MODULE 5.

PROTEINS

Course Outcomes: At the end of the course, the student shall be able to:
1 Describe the chemical structure that make up the components of living matter

2 Describe the interactions of these components that give rise to the organized
supramolecular structures, cells and multicellular tissues
3 Explain how living organisms extract energy from the surroundings to perpetuate life

4 Explain how chemical reactions are regulated inside living cells

5 Explain how organisms store and transmit genetic information to grow and to
reproduce accurately
6 Apply key concepts in biochemistry to explain its practical applications in the field of
nutrition, agriculture, medicine, pharmacy and allied fields
7 Demonstrate good leadership, critical and creative thinking skills and ability to
communicate scientific information clearly and concisely imbued with Augustinian
charism.
INTRODUCTION TO THE MODULE: Proteins are the polymeric biomolecules built up from amino
acid monomers. In proteins, amino acids are joined together by peptide bonds. A peptide bond
is formed in a nucleophilic addition – elimination reaction between the carboxylic group of one
amino acid and the amino group of a second amino acid resulting in an amide bond (with loss of
a water molecule, HOH). Each amino acid is termed as a residue because of the loss of a water
molecule. Both small peptides and large proteins are made up of a linear linkage of amino acid
residues joined by the peptide bond which is referred to as the primary structure of a peptide
or protein. The ends of a peptide or protein have a free amino group (known as the N-terminal
amino acid) and a free carboxyl group (known as the C-terminal amino acid). Proteins are
synthesized in the ribosome by translation of the mRNA from the N- to the C-terminus. There
are four hierarchies of protein structure starting with the primary structure, secondary
structure, tertiary structure, and quaternary structure determined by the structural features of
local segments of the protein, the protein folding and refolding unto itself to attain its native
conformation as well as the association of 2 or more polypeptides resulting in an oligomeric
protein complex. The native conformation determines the biologic function of a protein which
includes maintenance of structure, protection, catalysis, regulation, transport, storage, and
signal transduction. Loss of native conformation caused by denaturing agents such as
temperature, pH, various chemicals, and enzymes can lead to a loss of biologic function of a
protein.

LEARNING OUTCOMES:

1. Differentiate the organizational levels of proteins

2. Describe the effects of denaturation on the different levels of structure
3. Name some denaturing agents for proteins

DISCUSSION:

Proteins are polymers of amino acids joined together by peptide bonds. A peptide bond
is formed in a nucleophilic addition–elimination reaction, a condensation reaction between the
carboxylic group of one amino acid and the amino group of a second amino acid resulting in an
amide bond (with loss of a water molecule, HOH). Both small peptides and large proteins are
made up of a linear linkage of amino acid residues joined by the peptide bond. Two amino acids
joined by a peptide bond result in a dipeptide, three amino acids, a tripeptide, four amino acids
a tetrapeptide and so on. The peptide is named after the amino acid residues that comprise it
starting from the N-terminal end up to the C-terminal end. For instance, the tetrapeptide
composed of aspartic acid on the N-terminal end, followed by phenylalanine, cysteine and
lysine on the C-terminal end is called aspartyl phenylalanyl cysteinyl lysine and is represented
as asp-phe-cys-lys or D-F-C-K. The N-terminal is aspartic acid while the C-terminal is lysine. The
exact sequence of amino acids is called the primary structure. The amino group and carboxylic
group forming the peptide bond are not available for chemical reaction, thus the chemical
characteristics of the side chain R group determines the role the amino acid plays in the
protein. By applying the principles in determining the pI of individual amino acids, one can also
solve for the pI of a peptide or protein and, for that matter, determine the net charge of small
or large peptides or proteins. We learned how to perform these operations in the previous
module on amino acids and peptides.
 The peptide bond is an amide functional group consisting of the carbonyl group of 1
amino acid and the amino group of the adjacent amino acid. Because of resonance, the single
bond between the carbonyl C and the amino N in the amide bond (sp2-hybridized) possesses
partial double bond character. Thus, the peptide bond is a rigid, planar structure with restricted
rotation around the C-N bond. This explains why the peptide bond is stable under physiologic
conditions and why it is not easily broken; for example, it can be broken under drastic
hydrolysis conditions by treatment with 6N HCl at high temperature (110 oC) under vacuum for
at least 12 h. The restricted rotation around the peptide bond is important in the folding of the
protein backbone. The peptide bond also possesses a small electric dipole because of the
partial negative charge on the carbonyl oxygen and the partial positive charge on the amide
nitrogen, thus, it is able to participate in hydrogen bonding interactions with other peptide
bonds present in the protein.

Proteins are large, complex molecules, thus many different three-dimensional structures
(conformations) are possible for a protein; however, one or a few have biological activity and
these are called native conformations. Proteins possess four levels of structure, namely
primary structure, secondary structure, tertiary structure, and quaternary structure.

Primary structure is the exact order or sequence of the amino acids in the protein. The
primary structure determines the three-dimensional structure of the protein and its properties.
It is based on the sequence of the genes in DNA, transcribed into mRNA, and translated to
proteins in the ribosomes. Change in one or more amino acids in the primary structure of a
protein can result in drastic consequences on its structure and properties leading to a loss of
biological function.

