Amino acids and Protein

Unique characteristics of specific amino acids

Proline contains a secondary amine group, called an imine, instead of a primary amine group.
For this reason, proline is called an imino acid. Unlike the primary amino acids, free proline is a
secondary amine with the side chain curving back from the alpha carbon to bond to the amine
nitrogen. When incorporated into a protein, proline therefore lacks an amide proton and locks the
phi backbone angle into the static conformation of the pyrrolidine ring. The backbone near a
proline thus tends to be inflexible and may prevent certain secondary structures from forming,
particularly alpha helices.
Histidine is a rare amino acid most notable for the aromatic imidazole ring in its side chain.
The epsilon nitrogen of this ring is a weak base with a pKa near 6, and thus at phsiological pH
values the ring may either be neutral or positively charged depending upon the
microenvironment of the histidine. This chemical flexibility, coupled with its potential for both
ionic and hydrogen bonds, makes histidine a natural catalyst. It is therefore not surprising that
histidine is one of the most prevalent residues in enzyme active sites.
Arginine is one of the most chemically complex amino acids, and more than any other amino
acid is capable of exquisite molecular interactions. The unique moiety in arginine is the
guanidine group that terminates the long side chain. This group contains three nitrogens, one of
which is quaternary, making arginine the most basic of the amino acids with a virtually
permanent positive charge in living systems. Not surprisingly, arginine commonly mediates
interactions between proteins and the negatively charged backbones of DNA and RNA
Tryptophan is both the largest and rarest amino acid found in proteins. The side chain is
dominated by an indole group, a fused-ring aromatic heterocycle that is about 5 Angstroms wide
and 7 Angstroms long. The aromatic ring system is planar and rather hydrophobic, and thus
tryptophan is generally found buried in the hydrophobic core of proteins, often involved in
aromatic stacking interactions.
Glycine is chemically the simplest of the amino acids, since its side chain is simply a proton.
Thus, glycine is unique in that it contains two alpha protons (HA) and has no independent side
chain motion. The effective lack of a side chain endows glycine with a corresponding lack of
steric hinderance, making it an ideal residue for unusual conformations and for
accommodating dramatic changes in the direction of the polypeptide chain.

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Ninhydrin (triketohydrindene hydrate) reacts with α -
amino acids to produce CO2, NH3, and an aldehyde with
one less carbon than the parent amino acid. In most
cases, a blue or violet compound (Ruhemann’s purple)
is formed owing to reaction of the liberated NH 3 with
ninhydrin
Why proline and hydroxyproline is give yellow color
with ninhydrin?
Proline has aliphatic side chains with a distinctive cyclic
sturcture. The secondary amino (imino) group of proline
residues is held in a rigid conformation that reduces the
structural flexibility of polypeptide regions containing
proline. Proline does not give the ninhydrin reaction as
this reagent requires free alpha amino group (-NH2) but proline has an imino group (-NH). For
the amino acids which have a free -NH2 (amino) group, ninnydrin test is positive but is negative
for proline because it only has -NH (imino) group.
Which amino acids have side chains that are capable of forming isopeptide bonds?
An isopeptide bond is an amide bond that can form for example between the carboxyl group of
one amino acid and the amino group of another. At least one of these joining groups is part of the
side chain of one of these amino acids. Lysine has an amino group on its side chain and glutamic
acid has a carboxyl group on its side chain. These amino acids may join together or with some
other amino acids to form an isopeptide bond.
Oligopeptides:
Glutathione is a naturally occurring tripeptide comprised of three amino acids (cysteine,
glutamic acid, and glycine) acts as an antioxidant, a free radical scavenger and a detoxifying
agent. Glutathione (GSH), a cysteine-containing tripeptide (γglutamyl-cysteinyl-glycine) found
in eukaryotic cells at millimolar concentrations, has a number of important functions in cell
physiology. In its oxidized disulphide form, GSH protects cells from oxidative stress damage by
maintaining the intracellular redox state and modulating various intracellular functions via
metabolic interconversion. Glutathione helps inactivate oxidative compounds that could

