Proteins

Prepared by:
IC Duay, PhD
Assistant Professor
De La Salle University
searle.duay@dlsu.edu.ph
Proteins

• Most abundant among the four major biomolecules

• Constitute around 50% or more of the cell’s dry weight

• Structure can be disrupted by changes in environmental conditions

• Part of the process by which genetic information is translated into the machinery of
the cell

• White, crystalline, high-melting solids in pure form and under ordinary conditions
Proteins
Enzymes Antibodies Signaling

Structure Transport
Amino Acids

• Amino acid: a compound that contains both an amino

group and a carboxyl group
• α-Amino acid has an amino group attached to the carbon adjacent
to the carboxyl group

• α-carbon also bound to side chain group, R

• R gives identity to amino acid

• Two steroisomers of amino acids are designated L- or D-. Based on

similarity to glyceraldehyde.
Amino Acids
↓ pH, more H+, protonated

↑ pH, less H+, deprotonated

H2N AA1 AA2 AA3

AA6 AA5 AA4

AA7 AA8 AA9 COOH

Directionality: Amino terminus (N-term) to carboxy

terminus (C-term)

All α-carbons of canonical amino The termini can be protonated

acids, except for one, are chiral. ➢ Can be positively or negatively charged
depending on the local environment
Amino Acids
• Zwitterionic form at pH 7.4
• High melting points
• High solubility
• High dielectric constants
• Large dipole moments

• α-carbon follows an sp3 hybridization

• Chirality allows optical isomers (D or L)
• Glycine is an exception to chirality

• Naturally occurring optical isomer is the L-isomer

• Side-chain carbons in other amino acids designated

All α-carbons of canonical amino with Greek symbols, starting at a carbon (β, γ,
acids, except for one, are chiral. δ,…etc)
Condensation of Amino Acids

amide
nitrogen

R1` O R2`
O

H3N Cα C N Cα C

O
H H H

carbonyl
carbon
amide (or peptide) bond
Canonical Amino Acids

achiral branched branched

aromatic 2o amine
Canonical Amino Acids

Disulfide bridges Branched

Aromatic

Resonance pKa ~ 6
Canonical Amino Acids
Canonical Amino Acids
Canonical Amino Acids
Essential Amino Acids

• Phenylalanine
• Histidine
• Valine
• Arginine
• Tryptophan
• Leucine
• Threonine
• Lysine
• Isoleucine
• PVTTIMHALL
• Methionine
Side Chains
• Charged, hydrophilic
• Hardly get buried
• Cluster outside the protein, exposed in water solvent

• Nonpolar, hydrophobic
• Found buried in the protein
• Forms the core of the protein

• Molecular recognition relies on interactions with these residues

• Determine properties of the amino acid

• Structure and function of protein relies on the amino acid sequence

Important Structural Features

• All 20 are α-amino acids

• For 19 of the 20, the α-amino group is primary; for proline, it is secondary

• With the exception of glycine, the α-carbon of each is a stereocenter

• Isoleucine and threonine contain a second stereocenter

Uncommon Amino Acids

• Each derived from a common amino acid

by a modification
• hydroxylysine and hydroxyproline are found
only in a few connective tissues such as
collagen

• thyroxine is found only in the thyroid gland

Ionization of Amino Acids

• In amino acid, carboxyl group (-) and

amino group (+) are charged at neutral
pH.
• In free amino acids, α-carboxyl, and α-
amino groups have titratable protons.
Some side chains do as well
Ionization of Amino Acids

• Remember, amino acids without charged groups on side chain exist in neutral
solution as zwitterions with no net charge
• High melting point, high solubility, high dielectric constants, large dipole moments
Titration of Amino Acids

• When an amino acid is titrated, the titration

curve represents the reaction of each
functional group with the hydroxide ion
Titration of Histidine with NaOH
Acidity: α-COOH Groups

• The average pKa of an α-carboxyl group is 2.19, which makes them considerably
stronger acids than acetic acid (pKa 4.76)

• The greater acidity of the amino acid carboxyl group is due to the electron-
withdrawing inductive effect of the -NH3+ group
Basicity

• α-NH3+ groups: The average value of pKa for an α-NH3+ group is 9.47, compared
with a value of 10.76 for a 2° alkylammonium ion

• Guanidine Group
• The side chain of arginine is a considerably stronger base than an aliphatic amine
• Basicity of the guanidino group is attributed to the large resonance stabilization of the
protonated form relative to the neutral form

