Unit III

• Protein synthesis
• Secondary structure of the protein
• Structure and function
• Structural databases
• Protein visualizing tools
• Secondary structure prediction algorithms
Protein Synthesis
 DNA
Protein Synthesis
Transcription
▪ The production Prokaryotic mRNA
(synthesis) of Cell
Translation
polypeptide chains Ribosome

(proteins) Protein
▪ Two phases:
Transcription & Nuclear membrane

Translation Eukaryotic DNA

Cell
▪ mRNA must be Transcription
Pre-mRNA
processed before it
RNA Processing
leaves the nucleus of mRNA
eukaryotic cells Ribosome

Translation
Protein
.
Three Types of RNA
• Messenger RNA (mRNA) carries genetic
information to the ribosomes
(blueprint for the construction of a protein)

• Ribosomal RNA (rRNA), along with protein,

makes up the ribosomes
(construction site where the protein is made)

• Transfer RNA (tRNA) transfers amino acids to

the ribosomes where proteins are synthesized
(truck delivering the proper amino acid to the site at
the right time)
 Genes & Proteins
▪ Proteins are made of amino acids linked together by peptide bonds
▪ 20 different amino acids exist
▪ Amino acids chains are called polypeptides
▪ Segment of DNA that codes for the amino acid sequence in a protein are
called genes

Genetic Code:
▪ DNA contains a triplet code
▪ Every three bases on DNA stands for ONE amino acid
▪ Each three-letter unit on mRNA is called a codon
▪ Most amino acids have more than one codon!
▪ There are 20 amino acids with a possible 64 different triplets
▪ The code is nearly universal among living organisms
6
 Overview of Transcription
▪ During transcription in the
nucleus, a segment of DNA
unwinds and unzips, and the DNA
serves as a template for mRNA
formation
▪ RNA polymerase joins the RNA
nucleotides so that the codons in
mRNA are complementary to the
triplet code in DNA
▪ The transfer of information in the
nucleus from a DNA molecule to
an RNA molecule
▪ Only 1 DNA strand serves as the
template
▪ Starts at promoter DNA (TATA
box)
▪ Ends at terminator DNA (stop)
▪ When complete, pre-RNA
molecule is released
 RNA Polymerase
▪ Enzyme found in the nucleus
▪ Separates the two DNA strands by breaking
the hydrogen bonds between the bases
▪ Then moves along one of the DNA strands
and links RNA nucleotides together
DN
A
RNA
Polymerase

pre-mRNA
 Processing Pre-mRNA
• Also occurs in the nucleus
• Pre-mRNA made up of segments called introns & exons
• Exons code for proteins, while introns do NOT!
• Introns spliced out by splicesome-enzyme and exons re-join
• End product is a mature RNA molecule that leaves the nucleus to the cytoplasm

pre-RNA molecule
exon intron exon intron exon

intron intron

exon exon exon

splicesome splicesome

exon exon exon

Mature RNA molecule

 Messenger RNA (mRNA)
• Carries the information for a specific protein
• Made up of 500 to 1000 nucleotides long
• Sequence of 3 bases called codon
• AUG – methionine or start codon
• UAA, UAG, or UGA – stop codons

start
mR Acod
U G G G C U C C A U C G G C G C A U A A
NA on
codo codo codo codo codo codo codo
stop
prote methionine
n1 glycine
n2 serine
n3 isoleucine
n4 glycine
n5 alanine
n6 ncodon
7
in Primary structure of a protein
aa1 aa2 aa3 aa4 aa5 aa6

peptide bonds
 Transfer RNA (tRNA)
• Made up of 75 to 80 nucleotides amino acid
long attachment site

• Picks up the appropriate amino methionine

acid floating in the cytoplasm
• Transports amino acids to the
mRNA

• Have anticodons that are

complementary to mRNA codons

• Recognizes the appropriate codons

on the mRNA and bonds to them
with H-bonds

• Four ATP’s are required for each

amino acid added to the
polypeptide chain:Two to "charge"
the tRNA , one to carry the charged
tRNA to the ribosome and one to U A C
move the ribosome to the next anticodon
codon.
 Ribosomal RNA (rRNA)
• Made up of rRNA is 100 to 3000
nucleotides long
• Made inside the nucleus of a cell
• Associates with proteins to form
ribosomes

