AMINO ACIDS AND PROTEINS

Dr. D. Haritha
Assistant Professor
Dept. of LS1(FST)
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 For all the common amino acids except
glycine, the α carbon is bonded to four
different groups.
 Because of the tetrahedral arrangement of
the bonding orbitals around the α-carbon
atom, the four different groups can
occupy two unique spatial arrangements,
and thus amino acids have two possible
stereoisomers.
 Since they are non-superimposable mirror
images of each other, the two forms
represent a class of stereoisomers called
enantiomers.
 All molecules with a chiral center are
also optically active—that is, they rotate
the plane of plane-polarized light.
 Nearly all biological compounds with a chiral center occur naturally in only one stereo
isomeric form, either D or L.
 The amino acid residues in protein molecules are exclusively L stereoisomers.
 D-Amino acid residues have been found in only a few, generally small peptides,
including some peptides of bacterial cell walls and certain peptide antibiotics.
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 Aromatic R Groups Phenylalanine, tyrosine, and tryptophan, with their aromatic
side chains, are relatively nonpolar (hydrophobic). All can contribute to the
hydrophobic effect.
 Tryptophan and tyrosine, and to a much lesser extent phenylalanine, absorb ultraviolet
light at wavelength of 280 nm, a property exploited by researchers in the
characterization of proteins.
 Cysteine is readily oxidized to form a covalently linked dimeric amino acid called
cystine, in which two cysteine molecules or residues are joined by a disulfide bond.

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Positively Charged (Basic) R Groups

 Lysine, which has a second primary amino group at the ε position on its aliphatic
chain; arginine, which has a positively charged guanidinium group; and histidine,
which has an aromatic imidazole group.
 As the only common amino acid having an ionizable side chain with pKa near
neutrality, histidine may be positively charged (protonated form) or uncharged at pH
7.0.
 His residues facilitate many enzyme-catalyzed reactions by serving as proton
donors/acceptors.

Physical properties:
 Amino acids are generally colorless, crystalline solids with high melting points.
 They are soluble in water but vary in solubility in organic solvents depending on their
side chains.

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Amino Acids Can Act as Acids and Bases
 The amino and carboxyl groups of amino acids, along with the ionizable R groups of
some amino acids, function as weak acids and bases.
 When an amino acid lacking an ionizable R group is dissolved in water at neutral pH, it
exists in solution as the dipolar ion, or zwitterion (German for “hybrid ion”), which can
act as either an acid or a base.
 Substances having this dual (acid-base) nature are amphoteric and are often called
ampholytes.

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 Amino Acids Have Characteristic Titration Curves

 The plot has two distinct stages, corresponding to

deprotonation of two different groups on glycine.
 At very low pH, the predominant ionic species of
glycine is the fully protonated form, +H3N-CH2-
COOH.
 In the first stage of the titration, the -COOH group
of glycine loses its proton. At the midpoint of this
stage, equimolar concentrations of the proton-
donor (+H3N-CH2-COOH) and proton-acceptor
(+H3N-CH2-COO-) species are present.

 As the titration of glycine proceeds, another important point is reached at pH 5.97. Here there
is another point of inflection, at which removal of the first proton is essentially complete and
removal of the second has just begun.
 At this pH glycine is present largely as the dipolar ion (zwitterion) +H3N-CH2-COO-.
 The second stage of the titration corresponds to the removal of a proton from the group of
glycine. The pH at the midpoint of this stage is 9.60, equal to the pKa for the group.
 The titration is essentially complete at a pH of about 12, at which point the predominant form
of glycine is H2N-CH2-COO-. 7
 The pKa of any functional group is greatly affected by its chemical environment, a
phenomenon sometimes exploited in the active sites of enzymes to promote exquisitely
adapted reaction mechanisms.
 Within the buffering ranges of glycine, the Henderson-Hasselbalch equation can be used
to calculate the proportions of proton-donor and proton-acceptor species of glycine
required to make a buffer at a given pH.
 The relationship between its net charge and the pH of the solution can be predicted. The
characteristic pH at which the net electric charge is zero is called the isoelectric point or
isoelectric pH, designated pI.