• Imidazole Group
• The side chain imidazole group of histidine is a heterocyclic aromatic amine
Ionization vs pH

• Given the value of pKa of each functional group, we can calculate the ratio of each
acid to its conjugate base as a function of pH

• Consider the ionization of an α-COOH

pK a = 2.00
-
−COOH + H 2 O −COO + H3 O +

• Writing the acid ionization constant and rearranging terms gives

[ H 3 O + ] [ -COO - ] [ -COO - ] Ka
Ka = or =
[ -COO H] [ -COO H] [ H 3 O+ ]
Ionization vs pH

• Substituting the value of Ka (1 x 10-2) for the hydrogen ion concentration at pH 7.0
(1.0 x 10-7) gives
[ -COO - ] Ka 1.00 x 10-2
= = = 1.00 x 105
[ -COO H] [ H 3 O+ ] 1.00 x 10-7

• At pH 7.0, the α-carboxyl group is virtually 100% in the ionized or conjugate base
form, and has a net charge of -1
• We can repeat this calculation at any pH and determine the ratio of [α-COO-] to [α-
COOH] and the net charge on the α-carboxyl at that pH
Ionization vs pH

• We can also calculate the ratio of acid to conjugate base for an α-NH3+ group; for
this calculation, assume a value 10.0 for pKa

+ pK a = 10.00
−N H3 + H2 O −N H2 + H3 O +

• Writing the acid ionization constant and rearranging gives

[ -NH 2 ] Ka
=
[ -NH 3 + ] [H 3 O+ ]
Ionization vs pH

• Substituting values for Ka of an α-NH3+ group and the hydrogen ion concentration
at pH 7.0 gives
[ -NH 2 ] Ka 1.00 x 10-10
= + = = 1.00 x 10-3
[ -NH 3 ]
+ [H 3 O ] 1.00 x 10-7

• At pH 7.0, the ratio of α-NH2 to α-NH3+ is approximately 1 to 1000

• At this pH, an α-amino group is 99.9% in the acid or protonated form and has a
charge of +1
Henderson-Hasselbalch Equation

• We have calculated the ratio of acid to conjugate base for an α-carboxyl group and
an α-amino group at pH 7.0

• We can do this for any weak acid and its conjugate base at any pH using the
Henderson-Hasselbalch equation

[conjugate bas e]
pH = pK a + log
[weak acid]
Isoelectric pH

• Isoelectric pH, pI: the pH at which the majority of molecules of a compound in

solution have no net charge

• The pI for glycine, for example, falls midway between the pKa values for the
carboxyl and amino groups
pI = 1 ( p K −COOH + p K −N H + )
2 a a 3

= 1 (2.35 + 9.78) = 6.06

2
Isoelectric pH
Electrophoresis

• The process of separating compounds on the basis of their electric charge

• Electrophoresis of amino acids can be carried out using paper, starch, agar,
certain plastics, and cellulose acetate as solid supports

• In paper electrophoresis, a paper strip saturated with an aqueous buffer of

predetermined pH serves as a bridge between two electrode vessels
Peptide Bonds

• Individual amino acids can be linked

by forming covalent bonds.

• Peptide bond: the special name

given to the amide bond between the
α-carboxyl group of one amino acid
and the α-amino group of another
amino acid
Geometry of Peptide Bond

• The four atoms of a peptide bond and the two alpha carbons joined to it lie in a
plane with bond angles of 120° about C and N

• To account for this geometry, a peptide bond is most accurately represented as a

hybrid of two contributing structures (resonance structures)

• Partial double bond character of the peptide bond hinders rotations about it
Geometry of Peptide Bond

• Can be in cis or trans conformations

Geometry of Peptide Bond

• Proline residues favor the cis conformation

Peptides

• Peptide: the name given to a short polymer of amino acids joined by peptide
bonds; they are classified by the number of amino acids in the chain

• Dipeptide: a molecule containing two amino acids joined by a peptide bond

• Tripeptide: a molecule containing three amino acids joined by peptide bonds

• Oligopeptide: a molecule containing three to ten amino acids joined by peptide

bonds

• Polypeptide: a macromolecule containing many amino acids joined by peptide

bonds
Peptides with Physiological Activity
Protein Structure

• Many conformations are possible for proteins:

• Due to flexibility of amino acids linked by peptide bonds

• At least one major conformations has biological activity, and hence is considered
the protein’s native conformation
Different Structural Features of a Protein