Ribosomes
Made of a large and small subunit
Composed of rRNA (40%) and proteins (60%)
Have two sites for tRNAP and A
 Ribosomes P= Peptide site
A= Amino acid site

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G
Small
subunit
 Translation
• Synthesis of proteins in the cytoplasm
• Involves the following:
1. mRNA (codons)
2. tRNA (anticodons)
3. ribosomes
4. amino acids

• Three steps:
1. initiation: start codon (AUG)
2. elongation: amino acids linked
3. termination: stop codon (UAG, UAA, or UGA).
 mRNA Codons Join the
Ribosome

Large
subunit
P A
Site Site

mRNA
A U G C U A C U U C G
Small subunit
 Initiation
aa2
aa1

2-tRNA
1-tRNA
G A U
anticodon U A C
hydrogen A U G C U A C U U C G A
bonds codon mRNA
 Elongation
peptide bond
aa3
aa1 aa2

3-tRNA

1-tRNA 2-tRNA G A A
anticodon U A C G A U
hydrogen A U G C U A C U U C G A
bonds codon mRNA
 aa1 peptide bond
aa3
aa2

1-tRNA

U A C 3-tRNA
(leaves)
2-tRNA G A A

G A U
A U G C U A C U U C G A
mRNA

Ribosomes move over one codon

 peptide bonds
aa1 aa4

aa2 aa3

4-tRNA

2-tRNA 3-tRNA G C U

G A U G A A
A U G C U A C U U C G A A C U
mRNA
 peptide bonds
aa1 aa4
aa2

aa3

2-tRNA
4-tRNA
G A U
(leaves) 3-tRNA G C U

G A A
A U G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

 peptide bonds aa5
aa1
aa2
aa4
aa3

5-tRNA

U G A
3-tRNA 4-tRNA

G A A G C U
G C U A C U U C G A A C U
mRNA
 aa1 peptide bonds aa5
aa2
aa3
aa4

5-tRNA

3-tRNA U G A
G A A 4-tRNA

G C U
G C U A C U U C G A A C U
mRNA

Ribosomes move over one codon

 aa5
aa4 aa199 Termination
aa3 primary aa200
structure
aa2 of a protein

aa1

200-tRNA
terminator
or stop
codon
A C U C A U G U U U A G
mRNA
 End Product –The Protein!
• The end products of protein synthesis is a
primary structure of a protein
• A sequence of amino acid bonded together by
peptide bonds

aa5
aa3 aa4
aa2 aa199

aa1 aa200
24
Classification of Proteins
Based on the chemical nature, structure, shape, and
solubility, proteins are classified as:
1.Simple proteins: They are composed of only amino acid
residue. On hydrolysis, these proteins yield only constituent
amino acids. It is further divided into:
Fibrous protein: Keratin, Elastin, Collagen
Globular protein: Albumin, Globulin, Glutelin, Histones
2.Conjugated proteins: They are combined with non-protein
moiety. Eg. Nucleoprotein, Phosphoprotein, Lipoprotein,
Metalloprotein, etc.
3.Derived proteins: They are derivatives or degraded
products of simple and conjugated proteins. They may be
Primary derived protein: Proteans, Metaproteins,
Coagulated proteins
Secondary derived proteins: Proteosesn or albunoses,
peptones, peptides.
1. Primary Structure
Structure of a protein
3. Tertiary Structure
• The primary structure of a protein consists of the • The tertiary structure of a protein refers to its
amino acid sequence along the polypeptide chain. overall three-dimensional conformation.
Amino acids are joined by peptide bonds. • The types of interactions between amino acid
2. Secondary Structure residues that produce the three-dimensional
• Alpha-helix: The α-helix is a right-handed coiled shape of a protein include hydrophobic
strand. The side-chain substituents of the amino interactions, electrostatic interactions, and
acid groups in an α-helix extend to the outside. hydrogen bonds, all of which are non-covalent
Hydrogen bonds form between the oxygen of the and Covalent disulfide bonds also occur.
C=O bond of a aminoacid to hydrogen of the N-H 4. Quaternary Structure
group of the fourth amino acids below it in the helix. • Quaternary structure refers to the interaction of
• Beta Sheet:The hydrogen bonding in a ß-sheet is one or more subunits to form a functional
between strands (inter-strand) The sheet protein, using the same forces that stabilize the
conformation consists of pairs of strands lying side- tertiary structure.It is the spatial arrangement of
by-side. The carbonyl oxygens in one strand subunits in a protein that consists of more than
hydrogen bond with the amino hydrogens of the one polypeptide chain.
adjacent strand.
 Classes of Protein Structure
The function of a protein depends heavily on its final structure. Tertiary
and quaternary proteins are both functional proteins with a 3D
structure. However, the type of structure can vary significantly between
different proteins.
There are two main classes of 3D protein structure: globular
proteins and fibrous proteins.