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Amino Acids Differ in Their Acid-Base Properties

 First, all amino acids with a single α-amino group, a single α-carboxyl group, and an R
group that does not ionize have titration curves resembling that of glycine.
 pKa of the — COOH group in the range of 1.8 to 2.4, and pKa of the NH2 group in the
range of 8.8 to 11.0.
 Amino acids with an ionizable R group have more complex titration curves, with three
stages corresponding to the three possible ionization steps; thus they have three pKa
values.
 The additional stage for the titration of the ionizable R group merges to some extent with
that for the titration of the α-carboxyl group, the titration of the α-amino group, or both.

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Peptide Bonds Link Amino Acids Together in Proteins

 A peptide bond is defined as the covalent bond that links amino acids together to form
peptides, polypeptides, and proteins, created through a condensation reaction between
the α-carboxyl group of one amino acid and the α-amino group of another, resulting in
the loss of water.
 This bond exhibits partial double-bond character, making it planar and stable, with no
rotation possible around the bond itself.

 The product formed by linking two amino acids together is called a dipeptide. The
subsequent binding of additional amino acid units to this dipeptide via peptide bonding
generates tripeptides, tetrapeptides, pentapeptides etc.
 The polymers formed by more than 10 amino acids are designated polypeptide.
 A polypeptide chain is considered a protein when it has a molecular mass greater than
6000 Daltons (Da).
 A Da is the unit of atomic mass; it is 1/12 the mass of one atom of 12C. Frequently the
expression kilo dalton (kDa), 1000 Da is used. 10
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The Structure of Proteins

 A description of all covalent bonds

(mainly peptide bonds and disulfide
bonds) linking amino acid residues in a
polypeptide chain is its primary
structure. The most important element of
primary structure is the sequence of
amino acid residues.
 Secondary structure refers to
particularly stable arrangements of amino
acid residues giving rise to recurring
structural patterns.
 Tertiary structure describes all aspects
of the three-dimensional folding of a
polypeptide.
 When a protein has two or more
polypeptide subunits, their arrangement in
space is referred to as quaternary
structure.
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 The spatial arrangement of atoms in a protein or any part of a protein is called its
conformation.
 The possible conformations of a protein or protein segment include any structural state it
can achieve without breaking covalent bonds.
 The need for multiple stable conformations reflects the changes that must take place in
most proteins as they bind to other molecules or catalyze reactions.
 The conformations existing under a given set of conditions are usually the ones that are
thermodynamically the most stable —that is, having the lowest Gibbs free energy (G).
Proteins in any of their functional, folded conformations are called native proteins.
 A protein’s conformation is stabilized largely by weak interactions. In the context of
protein structure, the term stability can be defined as the tendency to maintain a native
conformation.
 For all proteins of all organisms, weak interactions are especially important in the
folding of polypeptide chains into their secondary, tertiary and quaternary structures.

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The Peptide Bond Is Rigid and Planar

 The α carbons of adjacent amino acid residues are separated by three covalent bonds,
arranged as N-Cα—C—N—Cα-C.
 peptide C—N bond is somewhat shorter than the C—N bond in a simple amine and
that the atoms associated with the peptide bond are coplanar.
 This indicated a resonance or partial sharing of two pairs of electrons between the
carbonyl oxygen and the amide nitrogen.
 The oxygen has a partial negative charge and the hydrogen bonded to the nitrogen has a
net partial positive charge, setting up a small electric dipole.
 The six atoms of the peptide group lie in a single plane, with the oxygen atom of the
carbonyl group trans to the hydrogen atom of the amide nitrogen.
 From these findings Pauling and Corey concluded that the peptide C—N bonds,
because of their partial double-bond character, cannot rotate freely.
 Rotation is permitted about the N—Cα and the Cα—C bonds.
 The rigid peptide bonds limit the range of conformations possible for a polypeptide
chain.