• Globular or spherical
• Water soluble
• Hydrophobic core
• Hydrophilic residues exposed to solvent

• Fibrous, elongated, threadlike

• Generally water insoluble
• Many hydrophobic residues end up being exposed to solvent
• Protein is tough (e.g. proteins in hair, nails, skin)

• Monomeric or polymeric
Hierarchical Organization of Proteins

• Primary Structure
• Amino acid sequence along a linear polypeptide
chain

• Secondary Structure
• Local conformation of the polypeptide chain
• α-helices, β-strands, loops
• Interfaces are composed of hydrophobic residues
• Packs to form structural domains

• Tertiary Structure
• 3D organization of the secondary structural
elements
• Protein folds
Hierarchical Organization of Proteins

• Quaternary Structure
• Multiple subunits associate to form a multimeric complex
• Can function independently or cooperatively
Primary Structure

• The 1˚ sequence of proteins determines its 3-D conformation

• Changes in just one amino acid in sequence can alter biological function, e.g.
hemoglobin associated with sickle-cell anemia

• Determination of 1˚ sequence is routine biochemistry lab work

Peptide Nomenclature

• C-terminal AA keeps its full amino acid name

• All other AAs end in –yl to replace the –ine or –ic acid
• Exceptions: tryptophyl, cysteinyl, glutaminyl, asparaginyl

• Naming sequence starts at the N-terminus

Secondary Structure

• 2˚ of proteins is hydrogen-bonded arrangement of

backbone of the protein

• Two bonds have free rotation:

• Bond between α-carbon and amino nitrogen in residue
• Bond between the α-carbon and carboxyl carbon of residue
Secondary Structure

• Many of the possible conformations about an α-carbon between two peptide

planes are forbidden because of steric crowding.
Secondary Structure

• Unfavorable orbital overlap/steric crowding precludes some combinations of φ and

ψ

• φ = 0°, ψ = 180° is unfavorable

• φ = 180°, ψ = 0° is unfavorable
• φ = 0°, ψ = 0° is unfavorable

• G. N. Ramachandran was the first to demonstrate the convenience of plotting phi,

psi combinations from known protein structures

• The sterically favorable combinations are the basis for preferred secondary
structures
Secondary Structure

• A Ramachandran diagram showing the sterically

reasonable values of the angles φ & ψ. The shaded
regions indicate favorable values of these angles. Dots in
purple indicate actual angles measured for 1000 residues
(excluding glycine, for which a wider range of angles is
permitted) in eight proteins.
α-Helices

• Coil of the helix is clockwise or right-handed

• There are 3.6 amino acids per turn

• Repeat distance is 5.4 Å

• Stabilized by hydrogen bonding between carbonyl oxygen of one residue

(namely i) to amine hydrogen of another residue (commonly i + 4)

• C=O----H-N hydrogen bonds are parallel to helical axis

• Main chain atoms are inside; side chains are exposed to the solvent
α-Helices
α-Helices

• Several factors can disrupt an α-helix

• Proline creates a bend because of (1) the restricted rotation due to its cyclic
structure and (2) its α-amino group has no N-H for hydrogen bonding

• Strong electrostatic repulsion caused by the proximity of several side chains of

like charge, e.g., Lys and Arg or Glu and Asp

• Steric crowding caused by the proximity of bulky side chains, e.g., Val, Ile, Thr
β-Sheets

• Polypeptide chains lie adjacent to one another; may be parallel or antiparallel

• R groups alternate, first above and then below plane

• Consists of β-strands connected laterally by at least two or three backbone H-

bonds

• C=O---H-N hydrogen bonds are between adjacent sheets and perpendicular to

the direction of the sheet
β-Sheets

• Forms a generally twisted, pleated sheet

• Can involve one or more polypeptide chains

• R-groups are usually small compact side

chains such as Gly, Ser, Ala

• Generally fibrous
β-Sheets

• β-bulge: a common non-repetitive irregular 2˚ motif in anti-parallel structure

Secondary Structure

• Turns, random coils

• Small regions that can form small loops
• Often contain glycine and proline
α-Helices and β-Sheets

• Supersecondary structures: the combination of α- and β-sections, as for

example
• βαβ unit: two parallel strands of β-sheet connected by a stretch of α-helix