Globular Proteins
Globular proteins are usually round and ball-shaped. They usually
have metabolic functions, for example, they may
be enzymes or antibodies. Haemoglobin is an example of a globular
protein.

Fibrous Proteins
Fibrous proteins are long and narrow and usually have a structural
function. Examples of fibrous proteins include collagen (found in
bones, muscle, and skin) and keratin (the material that makes up hair,
nails, and feathers).
 Functional Classification of Proteins
• Enzymes (biochemical catalysts)-In living organisms, almost all reactions are catalyzed
by specific proteins called enzymes. They have a high catalytic power, increasing the
rate of the reaction in which they are involved at least by factor 106. Eg: Glucose
isomerase
• Transport proteins -Many small molecules, organic and inorganic, are transported in the
bloodstream and extracellular fluids, across the cell membranes, Eg: hemoglobin, that
carries oxygen from the alveolar blood vessels to tissue capillaries;
• Storage proteins-Examples are: ferritin, that stores iron intracellularly in a non-toxic
form
• Mechanical support-Proteins have a pivotal role in the stabilization of many structures.
Examples are α-keratins, collagen and elastin.
• Movement-Example-the contraction of the muscle fibers (of which myosin is the main
component);
• Nerve transmission.-An example is the receptor for acetylcholine at synapses.
• Hormones- regulatory functions Example is insulin,
• Protection against harmful agents.-The antibodies or immunoglobulins are glycoproteins
that recognize antigens expressed on the surface of viruses, bacteria and other infectious
agents.
• Storage of energy-Proteins in particular the amino acids that constitute them, act as
energy storage, second in size only to the adipose tissue, that in particular conditions,
such as prolonged fasting, may become essential for survival
 Secondary structure of a protein
• The most common type of secondary structure in proteins is the α-helix.
• Fingernails and toenails are also made up of alpha-helices
• Beta Sheets form the core of globular proteins
• The end of a polypeptide chain can either be the N-terminus or the C-terminus. The N-
terminus is the end that contains the free amino group, and the C-terminus is the end
that contains the free carboxyl group.
• If two beta-strands run in the same direction, then it is a parallel beta-pleated sheet, and
if they run in opposing directions, then it is an antiparallel beta-pleated sheet.
 Clinical Implications of
Secondary Structure of Proteins
• The secondary structure of a protein can be altered by
either a mutation in the primary sequence of amino acids
that make up the protein or by extreme conditions that
force the proteins to denature or lose their shape.
• Spongiform encephalopathy, and Amyloidosis are two
classes of disease involving changes in the secondary
structure of proteins.
• Both involve the misfolding of proteins into Beta sheets,
and the presence of these proteins leading to tissue
damage.
• Structural databases
• Protein visualizing tools
• Secondary structure prediction algorithms
 Structural Databases
• Important in solving real problems in
molecular biology
• Protein Databank - PDB Established in
1972 at Brookhaven National Laboratory
(BNL)
• Sole international repository of
macromolecular structure data
• Moved to Research Collaboratory for
Structural Bioinformatics
http://www.rcsb.org
NDB: Nucleic Acid Structure
Database
• The NDB contains information about
experimentally-determined nucleic acids
and complex assemblies.
• Use the NDB to perform searches based on
annotations relating to sequence, structure
and function, and to download, analyze, and
learn about nucleic acids.
• http://ndbserver.rutgers.edu/
 PDB- Protein Structure database
• A protein database contains the information about 3D structure of
proteins.
• The PDB files contain experimentally decided 3D structures of
biological macromolecules.
• The structural information of a protein can be determined by X–ray
crystallography or Nuclear Magnetic Resonance (NMR)
spectroscopy methods.
• PDB allows searching for information regarding the structure,
sequence, function, visualize , download and to assess molecules.
• The PDB files also contains information of data collected, molecule
name, primary and secondary structure, ligand, atomic coordinates,
