Protein structure
• Proteins are formed of amino acids, bound by peptide bonds; the protein
structure is the result of two principal factors:
1. the character of the peptide bond
2. the nature of the side chain of amino acids
The peptide bond
• covalent bond - amide linkages between the α-carboxyl group of an amino acid
and the α-amino group of another one.
O O O H O
α α α Peptide bond
α
H
2HN CH C OH + H N CH C OH 2HN CH C N CH C OH
R1 R2 R1 R2
Dipeptid
- The peptide bond is rigid and planar with partial double bond in character.
- There is no freedom of rotation around the bond that connects the C and N atoms
and all 4 of the atoms shown in the figure lie in the same plane (coplanar).
- There is, by contrast, ample freedom of rotation around the remaining bonds of
the polypeptide backbone (Cα-Cp and N-Cα).
• This semi-rigidity has important consequences for orders of protein structure above
the primary level.
• It generally exists in trans configuration.
• Both -C=O and -NH groups of the peptide bond are polar (but uncharged) and can
be involved in hydrogen bond formation.
H H +
C C + C δ H
CP N CP N CP N
-
C - C δ C
O O O
I II III
The nature of side chains
The spatial disposition of the side chains has a determinant role in
- the protein spatial organisation,
- of its conformational stability and
- the achievement of its role in the cell.
I.e. in the case of soluble globular proteins, the polar groups are disposed in a
maximal number to the exterior (hydrophilia), and the hydrophobic groups are
disposed in a high number inside (hydrophobia).
The side chains of amino acids present a high diversity: hydrophobic, polar non-
ionisable, polar ionisable, presenting groups as:
–SH, -OH, -COOH, -NH2, -CONH2
The side chains have a decisive role in the establishment of protein conformation,
using the following mechanisms:
Chain orientation. Generally, the non-polar radicals (Val, Leu, Ile, Met, Phe) are
oriented inside the protein, while the polar ionisable radicals (Asp, Glu, His, Arg,
Lys) are directed at the surface of protein, allowing the protein to form bonds
with water.
• Bond formation between the side chains, as:
• Hydrogen bonds between the components of the peptide bonds (carbonyl and
imino)
• disulfide bridges between two cysteine residues
• ionic bonds between the ionized forms of -COOH and -NH2
• ester bonds between the groups –COOH and –OH
• hydrophobic bonds (van der Waals) between the side chains of hydrophobic
amino acids (Val, Leu, Ile, Phe, Trp)
• hydrogen bonds between the –OH groups of amino acids Ser, Thr and Tyr.
Such types of interactions depend on:
- pH value of the medium, which influences the ionization state of the groups of side
chains.
Peptides
• The polypeptide chain can be shorter or longer, function of the number of
amino acid residues in the sequence.
Peptides nomenclature
• all amino acid sequences are read from the N- to the C- terminal end of the
protein.
• The protein biosynthesis also starts from the N-terminal amino acid.
H 2N CH CO NH CH CO NH CH CO NH CH C OOH
R1 R2 R3 Rn
N–terminal C–terminal
amino acid amino acid
• Two amino acids can form two different dipeptides; it depends on the order in
which they are bound. For example, alanine and valine:
H2N-Ala-CO-NH-Val-COOH
H2N-Val-CO-NH-Ala-COOH
Denominate the following peptides:
Gly-Phe-Trp-Arg
Asp-Lys-Gly
Asn-Glu-Gly-Trp-Cys
Ser-Thr-Trp-Gly-Pro-Lys-Pro
Val-Ala-Ile-Trp-Phe-Val-Leu-Gly
Which is the peptide type?
How many water molecules are eliminated in order to form the peptide from
the corresponding aminoacids?
Which aminoacid resides at amino terminal and carboxy-terminal end of the
peptide in each case?
Which is the most hydrophobic one?
Peptides properties
• Peptide bond is not ionized at any pH of physiological interest.
• The formation of peptides, at pH 7.4 is accompanied by loosing of a positive
charge and a negative one, per peptide bond.
• Peptides are, however, molecules with electric charges at physiological pH,
due to the C- and N- terminal groups and to other functional groups, present
in polar amino acid residues.
• Therefore, the electrochemical properties of peptides are similar to those of
amino acids, each peptide having an isoelectric pH (pI).
• Peptide bonds are not broken by normal handling, not by conditions that
denature the proteins, such as heating or high concentration of urea.
• Prolonged exposure to strong acids or bases at high temperatures is
required to hydrolyze, nonenzymatically, these bonds.
Qualitative and quantitative
determination of peptides
• absorption at 280 nm due to their residues of
Trp and Tyr.
⇒identification of proteins in the
chromatographic procedures because, at the
same wavelength nucleic acids also absorb.
• Biuret reaction - colour complex of Cu2+ in
alkaline solution with two peptide bonds in the
protein.
• The method has a sensitivity of about 1-10 mg
protein.
• Lowry Method - colour complex of
Cu2+ with the peptide bonds, and
the reduction of a complex
reagent containing
phosphomolibdate (Folin-Ciocalteu
or Lowry reagent or phenol
reagent) by tyrosine and
tryptophan residues, present in
the protein.
• The method has a sensitivity of
about 10-200 μg of proteins.
• Coomassie Method. Coomassie
Brilliant Blue G250 reacts with
proteins to form a red-violet
complex.
• The method has a sensitivity of
about 1-10 μg of proteins.
Important natural peptides
Glutathione - γ-glutamyl-cysteinyl-glycine
H2N-CH-CH2CH2-CO-NH-CH-CO-NH-CH2COOH [G-SH]
COOH CH2SH
• Glutathione is required by several enzymes.
• Glutathione and the enzyme glutathione-reductase participate in the formation of the correct
disulfide bonds in many proteins and polypeptide hormones.
• Due to its cysteinyl-residue, glutathione has redox properties. The oxidation of G-SH doesn’t need
the presence of an enzyme, but Cu2+ and Fe3+.
GSH + HSG → G-S-S-G + 2H+ + 2e-
• It can be oxidized by the peroxides which appear in the cells due to oxidative normal processes or
promoted by some drugs
Glutathione peroxidase
G-SH + R-OOH → G-S-S-G + R-OH + H2O
• The reduction of the oxidized form can be done only in the presence of the enzyme glutathione-
reductase (NADPH or NADH-dependent on).
Glutathione reductase
G-S-S-G + NADPH + H+ → 2 G-SH + NADP+
• The reduced form (G-SH) is the biological active form of glutathione.
• Also, glutathione is a non enzymatic carrier of H+ and
• a component of the phase two of the detoxification process.
• It is also considered as a form of deposit for cysteine.
Polypeptidic hormones
Many important hormones are polypeptides: insulin, ACTH, parathormone,
calcitonin; bradykinin (a 9 amino acid-peptide) is a smooth muscle
hypotensive agent; angiotensin II (8 amino acids) is the most hypertensive
hormone. Thyrotropin-releasing hormone (TRH) is a tripeptide: the N-
terminal glutamate is cyclized to pyroglutamic acid, and the C-terminal
prolyl carboxyl is amidated.
Polypeptide antibiotics, elaborated by fungi, contain both D- and L-amino
acids and some others not present in proteins. Examples include tyrocidine
and gramicidine S, cyclic polypeptides that contain D-phenylalanine and
ornithine. 6-Aminopenicylanic acid, the main substance of penicillin and
forerunner for synthetic penicillins is an atypical dipeptide.
Endorphins and encephalins have a peptidic structure and they have a strong
pain-killer effect.