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potentially damage cellular structures, since the oxidation of GSH to GSSG is accompanied by
the reduction of another compound:
2 Glutathione (GSH) + X oxidized → Glutathione disulfide (GSSG) + X reduced
Aspartame was discovered in 1965 by James Schlatter a chemist. It is an artificial, non-
saccharide sweetener, L-aspertyl-L phenylalanine methyl ester that is a methyl ester of the
dipeptide of the amino acids aspartic acid and phenylalanine. Aspartame is a low calorie
sweetener used to sweeten a variety of low and reduced calorie foods and beverages including
low calorie tabletop sweetener as well as for use in gum, breakfast cereal and other dry products.
Aspartame provides energy of 4 calories per gram. Aspartame is unstable if subjected to prolong
heating and therefore cannot be used in baking or cooking. It also decomposes in liquids during
storage.
Significance of titration curve of amino acids
Titration curves are helpful in the identification of amino acids as follows:
1. The number of pKa values differentiates polar and nonpolar amino acids from charged amino
acids. 2. The position of the pKa values for charged amino acids allows one to identify positively
charged from negatively charged amino acids. 3. Comparisons between experimental and
literature pKa values can allow the identification of a specific amino acid.
Why do the pK values of ionizable groups differ between free amino acids and amino acid
residues in polypeptides?
The pK values of ionizable groups, including the N- and C-termini, usually differ from the pK
values for free amino acids. In the free amino acids, the pK values are much lower, because the
positively charged ammonium group electrostatically stabilizes the COO – group, in effect
making it easier for the carboxylic acid group to ionize. In the free amino acids, the pK values
are higher, due to the electron-withdrawing character of the nearby carboxylate group, which
makes it more difficult for the ammonium group to become deprotonated. In addition, the three-
dimensional structure of a folded polypeptide chain may bring polar side chains and the N- and
C-termini close together. The resulting electrostatic interactions between these groups may shift
their pK values up to several pH units from the values for the corresponding free amino acids.
Secondary Structure
The α helix is right-handed; that is, it turns in the direction that the fingers of a right hand curl
when its thumb points in the direction that the helix rises. The α helix has 3.6 residues per turn

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and a pitch (the distance the helix rises along its axis per turn) of 5.4 Å. The α helices of proteins
have an average length of ∼12 residues, which corresponds to more than three helical turns, and
a length of ∼18 Å. In the α helix, the backbone hydrogen bonds are arranged such that the
peptide C=O bond of the nth residue points along the helix axis toward the peptide N-H group of
the (n + 4)th residue. In 1951, the same year Pauling proposed the α helix, Pauling and Corey
postulated the existence of a different polypeptide secondary structure, the β sheet. Like the α
helix, the β sheet uses the full hydrogen-bonding capacity of the polypeptide backbone. In β
sheets, however, hydrogen bonding occurs between neighboring polypeptide chains rather than
within one, as in an α helix. Sheets come in two varieties: 1. The antiparallel 𝛃 sheet, in which
neighboring hydrogen-bonded polypeptide chains run in opposite directions. 2. The parallel 𝛃
sheet, in which the hydrogen-bonded chains extend in the same direction. Polypeptide segments
with regular secondary structure, such as α helices or the strands of β sheets, are often joined by
stretches of polypeptide that abruptly change direction. Such reverse turns or 𝛃 bends (so named
because they often connect successive strands of antiparallel β sheets) almost always occur at
protein surfaces.
Tertiary Structure
Globular proteins—each with a unique tertiary structure—are built from combinations of
secondary structural elements. The proportions of α helices and β sheets and the order in which
they are connected provide an informative way of classifying and analyzing protein structure.
Groupings of secondary structural elements, called supersecondary structures or motifs, occur in
many unrelated globular proteins:
1. The most common form of supersecondary structure is the 𝛃𝛂𝛃 motif, in which an α helix
connects two parallel strands of a β sheet. 2. Another common supersecondary structure, the 𝛃
hairpin motif, consists of antiparallel strands connected by relatively tight reverse turns. 3. In an
𝛂𝛂 motif, two successive antiparallel α helices pack against each other with their axes inclined.

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 𝛃𝛂𝛃 motif𝛃 hairpin motif 𝛂𝛂 motif

Quaternary Structure
Most proteins, particularly those with molecular masses >100 kD, consist of more than one
polypeptide chain. These polypeptide subunits associate with a specific geometry. The spatial
arrangement of these subunits is known as a protein’s quaternary structure. A multisubunit
protein may consist of identical or nonidentical polypeptide chains. Proteins with more than one
subunit are called oligomers, and their identical units are called protomers. A protomer may
therefore consist of one polypeptide chain or several unlike polypeptide chains. Hemoglobin, for
example, has the subunit composition α2β2. In this sense, hemoglobin is a dimer of αβ protomers.
There are several reasons why multisubunit proteins are so common. Defects can be repaired by
simply replacing the flawed subunit; the site of subunit manufacture can be different from the
site of assembly into the final product. In the case of enzymes, increasing a protein’s size tends
to better fix the three-dimensional positions of its reacting groups. Increasing the size of an
enzyme through the association of identical subunits is more efficient than increasing the length
of its polypeptide chain because each subunit has an active site.
Protein Stability (Forces holding the polypeptide together)
Protein stability depends primarily on hydrophobic effects and secondarily on electrostatic
interactions. The hydrophobic effect, which causes nonpolar substances to minimize their
contacts with water, is the major determinant of native protein structure. The combined
hydrophobic and hydrophilic tendencies of individual amino acid residues in proteins can be
expressed as hydropathies. The greater a side chain’s hydropathy, the more likely it is to
occupy the interior of a protein, and vice versa. Hydropathies are good predictors of which
portions of a polypeptide chain are inside a protein, out of contact with the aqueous solvent, and

