TOPIC 3

PART 2
PROTEINS
Topic Brief Outline
➢ Biological Functions of Protein
➢ Determining the Amino Acid Sequence in Proteins
➢ Hierarchal Structures of Protein
o Primary Structure
o Secondary Structure
o Tertiary Structure
o Quaternary Structure
➢ Classification of Proteins
o According to Solubility
o According to Shape
o According to Function
o According to Composition
➢ Protein Denaturation, Folding and Renaturation

Learning Goals
1. Enumerate the different biological functions of proteins,
2. Illustrate how amino acids are linked to from different level or organization in a protein.
3. Describe the different methods involved in determining the sequence of amino acid in
proteins.
4. Classify proteins according to shapes and functions.
5. Describe the characteristics of primary, secondary, tertiary and quaternary structure of
protein.
6. Describe protein denaturation and folding.

PROTEINS
1) From the Greek word proteios, which means “primary,” first used 1838 by the Swedish
Chemist Jöns Jakob Berzelius
2) Polymers of hundreds or thousands of amino acids linked by peptide bonds which can
appear as linear, folded into compact shapes (coils, zigzags, turns, and loops)
3) Abundant biomolecule, making up to 50% dry weight in cells
4) Most versatile macromolecule where their functions depend on their structure
5) Some properties of proteins that are key in several biological functions:
i. They are linear polymers built from amino acids’ monomer units;
ii. They consist of wide range of reactive functional groups such as alcohols,
thiols, thioesters, carboxylic acids, carboxamides and others;
iii. They interact with other proteins forming a complex structure synergistically
making them capable of achieving certain function which is difficult to do alone;
iv. They can form rigid structures that is useful as structural components in
connective tissues or exoskeleton of cells. They can be also flexible and
function as pivots, springs or levers.
Conformation

➢ is a spatial arrangement of atoms that depends on the rotation of a bond or bonds

➢ in proteins; conformation can change without breaking covalent bonds

Configuration

➢ L and D forms of amino acids

➢ Can be changed by breaking and re-forming covalent bonds.

Native confirmation / Native protein

➢ Single stable shape of proteins under physiological conditions

➢ Proteins in any of their functional, folded conformations

Biological Functions of Proteins

Proteins function in many biological processes; some of these functions are listed below:

a) Several proteins act as biochemical catalysts or enzymes in numerous cellular

reactions;
b) Some proteins linked to other molecules for characteristic transport and storage
functions;
c) Provide mechanical and electrochemical support;
d) Essential structural components of cells, provide support and shape to cells;
e) Monitor extracellular and intracellular settings, channeling information in membranes
of other molecules;
f) Some are in fact hormones or hormone receptors, that perform biochemical activities;
g) Cell surface proteins can bind to different ligands in modifying cell-cell interactions;
h) Some act as antibodies.

Protein Sequencing

Sequencing proteins is done by determining the sequences of amino acids in a protein.

This helps in determining the 3D structure of the protein, its mechanism of action, its molecular
functions, its amino acids changes that lead to diseases and also developing tests and
effective therapies to counter them.

Due to protein’s hundreds and thousands of amino acid units, it must be cleaved first into
individual polypeptides that can be overlapped and identified after through mass spectrometry
and other means of identifying from its mass-to-charge ratios of gas-phase protein fragments.

Edman Degradation Procedure, determined by Frederick Sanger in 1953; was used to

determine the sequence of amino acid residues in proteins. His first known protein sequence
was that of a bovine insulin. This breakthrough established that proteins have fixed amino
acid composition, sequence and molecular weight.
Bovine Insulin Amino Acid Sequence (Berg, et.al., 2015) with 2 polypeptide chains, A (21 AA
residues) & B (30 AA residues) linked together by 2 disulfide bridges (S-S)

Edman Degradation Procedure (left: Berg, et.al., 2015, right: Voet, et.al., 2016)

Each protein has a distinctive sequence of amino acid residues encoded by the
nucleotide sequence of DNA, which then form genetic information.
Six steps in determining the amino acid sequence of a protein:
1) Chain separation and purification of proteins with more than one polypeptide chain;
2) Intrachain disulfide bridges (S-S) between cysteine residues are cleaved;
3) N-terminal and C-terminal residues identification;
 4) Cleaving of polypeptide chains into smaller fragments using different proteolytic
enzymes or through acid hydrolysis; and determination of amino acid composition and
sequence of each fragment; some proteolytic enzymes are:
i. Trypsin
➢ digestive enzyme, commonly used for specific proteolysis
➢ only hydrolyze carbonyl groups of Arg or Lys
ii. Chymotrypsin
➢ Only hydrolyze carbonyl groups of aromatic AA – Phe, Tyr, and Trp
iii. Other endopeptidases
➢ Clostripain – cleaves only at Arg residues
➢ Lys-C – cleaves only at Lys residues
➢ Staphylococcal protease – cleaves acidic residues, Asp and Glu.
iv. Cyanogen bromide
➢ Highly specific chemical method of proteolysis
➢ Cleaves only at Met residues, where nucleophilic sulfur atom of Met
reacts with CNBr forming cyclic iminolactone which is easily hydrolyzed
with water forming peptide fragments with C-terminal homoserine
lactone residues.
5) Further cleaving of polypeptide chains until overlapping set of peptide fragments are
formed;
6) Reconstruction of overall amino acid sequence of protein from the sequences of the
overlapping fragments.