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 Peptide conformation is defined by three dihedral angles (also known as torsion angles)
called ϕ (phi), ψ(psi), and ω (omega), reflecting rotation about each of the three repeating
bonds in the peptide backbone.
 The N—Cα and Cα—C bonds can rotate, described by dihedral angles designated ϕ and
ψ, respectively.
 In principle, ϕ and ψ can have any value between -180° and +180°, but many values are
prohibited by steric interference between atoms in the polypeptide backbone and amino
acid side chains.
 The peptide bond is almost always (99.6% of the time) in the trans configuration,
constraining ω to a value of ±180°. For a rare cis peptide bond, ω = 0°

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 Ramachandran plot

 Allowed values for ϕ and ψ become evident when ψ is plotted versus ϕ in a

Ramachandran plot, introduced by G. N. Ramachandran.
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 Peptide bond conformations are defined by the values of ϕ and ψ.
 Conformations deemed possible are those that involve little or no steric interference,
based on calculations using known van der Waals radii and dihedral angles.
 The areas shaded dark blue represent conformations that involve no steric overlap if
the van der Waals radii of each atom are modeled as a hard sphere and that are thus
fully allowed.
 Medium blue indicates conformations permitted if atoms are allowed to approach each
other by an additional 0.1 nm, a slight clash.
 The lightest blue indicates conformations that are permissible if a very modest
flexibility (a few degrees) is allowed in the ω dihedral angle that describes the peptide
bond itself (generally constrained to 180°).
 The white regions are conformations that are not allowed.
 The asymmetry of the plot results from the L stereochemistry of the amino acid
residues. The plots for other L residues with unbranched side chains are nearly
identical.

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Protein Secondary Structure

 The term secondary structure refers to any chosen segment of a polypeptide chain and
describes the local spatial arrangement of its main chain atoms, without regard to the
positioning of its side chains or its relationship to other segments.
 A regular secondary structure occurs when each dihedral angle, ϕ and ψ, remains the
same or nearly the same throughout the segment.
 There are a few types of secondary structure that are particularly stable and occur
widely in proteins.
 The most prominent are the α-helix and β conformations; another common type is the β
turn.
 Where a regular pattern is not found, the secondary structure is sometimes referred to as
undefined or as a random coil.
 Triple-helix is another common secondary structure found in collagen proteins in
connective tissues.

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 The alpha helix (α-helix) has a right-handed spiral conformation, in which every
backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino
acid four residues before it in the sequence.
 The ideal alpha helix has the following properties:
 It completes one turn every 3.6 residues;
 It rises approximately 5.4 Â with each turn;
 It is a right-handed helix;
 It is held together by hydrogen bonds between the C=O of residue i and the NH of residue
i+4;
 It is typically slightly curved.

 Some amino acids are preferred in an alpha-helix.

Residues such as Ala, Glu, Leu and Met have a high
tendency to participate in a helix , while residues such as
Pro and Gly have a small such tendency.
 Of special interest is Proline, which cannot fit into a helix,
and introduces a kink.
 The helix has an overall dipole moment, the positive pole
is at the N-terminus and the negative pole is at the C-
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terminus.
 Five types of constraints affect the stability of an α helix:
1. the intrinsic propensity of an amino acid residue to form an α helix;
2. the interactions between R groups, particularly those spaced three (or four) residues
apart;
3. the bulkiness of adjacent R groups;
4. the occurrence of Pro and Gly residues; and
5. interactions between amino acid residues at the ends of the helical segment and the
electric dipole inherent to the α helix.
 The tendency of a given segment of a polypeptide chain to form an α helix therefore
depends on the identity and sequence of amino acid residues within the segment.