• αα unit: two antiparallel α-helices

• β-meander: an antiparallel sheet formed by a series of tight reverse turns connecting

stretches of a polypeptide chain

• Greek key: a repetitive supersecondary structure formed when an antiparallel sheet

doubles back on itself

• β-barrel: created when β-sheets are extensive enough to fold back on themselves
Schematic Diagrams of Supersecondary Structures
Tertiary Structure

• Global arrangement of secondary structure due to interactions among side

chains or other prosthetic groups (e.g. metals)

• Interactions matter such as:

• Salt bridges
• Disulfide bridges
• Polar interactions
Protein Folding

• Hydrophobic effect is the main driving force of protein folding

Protein Folding
Protein Folding

• Formation of α helices and β sheets satisfies the hydrogen bonding requirements

of the protein backbone
Protein Folding

• Secondary structures form wherever possible (due to formation of large numbers

of H bonds)

• Helices and sheets often pack close together

• Peptide segments between secondary structures tend to be short and direct

• Proteins fold so as to form the most stable structures. Stability arises from:
• Formation of large numbers of intramolecular hydrogen bonds
• Reduction in the surface area accessible to solvent that occurs upon folding
Fibrous Proteins

• Contain polypeptide chains organized approximately parallel along a single axis.

They
• consist of long fibers or large sheets
• tend to be mechanically strong
• are insoluble in water and dilute salt solutions
• play important structural roles in nature

• Examples are
• keratin of hair and wool
• collagen of connective tissue of animals including cartilage, bones, teeth, skin, and blood
vessels
Globular Proteins

• Proteins which are folded to a more or less spherical shape

• They tend to be soluble in water and salt solutions

• Most of their polar side chains are on the outside and interact with the aqueous
environment by hydrogen bonding and ion-dipole interactions

• Most of their nonpolar side chains are buried inside

• Nearly all have substantial sections of α-helix and β-sheet

Comparison of Shapes of Fibrous and Globular Proteins
Tertiary Structure

• The 3-dimensional arrangement of atoms in the molecule.

• In fibrous protein, backbone of protein does not fall back on itself, it is important
aspect of 3˚ not specified by 2˚ structure.

• In globular protein, more information needed. 3-d structure allows for the
determination of the way helical and pleated-sheet sections fold back on each
other.

• Interactions between side chains also plays a role.

Forces in 3˚ Structure

• Noncovalent interactions, including

• hydrogen bonding between polar side chains, e.g., Ser and Thr
• hydrophobic effect between nonpolar side chains, e.g., Val and Ile
• electrostatic attraction between side chains of opposite charge, e.g., Lys and Glu
• electrostatic repulsion between side chains of like charge, e.g., Lys and Arg, Glu and Asp

• Covalent interactions: Disulfide (-S-S-) bonds between side chains of

cysteines
Forces in 3˚ Structure
Tertiary Structure

• Helices and sheets make up the core of most globular proteins

• Most polar residues face the outside of the protein and interact with solvent

• Most hydrophobic residues face the interior of the protein and interact with each
other

• Packing of residues is close but empty space exists

Tertiary Structure

• Much of the surface is composed of loops and tight turns that connect the
helices and sheets of the core

• Thus the surface is a complex landscape of different structural elements

• These surface elements can interact with small molecules or with other proteins

• Special proteins called molecular chaperones aid in the correct and timely
folding of many proteins.
• They protect nascent proteins from the concentrated protein matrix in the cell and perhaps
accelerate slow steps.
Quaternary Structure

• The association of polypepetide monomers

into multi-subunit proteins
• dimers
• trimers
• tetramers

• Noncovalent interactions
• electrostatics, hydrogen bonds, hydrophobic
Quaternary Structure

• Advantages of quaternary structure

formation:
• Stability: reduction of surface to volume ratio
• Genetic economy and efficiency
• Bringing catalytic sites together
• Cooperativity
Denaturation

• The loss of the structural order (2°, 3°, 4°, or a combination of these) that gives a
protein its biological activity; that is, the loss of biological activity

• Denaturation can be brought about by

• heat
• large changes in pH, which alter charges on side chains, e.g., -COO- to -
COOH or –NH3+ to –NH2
• detergents such as sodium dodecyl sulfate (SDS) which disrupt hydrophobic interactions
• urea or guanidine, which disrupt hydrogen bonding
• mercaptoethanol, which reduces disulfide bonds
Denaturation
Alpha-Keratin

• Sequence consists of 311-314 residue alpha helical rod segments capped with
non-helical N- and C- termini

• The consensus amino acid sequence is a repeating heptamer of (-a-b-c-d-e-f-g-)n

where residues a and d are non polar.

