid constituents is determined by first hydrolysing the peptide bond, and then identifying the amino acids:

Tyr-Gly-Gly-Phe-Leu Gly2, Leu, Phe, Tyr

The amino acids are then purified by High Pressure Liquid Chromatography (HPLC). Detection is done by UV absorbance.

To detect those amino acids that cannot absorb UV (you know which ones…), the amino acid are derivatized, meaning they are chemically coupled with a compound that absorbs UV.

Quantification and identification of the amino acids is often done with the help of standards (analyzed with the same system but in a separate experiment);

6 M HCl


Analysis of amino acidsDetection of amino acids: ninhydrin reagent

While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids cannot;

Ninhydrin reacts with the amine group of amino acids, generating a purple product (yellow in the case of Pro).

The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids contained in the fractions of the IEX column.

O

O

OHOH

Ninhydrin Amino acid

2

O

O

N

O

O

CH

R

COO-

NH3+

CO2

Purple!!

R-HC=O


High Pressure Liquid Chromatography (HPLC)

PITC = phenylisothiocyanate PTC = phenylthiocarbamyl

http://www.protein.iastate.edu/aaa.html


High Pressure Liquid Chromatography (HPLC)

The relative amount of each amino acid is given by calculating the area under each curve.

http://www.protein.iastate.edu/aaa_figure3.html


Peptide - ionization

Each peptide will exist in different protonated forms, depending on the pH and its amino acid composition:

Terminal amino and carboxyl groups can be protonated/ionized as in the free amino acid;

The side chain can also be ionized, if an appropriate group is present;

The NH2 and COOH groups that are part of the peptide bond are NOT ionized.

Therefore, there will be a pH where a given peptide/protein will carry no net charges: this pH value will be the isoelectric point of the peptide/protein in question.

Example: Ionization of the peptide GAVFD at pH 2, 6 and 12.


Example of peptides1. Aspartame: artificial sweetener

2. Oxytocin: stimulates uterine contractions

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2

Glycinamide residue: 2HN-CH2-CONH2

S S

Disulfide bond

H3N+-CH-C-NH-CH-C-OCH3

COO-

CH2

CH2

O

OAsp-Phe-methyl ester


Example of peptides3.Insulin

Intrachain disulfide bond

Interchain disulfide bonds


Example of peptides4.Cystic Fibrosis Transductance Regulator

Single polypeptide chain of 1480 amino acids;

Responsible for the transport of chloride ions across the cell membrane;

Mutation of F508 yields a non-functional protein and cystic fibrosis.

What is the approximate Mr of CFTR?


General properties of proteins1. Proteins differ in their Mr.

ProteinMr

(kDa)#

residues# chains

Insulin 5.7 51 2Cytochrome c 13 104 1Ribonuclease A 13.7 124 1Lysozyme 13.9 129 1Myoglobin 16.9 153 1Chymotrypsin 21.6 241 3Chymotrypsinogen 22 245 1Hemoglobin 64.5 574 4Serum albumin 68.5 550 1Hexokinase 102 800 2Immunoglobulin G 145 1,320 4RNA polymerase 450 4,100 5Apolipoprotein B 513 4,536 1Glutamate dehydrogenase

1,000 8,300 40

Source: Biochemistry. Lehninger.


Multimeric proteins Proteins with more than one polypeptide

chains are called multimeric proteins;

Different types of multimeric proteins exist: Homo/oligomeric: 2 or more copies of the

same polypeptide chain; Heteromeric: different polypeptide chains

make up the proteins.

The different polypeptides of a multimeric protein (i.e. the protein’s subunits) can be held together in different ways:

Disulfide bonds Hydrogen bonds Hydrophobic interactions Electrostatic interactions

Multimeric proteins most often require all their parts in order to be functional.

Very often proteins can change partners, providing them leading to their in/activation or giving them a different function.

Monomer/subunit

Homodimer

1 1

Heterodimer

1 2

Hydrogen bonds:N-H ----- O-H N-H ----- NO-H ----- O=C N-H ----- O=C

Hydrophobic interactions:-CH3 CH3- CH3

Electrostatic interactions:COO- ----- H3

+N


Importance of multimeric proteins – planar cell polarity


Importance of multimeric proteins – planar cell polarity

Nature Genetics 38, 21 - 23 (2006)


General properties of proteins2. Proteins differ in their pI.

Protein pI

Pepsin 1

Egg albumin 4.6

Serum albumin 4.9

Urease 5

-lactoglobulin 5.2

Hemoglobin 6.8

Myoglobin 7

Chymotrypsinogen 9.5

Cytochrome c 10.7

Lysozyme 11

Source: Biochemistry. Lehninger.


General properties of proteins3. Frequency of amino acid composition

Number of residues per molecule of protein

Amino Acid

Human cytochrome c

Bovine chymotrypsinogen

Ala 6 22

Arg 2 4

Asn 5 15

Asp 3 8

Cys 2 10

Gln 2 10

Glu 8 5

Gly 13 23

His 3 2

Ile 8 10

Number of residues per molecule of protein

Amino Acid

Human cytochrome c

Bovine chymotrypsinogen

Leu 6 19

Lys 18 14

Met 3 2

Phe 3 6

Pro 4 9

Ser 2 28

Thr 7 23

Trp 1 8

Tyr 5 4

Val 3 23

Total 104 245


General properties of proteins3. Frequency of amino acid composition


General properties of proteins4. Proteins can include other chemical groups in addition to amino acids

Class Prosthetic group

Example

Lipoprotein Lipids 1-lipoprotein (blood)

GlycoproteinCarbohydrates (sugars)

Immunoglobulin G (blood)

PhosphoproteinPhosphate groups

Casein (milk)

HemoproteinHeme (iron porphyrin)

Hemoglobin

FlavoproteinFlavin nucleotides

Succinate dehydrogenase

Metalloprotein

Fe

Zn

Ca

Cu

Ferritin

Alcohol dehydrogenase

Calmodulin

Plastocyanin


General properties of proteins5. Proteins have a specific shape Each polypeptide spontaneously

adopts a shape or conformation.

This conformation is unique to each protein;

A protein in its correct conformation is said to be native;

Disruptions in the conformation (e.g. by heating) denatures the protein and usually leads to its inactivation.Globular proteins

Fibrillar (rod-like) proteins

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CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Peptides - General structure and properties