Biomolecules: Proteins

Protein
• is a large biomolecule made up of numerous amino acids linked by amide bonds
• most abundant molecules in the cells after water –about 15% of a cell’s overall mass
• Elemental composition:
Always present: Carbon (C), Hydrogen (H), Nitrogen (N), Oxygen (O),
Some: Sulfur (S),
Iron (Fe), phosphorus (P) and some other metals (specialized proteins)
• average nitrogen content of proteins is 15.4% by mass

Functions of proteins
1.Structure: collagen and keratin are the chief constituents of skin, bone, hair, and nails.
2. Catalysts: virtually all reactions in living systems are catalyzed by proteins called enzymes.
3. Movement: muscles are made up of proteins called myosin and actin.
4. Transport: hemoglobin transports oxygen from the lungs to cells; other proteins transport molecules
across cell membranes.
5. Hormones: many hormones are proteins, among them insulin, oxytocin, and human growth hormone.
6. Protection: blood clotting involves the protein fibrinogen; the body used proteins called antibodies to
fight disease.
7. Storage: casein in milk and ovalbumin in eggs store nutrients for newborn infants and birds; ferritin, a
protein in the liver, stores iron.
8. Regulation: certain proteins not only control the expression of genes, but also dictates when gene
expression takes place.

Proteins are divided into two major types:

• Globular proteins- soluble in water and are used mainly for non structural purposes
• Fibrous proteins- water insoluble and are used mainly for structural purposes

What are amino acids

• Amino acid: a compound that contains both an amino group and a carboxyl group. There are 20 primordial
amino acids. They are all in the α-L configuration. All of these differs in the R group

Amino Acids - building blocks of protein Chirality of Proteins

• Four different groups are attached to the α-carbon
atom in all of the standard amino acids except
glycine
*In glycine R-group is hydrogen

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Chirality of Proteins
• Therefore 19 of the 20 standard amino acids contain a chiral center
• Chiral centers exhibit enantiomerism (left- and right-handed forms)
• Each of the 19 amino acids exist in left and right handed forms
• The amino acids found in nature as well as in proteins are L isomers.
• Bacteria do have some D-amino acids
• With monosaccharides nature favors D-isomers

The rules for drawing Fischer projection formulas for amino acid structures
The — COOH group is put at the top, the R group at the bottom to position the carbon
Designation of Mirror
chain vertically
handedness in standard amino acid
The — NH2 group is in a horizontal position.
structures
Positioning — NH2 on the left - L isomer
Positioning — NH2 on the right - D isomer.

The 20 Amino Acids

• are those incorporated into polypeptides during protein synthesis
• They fall into five distinct groupings as clusters of 7, 5, 3, 3, and 2: the aliphatics, polars, aromatics, basics,
and acidics
• Within each cluster, each amino acid has particular unique characteristics

Other ways to group the 20 amino acids

• Which and how many…
• … have hydroxyl groups?
• … contain sulfur?
• … contain 5- or 6-membered rings?
• … have pure hydrocarbon side chains?
• … have nitrogen in their side chains?
• Which is biggest? …smallest? …most acidic? …most basic?

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PI value of an amino acid
Isoelectric point, pI: The pH at which the majority of molecules of a compound in solution have no net
charge.

Ionization vs pH
• The net charge on an amino acid depends on the pH of the
solution in which it is dissolved.
• If we dissolve an amino acid in water, it is present in
the aqueous solution as its zwitterion.
• If we now add a strong acid such as HCl to bring the
pH of the solution to 2.0 or lower, the strong acid
donates a proton to the -COO- of the amino acid
turning the zwitterion into a positive ion.

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 Zwitterion
*Protonation of zwitterion

Computing the isoelectric point of amino acids

Some Properties of Amino Acids found in Proteins
Name Abbrev pKa of pKa of pKa of ionizing side chain
α-COOH gp α-NH3+ gp
Alanine Ala 2.3 9.7 -
Arginine Arg 2.2 9.0 12.5
Asparagine Asn 2.0 8.8 -
Aspartic acid Asp 2.1 9.8 3.9
Cysteine Cys 1.8 10.8 8.3
Glutamine 2.2 9.1 -
Glutamic 2.2 9.7 4.2
Glycine 2.3 9.6 -
Histidine 1.8 9.2 6.0
Isoleucine 2.4 9.7 -
Computing the PI Values of Amino Acids

Aspartic acid Asp 2.1 9.8 3.9

3+
*At pH below 2.1 NH2 protonated to NH and both COOH are all protonated,
charge is +1
*At pH 3.9 NH2 is protonated to NH3+, COOH1 is deprotonated to COO- while
COOH2 is deprotonated to COO- charge is -1
*At pH above 9.8 NH3+ is deprotonated to NH2, both COO- is deprotonated
to COO- charge -2
* Find Ka when +1 and Ka when -1. Add the Ka values and divide by 2
Comp. PI= (2.1 + 3.9) /2 = 3.0

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Peptides
• In 1902, Emil Fischer proposed that proteins are long chains of amino acids joined by amide bonds.
○ peptide bond: The special name given to the amide bond between the a-carboxyl group of one
amino acid and the a-amino group of another.