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which portions are outside. The association of two ionic protein groups of opposite charge (e.g.,
Lys and Asp) is known as an ion pair or a salt bridge. About 75% of the charged residues in
proteins are members of ion pairs that are located mostly on the protein surface. Despite the
strong electrostatic attraction between the oppositely charged members of an ion pair, these
interactions contribute little to the stability of a native protein.
Ramachandran plot
A Ramachandran plot, often referred to as a φ,ψ plot was developed in 1963 by G. N.
Ramachandran and colleagues to be able to understand the geometrical features of the peptide
backbone. It was demonstrated that the backbone dihedral angles ψ against φ of amino acid
residues in the protein structure are unique for specific secondary structural elements. The plot is
usually used to determine which conformations are possible for a protein but its usage is
very important in structure validation of proteins. Since some regions of the map are
forbidden due to geometrical restraints in the backbone, the calculations of the dihedral angles
serve as a measure of the structural integrity of the protein structure. Ramachandran plot can be
used to understand the relative distribution of alpha helices and beta sheets within a given protein
depending on the phi and psi angles of the component amino acid residues. The allowable
regions are the ones, which represent the conformations wherein atoms of the polypeptide would
not have any steric clashes. Disallowed regions generally involve steric hindrance between the
side chain and main chain atoms. Glycine stands out as an exception and has no side chain
and therefore can adopt phi and psi angles in all four quadrants of the Ramachandran
plot.
Protein domain
A discrete structural unit that is assumed to fold independently of the rest of the protein. It have
its own function. Can be composed of 20 or so amino acid residues to up to hundreds of them.
Made up of multiple secondary structure units (alpha helices, beta sheets, etc.). A protein domain
is a conserved part of a given protein sequence and structure that can evolve, function, and exist
independently of the rest of the protein chain. Each domain forms a compact three-dimensional
structure and often can be independently stable and folded. Many proteins consist of several
structural domains.
Motif

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Short, conserved regions and frequently are the most conserved regions of domains. Motifs are
critical for the domain to function In enzymes, they may contain the active sites. Another
example of motifs would be nuclear localization sequences.
Collagen
Collagens is the most abundant protein in the human body and accounts for 90% of bone matrix
protein content. They are characterized by the formation of triple helices in which three
polypeptide chains are wound tightly around one another in a rope-like structure. The triple
helix domains of the collagens consist of repeats of the amino acid sequence Gly-X-Y. A
glycine (the smallest amino acid) is required in every third position, so that the polypeptide
chains can pack together close enough to form the collagen triple helix. Proline is frequently
found in the X position and hydroxyproline in the Y position; because of their ring structure
these amino acids stabilize the helical conformations of the polypeptide chains.
Why is hydroxyproline important component of collagen?
Hydroxyproline has the same structure as the amino acid proline, with an hydoxyl (OH) group
attached to the gamma carbon atom on the pyrrolidine ring, as below. As you stated, it is an
important component of the collagen triple helix, for which the primary structure is repeats of
GXY, where X and Y are proline and hydroxyproline. This sequence contains many pyrrolidine
rings (on the proline and hydroxyproline residues), which would create a great deal of steric
clashing in a conventional helix, forcing the structure into a much more extended helical form
(compared to the alpha-helix). It is most often found in the Y position of the collagen Gly-X-Y
repeat where it stabilizes the collagen triple helix by forming hydrogen bonds with neighboring
collagen α-chains. In addition, the extra OH on the
hydroxyproline participates in hydrogen bonding to the
other chains in the triple helix, stabilising the structure.
Glycine and proline are commonly present in turns
Proline has cyclic side chain therefore rotation around
bond is constrained by its inclusion in the pyrrolidine ring and hence Proline is the most
conformationally restricted amino acid residue. Proline are commonly present in turns. The
presence of a built-in bend in the case of proline allow the polypeptide backbone to fold into a
tight U-shaped structure. Glycine– has no Beta carbon atom, i.e. no side chain. Therefore it is the
least sterically hindered as compared to other amino acids. This fact permits it to cover a large