Specificity of Polypeptide Cleavage Procedures (top: Garrett and Grisham, 2017; bottom:
Voet, et.al., 2016).
Trypsin cleavage on Arg and Lys residues (Garret and Grisham, 2017)

Cyanogen bromide (CNBr) cleaving at

Met residues (left: Voet, et.al., 2017;
right: Garret and Grisham, 2017).
Overview of protein sequencing (Voet, et.al., 2016)
When amino acids of proteins are already sequenced, they are then analyzed to measure the
mass-to-charge (m/z) ratio for ions in the gas phase in identification of the protein. Mass
spectrometry is a technique to do this, some newer techniques are:
i. Electrospray Ionization (ESI-MS)
ii. Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS)
iii. Tandem Mass Spectrometry

Hierarchal Structure of Proteins

Levels of Protein Structure, (left) Moran and Horton, (right) Voet, et. al., 2016.

A. Primary structure
➢ linear sequence of amino acids in a polypeptide chain
➢ the amino acid sequence of a protein.
B. Secondary (20) structure
➢ regular repetitive arrangement of local regions of the polypeptide backbone
➢ structural pattern of local segments of a polymer (spatial arrangement)
➢ local conformations maintained by H-bonds between amide hydrogens and
carbonyl oxygens of the peptide backbone
➢ can be formed into:
o α-helix
▪ rodlike structure where a tightly coiled backbone forms the inner
part of the rod and the side chains extend outward in a helical array
▪ each carbonyl oxygen (residue n) of the polypeptide backbone is H-
bonded to the backbone amide hydrogen of the 4th residue further
toward the C-terminus (residue n+4)
▪ the backbone dihedrals are angles at: φ= -57o and ψ=-47o
▪ side chains of the amino acids in an α-helix point outward from the
cylinder of the helix and they are not involved in the hydrogen bonds
that stabilize the α helix
 α helix structure (top: Berg, et.al., 2015 (a) ribbon model, (b) ball-and-stick model sideview, (c) end
view, (d) space-filling view); (bottom: Garrett and Grisham, 2017 (a) ball-and-stick model, (b) peptide
planes arrangement in the helix, (c) space-filling model, and (d) ribbon model with polypeptide
backbone.
α helix hydrogen bonding, CO group of residue i forms a hydrogen bond with the NH group
of residue i + 4 (Berg, et.al., 2015)

(left) Torsion angles of the polypeptide backbone, (right) Steric interference between
adjacent peptide groups (Voet, et.al., 2016).

o β pleated sheets
▪ backbone of the polypeptide chain is extended into a zigzag
structure arranged side by side to form a series of pleats.
▪ H-bonding occurs between adjacent polypeptide chains
▪ 2 varieties of sheets:
i. Antiparallel β sheet – neighboring hydrogen-bonded
polypeptide chains run in opposite directions
ii. Parallel β sheet – the hydrogen-bonded chains extend in
the same direction
iii. Mixed β sheet – with parallel and antiparallel varieties
Antiparallel β sheets (left) Nelson and Cox, 2017 and (right) Voet, et.al., 2016

Parallel β sheets (left) Nelson and Cox, 2017 and (right) Voet, et.al., 2016

a) Antiparallel; b) parallel
(Garrett and Grisham, 2017)

Mixed β-sheet (Berg, et.al., 2015

 o β turn
▪ connects two antiparallel β sheet strands, to reorient and produce
compact structures
▪ also called tight turn, reverse turn or β bend
▪ 1800 turn
▪ C-O one residue forms a hydrogen bond with N-H residue at the 3rd
position down the chain. The H-bond makes the structure stable.
▪ 3 types
i. Type I – carbonyl oxygen (C-O) is inwards
ii. Type II – carbonyl oxygen (C-O) is outwards

Structures of two kinds of b-turns (a) Type I, (b) Type II; (top) Garrett and Grisham, 2017,
(bottom) Miesfeld and McEvoy, 2017.