Peptide Bond/link The Peptide Bond

• is usually found in the trans conformation
• has partial (40%) double bond character
• is about 0.133 nm long - shorter than a
typical single bond but longer than a
double bond
• Due to the double bond character, the six
atoms of the peptide bond group are
always planar!
• N partially positive; O partially negative

Polypeptides
• -Short polymers of amino acids
• -Each unit is called a residue
• -2 residues - dipeptide
• -3 residues - tripeptide
• -12-20 residues - oligopeptide
• - many - polypeptide

Writing Peptides
• By convention, peptides are written from the
left, beginning with the free -NH3+ group and
ending with the free -COO- group on the right.
• C-terminal amino acid: the amino acid at the
end of the chain having the free -COO- group.
• N-terminal amino acid: the amino acid at the
end of the chain having the free -NH3+ group.

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 Peptides and Proteins
• Hemoglobin, for example, has an almost equal number
of acidic and basic side chains; its pI is 6.8.
• Serum albumin has more acidic side chains; its pI is 4.9.
• Proteins are least soluble in water at their isoelectric
points and can be precipitated from solution at this pH.

Proteins behave as zwitterions.

Proteins also have an isoelectric point, pI.
• At its isoelectric point, the protein has no net charge.
• At any pH above (more basic than) its pI, it has a net negative charge.
• At any pH below (more acidic than) its pI, it has a net positive charge.

Levels of Protein Structure

• Primary structure: the sequence of amino acids in a polypeptide chain; read from the N-terminal amino acid
to the C-terminal amino acid.

• Just how important is the exact amino acid sequence?

Human insulin consists of two polypeptide chains having a total of 51 amino acids; the two chains are connected
by two interchain disulfide bonds.

Insulin consists of two polypeptide chains, A and B, held together by two disulfide bonds. The A chain has 21
residues and the B chain has 30 residues.

In the table are differences between four types of insulin .

• Secondary structure: conformations of amino
acids in localized regions of a polypeptide
chain; examples are a-helix, b-pleated sheet,
and random coil.
• A protein’s secondary structure consists of
regular, repeated patterns in different regions
in a polypeptide chain.
• This shape is influenced primarily by hydrogen
bonds arising from the amino acid sequence
(the primary structure).
The two common secondary structures are the alpha
helix and the beta pleated sheet.
• b pleated sheets form from peptide regions
that lie parallel to each other.
Sometimes the parallel regions are in the same
peptide, sometimes they are from different peptide
strands.

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• -Pleated sheet: a type of secondary structure in which two polypeptide chains or sections of the same polypeptide
chain align parallel to each other; the chains may be parallel or antiparallel.

• In a section of b-pleated sheet;

• The six atoms of each peptide bond lie
in the same plane.
• The C=O and N-H groups of peptide
bonds from adjacent chains point
toward each other and are in the same
plane so that hydrogen bonding is
possible between them.
• All R- groups on any one chain
alternate, first above, then below the
plane of the sheet, etc.

-Helix: a type of secondary structure in which a section of polypeptide chain coils into a spiral, most commonly a
right-handed spiral.

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• In a section of a-helix;
• There are 3.6 amino acids per turn of the helix.
• The six atoms of each peptide bond lie in the same plane.
• N-H groups of peptide bonds point in the same direction, roughly parallel to the axis of the helix.
• C=O groups of peptide bonds point in the opposite direction, also roughly parallel to the axis of the helix.
• The C=O group of each peptide bond is hydrogen bonded to the N-H group of the peptide bond four amino
acid units away from it.
• All R- groups point outward from the helix.

Tertiary Structure
• Tertiary structure is the three-dimensional shape of the completed polypeptide. The primary determinant of
the tertiary structure is the interaction between R groups. Other factors can include the location of disulfide
bridges, which form between cysteine residues

Examples of bonds contributing to the tertiary structure of a

protein

• Secondary structures form wherever possible (due to formation of large numbers of H-bonds)
• Helices and sheets often pack close together

Important principles to a tertiary structure

• The backbone links between elements of secondary structure are usually short and direct
• Proteins fold to make the most stable structures (make H-bonds and minimize solvent contact)

Tertiary structure is stabilized in four ways:

• Covalent bonds, as for example, the formation of disulfide bonds between cysteine side chains
○ Hydrogen bonding between polar groups of side chains, as for example between the -OH groups of
serine and threonine.
○ Salt bridges, as for example, the attraction of the -NH3+ group of lysine and the -COO- group of aspartic
acid.
○ Hydrophobic interactions, as for example, between the nonpolar side chains of phenylalanine and
isoleucine.
Quaternary Structure
• the arrangement of polypeptide chains into a noncovalently bonded aggregation.
○ The individual chains are held in together by hydrogen bonds, salt bridges, and hydrophobic
interactions.
• Hemoglobin
○ Adult hemoglobin: two alpha chains of 141 amino acids each, and two beta chains of 146 amino acids
each.
○ Each chain surrounds an iron-containing heme unit.
○ Fetal hemoglobin: two alpha chains and two gamma chains; fetal hemoglobin has a greater affinity for
oxygen than does adult hemoglobin.