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 A Beta strand (β-strand) is a stretch of polypeptide chain, typically 3 to 10 amino acids
long, with its backbone in an almost fully extended conformation.
 The two neighboring chains are parallel if both have N-terminus on the same side, as
shown on the right, and antiparallel if the two adjacent chains have N-terminus on the
opposite sides
 Two or more parallel or anti-parallel adjacent polypeptide chains of beta strand
stabilized by hydrogen bonds form a beta sheet.
 Amino acids such as tryptophan, tyrosine, and phenylalanine, which have large ring
structures in their R groups, are often found in β pleated sheets, perhaps because the β
pleated sheet structure provides plenty of space for the side chains.
 Some amino acid residues act as the connecting elements that link successive runs of α
helix or β-conformation.
 Particularly common are β turns that connect the ends of two adjacent segments of an
antiparallel β sheet.
 The structure is a 180° turn involving four amino acid residues, with the carbonyl
oxygen of the first residue forming a hydrogen bond with the amino-group hydrogen of
the fourth.
 The peptide groups of the central two residues do not participate in any inter-residue
hydrogen bonding.
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 Gly and Pro residues often occur in β turns, the former because it is small and flexible,
the latter because peptide bonds involving the imino nitrogen of proline readily assume
the cis configuration .
 Type I turns occur more than twice as frequently as type II with Pro at 2nd position in
both type of turns. The most prominent bias is the presence of Gly at position 3 in more
than 75% of type II turns.
 Considerably less common is the γ turn, a three-residue turn with a hydrogen bond
between the first and third residues.

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Super secondary structures or Motif

 Super secondary motifs are the structures that are formed by the combination of
secondary structures containing alpha-helices and beta-pleated sheets. These structures
are found in the globular proteins and are joined by loop, turn, or hairpin.
 Super secondary structures are in between secondary structures and tertiary structures of
proteins.
 Proteins are composed of Motifs (when secondary structures combine, they make a
motif that does not function independently) and Domains (when specific motifs and
secondary structures combine, they form a domain that has a particular function and may
exist independently).

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Zinc-finger motif Greek key motif

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 Protein Tertiary Structure

 The overall three-dimensional arrangement of all atoms in a protein is referred to as the

protein’s tertiary structure.
 This complex folding integrates the spatial arrangement of secondary structural
elements, such as α-helices and β-sheets, with the precise orientation of all amino acid
side chains (R groups).
 The tertiary structure is the fully folded, functional state of the protein essential for
specific biological activity.
 Tertiary structures frequently contain 1 or more protein domains—self-stabilizing
regions of a polypeptide that fold independently into compact 3-dimensional units.
 Domains typically range from 50 to 250 amino acids, and each domain forms a
hydrophobic core constructed from secondary structures connected by loops.
 Domain architecture—including number, type, and arrangement—critically determines
protein function, interaction potential, and the accessibility and specificity of active
sites, influenced by domain orientation and flexibility within the tertiary structure

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Forces Stabilizing Tertiary Structure

 The tertiary structure of a protein, representing its intricate 3-dimensional conformation,

is stabilized by a network of interactions involving amino acid side chains and the
polypeptide backbone.
 These interactions frequently occur between residues distant in the linear sequence and
are essential for maintaining the folded conformation.
 Stabilizing forces are classified as noncovalent—including hydrogen bonds, ionic
interactions, van der Waals forces, and hydrophobic effects—and covalent, such as
disulfide bonds

Function

 Tertiary structure defines the specific geometry and chemical environment of binding
sites for ligands, substrates, and interaction partners, which are essential for proper
activity and regulation.
 Disruption of the native conformation, as occurs with misfolding, can lead to loss of
function or toxic gain of function, contributing to a spectrum of protein-misfolding
diseases, including neurodegenerative and systemic disorders.

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 Protein Quaternary Structure

 Some proteins are made up of more than one amino acid chain, giving them
a quaternary structure.
 Protein quaternary structure refers to the arrangement and interaction of multiple
polypeptide chains or subunits in a protein complex, which can lead to significant
functional changes resulting from small variations in the amino acid sequence.
 The subunits, or protomers, are usually present as an even number, most often two or
four. The terms dimer, trimer, tetramer, oligomer, and multimer refer to structures with
two, three, four, few/several, and multiple subunits, respectively.