The secondary structure of a protein is the result of hydrogen bonding interactions of

the backbone of the protein. Free rotation around two single bonds (the C-N and the C-C
bonds) within the amino acid residue in combination with the peptide bond is a determinant of
the three-dimensional structure and conformation of the protein. Rotation (-180 o to 180o)
around the C-N bond, the phi (angle, and the C-C bond, the psi (angle, also called
Ramachandran angles (after their originator G. N. Ramachandran) are used to describe the
conformation of the protein backbone. Two kinds of secondary structures are often
encountered in a protein, the alpha () helix and beta ()-pleated sheet. The  helix involves
one polypeptide chain. The -pleated sheet can involve different segments of a long chain
(intrachain bonds) or segments from different polypeptide chains (interchain bonds). If the
chains run in the same direction (from the N-terminal end to the C-terminal end) the  pleated
sheet is described as parallel. If the chains run in opposite direction, it is described as anti-
parallel. The  helix is stabilized by hydrogen bonding interaction between C-O group of one
amino acid residue and the N-H group of an amino acid four residues away from it in the
sequence. The hydrogen bonds impart maximum strength and make the  helix very stable.
There are 3.6 residues per turn of the helix and a pitch (the linear distance between the
corresponding units in successive turns) is 5.4 angstroms (A). There are other secondary
structures possible such as random coil, beta () bends, non-repetitive secondary structures (
bulge) and supersecondary structures or domains (combinations of helix,  meander,
barrel, Greek key motif), but the  helix and  pleated sheets are the most important.

Proline residues create a bend in the structure because of their rigid, cyclic structure
cannot participate in the  helix or  pleated sheet. Furthermore, strong electrostatic
repulsions due to close proximity of several charged groups (negatively charged acidic residues
such as aspartic acid and glutamic acid or positively charged basic residues such as lysine,
arginine, or histidine) can destabilize the helix. In addition, steric repulsion caused by bulky R
groups such as the branched chain amino acids leucine, isoleucine, and valine can also result in
subtle changes in the secondary structure of a protein.

The tertiary structure of proteins refers to the three-dimensional structure resulting

from the folding and refolding of the protein. It is determined by X-ray crystallography and
nuclear magnetic resonance (NMR) spectroscopy. The tertiary structure is stabilized by
disulfide bonds, hydrogen bonds, hydrophobic interactions, and electrostatic or ionic
interactions. Some proteins are fibrous proteins where  helices and  pleated sheet make a
rigid polypeptide that is usually long and thin. Many structural proteins are fibrous proteins
such as collagen, keratin, and proteins that comprise the cytoskeleton. Other proteins are
globular proteins which contain short stretches of  helices,  pleated sheets and random coils
and fold into compact and flexible spherical biomolecules. Myoglobin, the globular protein
which binds oxygen in the muscle, was the first protein for which the tertiary structure was
determined by X-ray crystallography. Many enzymes are globular proteins.

The quaternary structure of proteins pertains to proteins with two or more polypeptide
chains which may be identical or different. Some proteins may be dimers, trimers, or tetramers
consisting of two, three, or four subunits, respectively. Others may contain more subunits
sometimes numbering more than a dozen in multi-subunit molecular complexes. A classic
example is hemoglobin, the heme protein that carries oxygen in the blood, which is a tetramer
made up of 2 identical  chains and 2 identical  chains. The protein also shows positive
cooperativity meaning when one molecule of oxygen is bound, the binding of the succeeding
oxygen molecules becomes easier. In contrast, myoglobin does not show cooperativity because
it is a monomeric molecule.

Certain conditions can lead to a loss of biological activity or denaturation. Elevated

temperature, drastic changes in pH, certain chemicals, and enzymes can denature proteins.
These conditions cause the destruction of the secondary and tertiary structures of protein by
breaking hydrogen bonds and interfering with hydrophobic and electrostatic interactions.
Enzymes such as proteolytic enzymes cause the loss of primary structure by cleaving peptide
bonds. Chemicals such as sodium dodecyl sulfate (SDS, a detergent) and urea can interfere with
hydrophobic interactions and guanidinium chloride interferes with electrostatic or ionic
interactions. -mercaptoethanol can disrupt disulfide bridges in proteins. Denaturation due to
treatment of the protein with these chemicals is reversible (renaturation) meaning the protein
can fold back into its native conformation and recover its biological activity or function.

POWERPOINT : MODULE 5. PROTEINS

VIDEO: WHAT IS A PROTEIN? Learn about the 3D shape and function of macromolecules

(https://youtu.be/qBRFIMcxZNM) (3 min 39 sec)

VIDEO: CONFORMATIONAL STABILITY: PROTEIN FOLDING AND DENATURATION

(https://youtu.be/dNHtdiVjQbM) (7 min 44 sec)

ACTIVITY:

ACTIVITY 5. PROTEINS

SHORT QUIZ

SQ MODULE 5. PROTEINS (10 POINTS)

TEXTBOOK:

Campbell, Mary K., Farrell, Shawn O., and McDougal, Owen (2018). Biochemistry, Cengage
Learning 9th edition (other editions 8th , 2015 and 7th , 2012)

OTHER REFERENCES:

1. Bettelheim et al. (2019), Biochemistry, C & E Publishing, Inc.

2. Ferrier, D.(2017), Lippincott’s Illustrated Reviews: Biochemistry, 7th ed. Lippincott
Williams & Wilkins: Baltimore
3. Garrett, R. and Grisham, C. (2017), Biochemistry, 6th ed. BROOKS/COLE:Cengage
Learning: Boston
4. McKee & McKee(2019), Biochemistry: An Introduction, 7nd Ed., WCB-Mcraw-Hill
5. Nelson and Cox (2017), Lehninger Principles of Biochemistry, 7th Ed., Macmillan Learning
6. Stoker (2017), Biochemistry, 3rd Ed.,Cengage Learning