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• Hemoglobin is an example of such a protein; it has four subunits.

Primary, secondary, tertiary and quaternary structures of proteins

Other Chemical Groups in Proteins

Proteins may be "conjugated" with other chemical groups
• If the non-amino acid part of the protein is important to its function, it is called a prosthetic group.
• Be familiar with the terms: glycoprotein, lipoprotein, nucleoprotein, phosphoprotein, metalloprotein,
hemoprotein, flavoprotein.

Sequence Determination
Frederick Sanger was the first - in 1953, he sequenced the two chains of insulin.
• Sanger's results established that all of the molecules of a given protein have the same sequence.
• Proteins can be sequenced in two ways:
- real amino acid sequencing
- sequencing the corresponding DNA in the gene

Determining the Sequence

A six Step Strategy 4. Determine N- and C-terminal residues
1. If more than one polypeptide chain, separate. 5. Cleave each chain into smaller fragments and
2. Cleave (reduce) disulfide bridges determine the sequence of each chain
3. Determine composition of each chain 6. Repeat step 5, using a different cleavage procedure
to generate a different set of fragments.
Denaturation
• Denaturation: the process of destroying the native conformation of a protein by chemical or physical means.
○ Some denaturations are reversible, while others permanently damage the protein.
• Denaturing agents include:
○ Heat: heat can disrupt hydrogen bonding; in globular proteins, it can cause unfolding of polypeptide
chains with the result that coagulation and precipitation may take place.

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• 6 M aqueous urea: disrupts hydrogen bonding.
• Surface-active agents: detergents such as sodium dodecylbenzenesulfate (SDS) disrupt hydrogen bonding.
• Reducing agents: 2-mercaptoethanol (HOCH2CH2SH) cleaves disulfide bonds by reducing -S-S- groups to -SH
groups.
• Heavy metal ions: transition metal ions such as Pb 2+, Hg2+, and Cd2+ form water-insoluble salts with -SH groups; Hg2
+
for example forms -S-Hg-S-.
• Alcohols: 70% ethanol, for example, which denatures proteins, is used to sterilize skin before injections.

• Changes in temperature, pH, salt concentrations, and oxidation or reduction conditions can change the shape of
proteins.

Nomenclature of proteins
• 20 Primordial Amino Acids

Name 3 letter abbreviation 1 letter abbreviation

Alanine Ala A
Argenine Arg R
Asparagine Asn N
Aspartic acid Asp D
Write the name using the three letter
Cysteine Cys C symbol but change the ending to “yl”
Glutamic acid Glu E
The amino acid in the end assumes its
Glutamine Gln Q original name
Glycine Gly G e.g.
Histidine His H • Ala-Gly-Lys
Isoleucine Ile I • Alanylglycyllysine
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val Z

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Essential and non-essential amino acids
• An essential amino acid is an amino acid that cannot be synthesized de novo by the organism (usually
referring to humans), and therefore must be supplied in the diet.
• Eight amino acids are generally regarded as essential for humans: phenylalanine, valine, threonine,
tryptophan, isoleucine, methionine, leucine, and lysine. Additionally, cysteine (or sulphur-containing amino
acids), tyrosine (or aromatic amino acids), histidine and arginine are required by infants and growing
children.
• Non essential amino acids includes
• arginine, cysteine, glycine, glutamine, histidine, proline, serine and tyrosine aspartate , asparagine

Ketogenic , Glucogenic
• Ketogenic amino acids- are amino acids capable of yielding ketone bodies
• e.g. Leucine
• Glucogenic amino acids- are amino acids capable of yielding glucose
• e.g. Alanine

Biochemically Active Small Peptides

• Many relatively small peptides are biochemically active:
○ Hormones
○ Neurotransmitters
○ Antioxidants
• Small Peptide Hormones:
○ Best-known peptide hormones: oxytocin and vasopressin
○ Produced by the pituitary gland
○ nonapeptide (nine amino acid residues) with six of the residues held in the form of a loop by a
disulfide bond formed between two cysteine residues

Small Peptide Neurotransmitters

• Enkephalins are pentapeptide neurotransmitters produced by the brain and bind receptor within the brain
• Help reduce pain
• Best-known enkephalins:
○ Met-enkephalin: Tyr–Gly–Gly–Phe–Met
○ Leu-enkephalin: Tyr–Gly–Gly–Phe–Leu

Small Peptide Antioxidants

• Glutathione (Glu–Cys–Gly) – a tripeptide – is present is in high levels in most cells. Regulator of oxidation–
reduction reactions.
• Glutathione is an antioxidant and protects cellular contents from oxidizing agents such as peroxides and
superoxides-(Highly reactive forms of oxygen often generated within the cell in response to bacterial
invasion)
*Unusual structural feature – Glu is bonded to Cys through the side-chain carboxyl group.

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