• This structure promotes association of helices to form coiled coils

• Alpha-keratin is strong, inextensible, insoluble and chemically inert.

Alpha-Keratin

• The protein is relatively rich in cysteine residues and disulfide bridges

• Proteins of horn and nails are hard and brittle and have up to 22% Cys ; those of
hair, skin and wool contain 14% cysteine and are softer, more flexible and elastic

• In the alpha keratins of hair and wool, 3 or 7 α-helices may be coiled around each
other to form ropes held together by S-S linkages.
Beta-Keratin

• Silk fibroin is strong, inextensible, insoluble and chemically inert; extension

resisted by full strength of covalent bonds

• In silk fibroin, antiparallel beta pleated sheets contain sequences of (-Ser-Gly-Ala-

Gly-)n interrupted by regions containing bulkier residues.

• Since residues of a β sheet extend alternately above and below the plane of the
sheet, this places all glycines on one side and all alanines and serines on other
side
Beta-Keratin

• This allows Gly on one sheet to mesh

with Gly on an adjacent sheet (same for
Ser)

• In sum, the fiber is composed of

microcrystalline arrays alternating with
amorphous regions.

• Fiber strengths result from extensive H

bonding of adjacent chains and the
cumulative effect of van der Waal’s
interactions among stretched chains
Collagen Triple Helix

• Consists of three polypeptide chains wrapped around each other in

a ropelike twist to form a triple helix called tropocollagen; MW
approx. 300,000

• 30% of amino acids in each chain are Pro and Hyp (hydroxyproline);
hydroxylysine also occurs

• Every third position is Gly and repeating sequences are X-Pro-Gly

and X-Hyp-Gly
Collagen Triple Helix

• Each polypeptide chain is a helix but not an α-helix

• The three strands are held together by hydrogen bonding involving

hydroxyproline and hydroxylysine

• With age, collagen helices become cross linked by covalent bonds

formed between Lys and His residues
Myoglobin

• A single polypeptide chain of 153 amino

acids

• A single heme group in a hydrophobic

pocket

• 8 regions of α-helix; no regions of β-sheet

• Most polar side chains are on the surface

Myoglobin

• Nonpolar side chains are folded to the

interior

• Two His side chains are in the interior,

involved with interaction with the heme
group

• Fe(II) of heme has 6 coordinates sites; 4

interact with N atoms of heme, 1 with N
of a His side chain, and 1 with either an
O2 molecule or an N of the second His
side chain
Oxygen Binding Site of Myoglobin
Structure of Hemoglobin
Oxygen Binding in Myoglobin

• Oxygen binds at an angle in free heme, in contrast

to carbon monoxide

• Distal histidine (His E7) provides steric hindrance

for CO to bind to the heme in myoglobin

• Difference in structure of heme upon oxygen

binding
Oxygen Binding in Hemoglobin

• A tetramer of two α-chains (141 amino acids

each) and two β-chains (153 amino acids
each); α2β2

• Each chain has 1 heme group; hemoglobin

can bind up to 4 molecules of O2

• Binding of O2 exhibited by positive

cooperativity; when one O2 is bound, it
becomes easier for the next O2 to bind
Oxygen Binding in Hemoglobin

• The function of hemoglobin is to transport

oxygen

• The structure of oxygenated Hb is different

from that of unoxygenated Hb

• H+, CO2, Cl-, and 2,3-bisphosphoglycerate

(BPG) affect the ability of Hb to bind and
transport oxygen
Oxygen Binding in Hemoglobin

• Bohr effect: the affinity of hemoglobin for O2

depends on pH

• Hemoglobin in blood is also bound to 2,3-

bisphosphoglycerate

• Fetal hemoglobin binds less strongly to BPG

and has greater affinity for O2 than does
maternal hemoglobin. This allows for efficient
transfer of O2 from mother to fetus.
Conformation Changes That Accompany Hb Function

• Structural changes occur during binding of small molecules

• Characteristic of allosteric behavior

• Hb exhibits different 4˚ structure in the bound and unbound oxygenated forms

• Other ligands involved in cooperativity effect of Hb can affect protein’s affinity for
O2 by altering structure
Allosteric Effectors of Hemoglobin
Proteins

Prepared by:
IC Duay, PhD
Assistant Professor
De La Salle University
searle.duay@dlsu.edu.ph