Molecule with protein containing β

turns (Miesfeld and McEvoy, 2017).
C. Tertiary (3o) structure
➢ Overall three-dimensional arrangement of all atoms in a protein
➢ Polypeptide folded into closely packed 3D structure, where amino acid residues far
from each other at their primary structure are brought together permitting
interactions among their side chains

D. Quaternary (4o) structure

➢ refers to the spatial arrangement and organization of subunits and the nature of
their interaction. One subunit is one polypeptide chain
➢ non-covalent interaction via electrostatic attractions, hydrogen bonds, and
hydrophobic interactions
➢ types of subunits are:
i. monomer – protein with only one (1) polypeptide chain
ii. dimer – simplest, with two (2) identical subunits
iii. oligomer – multi-subunit protein, generic term for small number of subunits
1. homodimer – 2 identical copies of a polypeptide; 3–trimer, 4-
tetramer, so on.
2. Heterodimer – two non-identical subunits

➢ Multi-subunit rotational symmetries

i. Cyclic symmetry
▪ subunits are arranged around a single rotation axis
▪ 2 subunits – twofold rotation axis at 1800
▪ 3 subunits – threefold rotation axis at 1200
ii. Dihedral symmetry
▪ occurs when an n-fold rotation axis intersects a twofold rotation
axis at right angles

Different Rotation Symmetries of proteins’ multi-subunits (Voet, et.al., 2016)

 Protein Denaturation / Folding

Protein Denaturation / Folding

➢ Occurs in secondary to quaternary structures of proteins
➢ Folding is from its denatured state to native state
➢ Is the change or loss of protein structure and function due to disruption of proteins’ weak
bond through;
o Heating
o Chemical denaturants (i.e. urea, guanidinium chloride)
o pH changes
➢ folding of proteins is a cooperative process, following a progressive stabilization pathway
of intermediates to form a unique conformation in the native form

Sample of Protein folding pathway (top: Berg, et. al., 2015; bottom: Miesfeld and McEvoy, 2017 ).

➢ folding is a rapid process

Folded to unfolded state transition (Most proteins show a sharp transition from the folded to
the unfolded form on treatment with increasing concentrations of denaturant) (left) Berg, et.al.,
2015, (right) Miesfeld and McEvoy, 2017
 ➢ 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, etc. Example is a cooked egg.

Protein in egg whites are denatured due to heat during cooking (Garrett and Grisham, 2017)

➢ 54% of the mass of an egg white is a protein ovalbumin. Opening an egg releases
transparent and viscous fluid; when cooked in turns into white, solid mass. The proteins
in the egg whites have been denatured and aggregated into white solid mass
➢ Folding protein thermodynamics is from a high-energy, high-entropy state to a low-
energy, low-entropy state, represented by a folding funnel

Energy-Entropy diagram in Protein Folding (Voet, et.al., 2016)

➢ The unfolded states are characterized by a high degree of conformational entropy and
relatively high free energy. As folding proceeds, the narrowing of the funnel represents
a decrease in the number of conformational species present. Small depressions along
the sides of the free-energy funnel represents semi-stable intermediates that can
briefly slow the folding process. At the bottom of the funnel, an ensemble of folding
intermediates has been reduced to a single native conformation (or one of a small set
of native conformations).
Diseases Cause by Protein Misfolding
➢ Proteins may fold incorrectly; they may interact with other proteins and form
aggregates due to the hydrophobic regions of proteins that are exposed during folding.
➢ Soluble and insoluble aggregates formed are toxic to cells.

Protein aggregation due to misfolding (Campbell and Farrell, 2015)

➢ If misfolded proteins are not refolded or removed, they disrupt other normal protein
functions and cause certain diseases.

Some diseases cause by protein misfolding and their mechanisms (Garrett and Grisham, 2017)
REFERENCES
Berg, J. M., et al. 2015, Biochemistry 8th edition. W. H. Freeman and Company
Campbell, M.K. and Farrell, S. O. 2015. Biochemistry 8th edition. Cengage Learning
Garret, R.H. and Grisham, C.M. 2017. Biochemistry 6th edition. Brooks/Cole Cengage
Learning
Miesfeld, R. L. and McEvoy, M. M., 2017. Biochemistry. W. W. Norton & Company, Inc.
Moran, L.A. et al. 2012. Principles of Biochemistry 5th Edition. Pearson Education Inc.
Nelson, D. L. and Cox, M. M., 2004 Lehninger Principles of Biochemistry 4th edition. W. H.
Freeman and Company
Nelson, D. L. and Cox, M. M., 2017 Lehninger Principles of Biochemistry 7th edition. W. H.
Freeman and Company
Voet, D., et. al. 2016. Fundamentals of Biochemistry: Life at the Molecular Level. John-Wiley
& Sons, Inc.
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