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CHMI 2227EBiochemistry I
Peptides-General structure and properties
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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
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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.
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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.
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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).
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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
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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
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High Pressure Liquid Chromatography (HPLC)
PITC = phenylisothiocyanate PTC = phenylthiocarbamyl
http://www.protein.iastate.edu/aaa.html
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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
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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.
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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
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Example of peptides3.Insulin
Intrachain disulfide bond
Interchain disulfide bonds
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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?
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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.
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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
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Importance of multimeric proteins – planar cell polarity
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Importance of multimeric proteins – planar cell polarity
Nature Genetics 38, 21 - 23 (2006)
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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.
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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
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General properties of proteins3. Frequency of amino acid composition
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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
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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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