 The subunits may be all identical or all different or several

each of two (or more) different types.
 For example, the lactose repressor consists of four identical
subunits, whereas hemoglobin has two α-subunits and two
β-subunits.
 Hydrogen bonds, electrostatic attractions, hydrophobic
interactions, and disulfide bridges are the forces responsible
for maintaining the position of the different subunits within Hemoglobin - An Example of a
the multimeric protein. Heterotetramer

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 Importance of hydrophobic interactions in proteins

 When water surrounds a hydrophobic molecule, the optimal arrangement of hydrogen

bonds results in a highly structured shell, or solvation layer, of water around the
molecule.
 The increased order of the water molecules in the solvation layer correlates with an
unfavorable decrease in the entropy of the water.
 However, when nonpolar groups cluster together, the extent of the solvation layer
decreases, because each group no longer presents its entire surface to the solution. The
result is a favorable increase in entropy.
 This increase in entropy is the major thermodynamic driving force for the association of
hydrophobic groups in aqueous solution.
 Hydrophobic amino acid side chains therefore tend to cluster in a protein’s interior, away
from water (think of an oil droplet in water).
 The hydrophobic effect is clearly important in stabilizing conformation; the interior of a
structured protein is generally a densely packed core of hydrophobic amino acid side
chains.

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 The term hydrophobic effect refers to the unusual behavior of water towards nonpolar
solutes.
 The insertion of a hydrophobic molecule into an aqueous environment induces a diffusely
ordered water shell surrounding the molecule, akin to the formation of clathrates around
noble gases and simple hydrocarbons.
 This shell forms because the hydrophobic compound cannot form hydrogen bonds to the
water that surrounds it. Consequently those water molecules have a more restricted set of
neighbors with whom to fulfil their hydrogen bonding capacities.
 This reduces their degrees of rotational freedom and thus leads to a reduction in entropy
which is thermodynamically unfavorable.
 To minimize this entropy loss, the protein folds in such a way that hydrophobic residues
are buried inside, away from water.
 As a result, water molecules are released and become disordered again, increasing
entropy of the system, which is thermodynamically favorable.

Δ𝐺𝑓𝑜𝑙𝑑𝑖𝑛𝑔 = Δ𝐻𝑓𝑜𝑙𝑑𝑖𝑛𝑔−𝑇Δ𝑆𝑠𝑜𝑙𝑣𝑒𝑛𝑡
 Increase in entropy of water molecules upon burial of hydrophobic molecules is the main
contributor to the hydrophobic effect during protein folding.
Importance of hydrogen bonding of proteins

 Any polar or charged groups in the protein interior have suitable partners for hydrogen
bonding or ionic interactions.
 One hydrogen bond seems to contribute little to the stability of a native structure, but
the presence of hydrogen-bonding groups without partners in the hydrophobic core of a
protein can be so destabilizing that conformations containing these groups are often
thermodynamically untenable.
 The favorable free energy change resulting from the combination of several such
groups with partners in the surrounding solution can be greater than the free-energy
difference between the folded and unfolded states.
 In addition, hydrogen bonds between groups in a protein form cooperatively (formation
of one makes formation of the next one more likely) in repeating secondary structures
that optimize hydrogen bonding, as described below.
 In this way, hydrogen bonds often have an important role in guiding the protein folding
process.

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 Protein Denaturation

 Protein denaturation is defined as the process in which the proper conformation of a

protein is altered due to physical agents or chemicals, leading to the disruption of its
secondary, tertiary, and quaternary structures, resulting in the loss of its functional
properties.
 This process often involves the unfolding of the polypeptide chain and can be
irreversible or reversible, depending on the intensity of the molecular disruption.
 Protein conformation can be altered when proteins are subjected to physical agents, such
as heat, radiation, repeated freezing, high pressure, or chemicals (including acids, alkalis,
organic solvents, and concentrated urea solutions.

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