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range of area in the plot. Therefore, glycine residues polypeptide chain often assumes
conformations that are forbidden to other residues. So glycine frequently occurs in turn regions
of proteins where any other residue would be sterically hindered.
Protein Sequencing
The complete amino acid sequence of a protein includes the sequence of each of its subunits, if
any, so the subunits must be identifi ed and isolated before sequencing begins. Each polypeptide
chain (if it is not chemically blocked) has an N-terminal residue. Identifying this “end group”
can establish the number of chemically distinct polypeptides in a protein. The N-terminus of
a polypeptide can be determined by several methods. The fluorescent compound 5-
dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride) reacts with primary amines to
yield dansylated polypeptides. The treatment of a dansylated polypeptide with aqueous acid at
high temperature hydrolyzes its peptide bonds. This liberates the dansylated N-terminal residue,
which can then be separated chromatographically from the other amino acids and identified by its
intense yellow fluorescence.

The protein must be broken down into fragments small enough to be individually sequenced, and
the primary structure of the intact protein is then reconstructed from the sequences of
overlapping fragments. Polypeptides that are longer than 25 to 100 residues cannot be directly

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sequenced and must therefore be cleaved, either enzymatically or chemically, to specific
fragments that are small enough to be sequenced. Various endopeptidases can be used to
fragment polypeptides. The digestive enzyme trypsin has the greatest specificity and is therefore
the most valuable member of the arsenal of endopeptidases used to fragment polypeptides. It
cleaves peptide bonds on the C side (toward the carboxyl terminus) of the positively charged
residues Arg and Lys if the next residue is not Pro.

Once the peptide fragments formed through specific cleavage reactions have been isolated, their
amino acid sequences can be determined. This can be accomplished through repeated cycles of
Edman degradation. In this process (named after its inventor, Pehr Edman),
phenylisothiocyanate (PITC; also known as Edman’s reagent) reacts with the N-terminal
amino group of a polypeptide under mildly alkaline conditions to form a phenylthiocarbamyl
(PTC) adduct. This product is treated with anhydrous trifluoroacetic acid, which cleaves the N-
terminal residue as a thiazolinone derivative but does not hydrolyze other peptide bonds. Edman
degradation therefore releases the N-terminal amino acid residue but leaves intact the rest of the
polypeptide chain. The thiazolinone-amino acid is selectively extracted into an organic solvent
and is converted to the more stable phenylthiohydantoin (PTH) derivative by treatment with
aqueous acid. This PTH-amino acid can later be identified by chromatography.
The two steps of the peptide cleavage process take place under different conditions. Hence, the
amino acid sequence of a polypeptide chain can be determined from the N-terminus inward by
subjecting the polypeptide to repeated cycles of Edman degradation and, after every cycle,
identifying the newly liberated PTH-amino acid.
Up to 100 residues can be identified before the cumulative effects of incomplete reactions, side
reactions, and peptide loss make further amino acid identification unreliable.

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Peptides Can Be Sequenced by Mass Spectrometry
Mass spectrometry has emerged as the dominant technique for characterizing and sequencing
proteins. In a technique known as protein mass fingerprinting, an unidentified protein (e.g.,
extracted from a spot in a 2D gel) is cleaved into defined fragments and their masses are
determined. The members of a database of proteins of known sequences are theoretically
subjected to the same cleavage method and the masses of the resulting peptides are calculated. A
match between the experimental and a theoretical set of masses identifies the protein. Short
polypeptides (< 25 residues) can be directly sequenced through the use of a tandem mass
spectrometer.
By comparing the molecular masses of successively larger members of a family of fragments,
the molecular masses and therefore the identities of successive amino acid residues in a
polypeptide are determined. Computerization of the mass-comparison process has reduced
the time required to sequence a short polypeptide to only a few minutes (one cycle of Edman
degradation may take an hour). However, mass spectrometry cannot distinguish the isomeric
residues Ile and Leu because they have exactly the same mass, and it cannot always reliably

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distinguish Gln and Lys residues because their molecular masses differ by only 0.036 D. Mass
spectrometry can be used to sequence peptides with chemically blocked N-termini (which
prevents Edman degradation) and to characterize other posttranslational modifications such
as the addition of phosphate or carbohydrate groups.
After a protein’s amino acid sequence has been determined, the information is customarily
deposited in a public database. Databases for proteins as well as DNA sequences are accessible
via the Internet. Electronic links between databases allow rapid updates and cross-checking of
sequence information. The entry includes a description of the protein, its function (if known), its
sequence, and features such as disulfide bonds, posttranslational modifications, and binding sites.
Armed with the appropriate software (which is often publicly available at the database sites), a
researcher can search a database to find proteins with similar sequences in various organisms.
Protein Folding
The protein folding begins with the formation of local segments of secondary structure (α helices
and β sheets). Because native proteins contain compact hydrophobic cores, it is likely that the
driving force in protein folding is what has been termed a hydrophobic collapse. The collapsed
state is known as a molten globule, a species that has much of the secondary structure of the
native protein but little of its tertiary structure. Then the secondary structure becomes stabilized
and tertiary structure begins to form. During this intermediate stage, the native like elements are
thought to take the form of subdomains that are not yet properly docked to form domains. In the
final stage of folding, which for small, single-domain proteins occurs over the next few seconds,
the protein undergoes a series of complex rearrangements in which it attains its relatively stable
internal side chain packing and hydrogen bonding while it expels the remaining water molecules
from its hydrophobic core. In multidomain and multisubunit proteins, the respective units then
assemble in a similar manner, with a few slight conformational adjustments required to produce
the protein’s native tertiary or quaternary structure. Thus, proteins appear to fold in a hierarchical
manner, with small local elements of structure forming and then coalescing to yield larger
elements, which coalesce with other such elements to form yet larger elements.
Folding is a cooperative process, with small elements of structure accelerating the formation of
additional structures. A folding protein must proceed from a high-energy, high-entropy state to a
low-energy, low-entropy state. This energy–entropy relationship is known as a folding funnel.
An unfolded polypeptide has many possible conformations (high entropy). As it folds into an

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ever-decreasing number of possible conformations, its entropy and free energy decrease. The
energy–entropy diagram is not a smooth valley, but a jagged landscape. The minor clefts and
gullies on the sides of the funnel (false energy minima) represent partially folded conformations
that are temporarily trapped until, through random thermal activation or the action of molecular
chaperones, they overcome an “uphill” free energy barrier and can then proceed to a lower
energy conformation. Evidently, proteins have evolved to have efficient folding pathways as well
as stable native conformations.
The surface of the folding funnel represents all possible conformations that the polypeptide can
assume with each point on it corresponding to a specific conformation. The height of each point
is indicative of the conformation’s energy, and
the funnel’s width at that energy is indicative of
the polypeptide’s entropy. The unfolded
polypeptide proceeds from a high-energy, high-
entropy, disordered state to a low-energy, low-
entropy, native conformation. The folding can
occur via multiple trajectories, each starting
from a different unfolded structure at the top of
the energy landscape, and that it has many high-
energy, partially folded structures (occupying Energy–entropy diagram for protein folding
the false energy minima) and only a few low-energy, native structures.
Molecular chaperones are essential proteins that bind to unfolded and partially folded
polypeptide chains to disrupt the improper association of exposed hydrophobic segments that
would otherwise lead to non-native folding as well as polypeptide aggregation and precipitation.
In essence, molecular chaperones function to lift folding polypeptides out of the false minima in
their folding funnels. This is especially important for multidomain and multisubunit proteins,
whose components must fold fully before they can properly associate with each other. There are
several classes of molecular chaperones in both prokaryotes and eukaryotes, including the
following:
The Hsp70, a family of highly conserved 70-kD proteins in both prokaryotes and eukaryotes. In
association with the cochaperone protein Hsp40, they facilitate the folding of newly synthesized
proteins and reverse the denaturation and aggregation of proteins.

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Trigger factor, a ribosome-associated chaperone in prokaryotes that prevents the aggregation of
polypeptides as they emerge from the ribosome.
The chaperonins, which form large, multisubunit, cagelike assemblies in both prokaryotes and
eukaryotes. They bind improperly folded proteins and induce them to refold inside an internal
cavity.
The Hsp90, a family of 90-kD eukaryotic proteins that mainly facilitate the late stages of folding
of proteins involved in cellular signaling.
Most molecular chaperones are ATPases; that is, enzymes that catalyze the hydrolysis of ATP
(adenosine triphosphate) to ADP (adenosine diphosphate) and Pi (inorganic phosphate): ATP +
H2O → ADP + Pi; The ATP complex of the chaperone binds the unfolded or aggregated
substrate protein, whereas its ADP complex releases it. Thus, the favorable free energy change of
ATP hydrolysis drives the chaperone’s bind-and-release reaction cycle.

Reaction cycle of the GroEL/ES chaperonin

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