crystallographic structure factors, NMR experimental data etc..
• The data are submitted by scientists from all over the world. PDB is
maintained by Worldwide Protein Data Bank. All data in PDB are
accessible to public
• Each entry in the PDB is provided with a unique
identification number called the PDB ID. It is a 4 letter
identification number which consist of both alphanumeric
characters.
• Without a proper tool, the PDB file will be read as a text
file that lists each atom and its numerical coordinates in 3-
D space.
• There are databases which contain data derived from PDB.
For example
• Structural Classification of Proteins (SCOP) that groups different
protein structures,
• HSSP (Homology-Derived Secondary Structure of Proteins) for 3D-
structure and 1D- sequence of the protein,
• CATH for protein structure classification according to their evolution
etc.
 Protein visualizing tools
Uses
• To view the structures that are encoded by
these atomic coordinate files (which have
the extension .pdb), and
• to be able to manipulate the images to view
the molecules from various perspectives,
requires a molecular graphics visualization
tool.
 Protein visualizing tools
Rasmol/RasTop-
• RasMol is a molecular graphics program intended for the
visualisation of proteins, nucleic acids and small
molecules.
• The program reads in a molecule coordinate file and
interactively displays the molecule on the screen in a
variety of colour schemes and molecule representations.
• Currently available representations include depth cued
wireframes, 'Dreiding' sticks, spacefilling (CPK) spheres,
ball and stick, solid and strand biomolecular ribbons, atom
labels and dot surfaces.
 Pymol
• PyMOL is a molecular graphics system
with an embedded Python interpreter
designed for real-time visualization and
rapid generation of high-quality molecular
graphics images and animations.
• PyMol is a powerful molecule visualization software with
the following main features:
• Able to produce high-quality graphics ready for
publications.
• Able to create movies.
• Able to measure bond distances and angles.
• Has an extensive help system.
• Structures can be sliced, diced, and reassembled on the fly
and written out to standard files.
• Both command line interface and graphical user interface
are provided.
• Python API is provided to access all functionalities.
 Online Visualizer
• A PDB CODE (e.g. 1B8G)
• Go to the Structure Home Page-
https://www.ncbi.nlm.nih.gov/sites/entrez?db=Structure
• Enter the PDB code in the search box and press the Go button.
• Click a structure image to access its record page
• Scroll to the molecular graphic section and click on the spin icon to
load an interactive view of the structure within the web page.
Alternatively, click on the launch icon to open the advanced (full
feature) version of iCn3D, NCBI's web-based 3D structure viewer, in a
separate window.
• One protein, one true structure, eight ways to
look at it using a molecular visualization tool
• Pink is for helix and yellow for sheet
• Ball and stick model - 1a show the main atoms in all of
the protein’s amino acids.
• Space filling model - In 1b you can see how much space
each atom actually occupies. This is closest to the protein's
actual shape.
• Backbone model - In 1c, all the details of the different
amino acids have been removed and all that remains is a
stick drawing showing where the protein's backbone goes.
• Ribbon (or cartoon) model - In 1d the individual amino
acid structures have again been removed, but the flattened
ribbon areas highlight places where the amino acids come
together to form spirals (helices) or sheets. This makes it
easier to visualize important secondary structures in the
protein.
 Secondary structure prediction
algorithm
• The secondary structure prediction methods
can be either
• ab initio based, which make use of single
sequence information only, or
• homology based, which make use of
multiple sequence alignment information.
• The ab initio methods, which belong to
early generation methods, predict secondary
structures based on statistical calculations of
the residues of a single query sequence.
• The homology-based methods do not rely
on statistics of residues of a single
sequence, but on common secondary
structural patterns conserved among
multiple similar sequences.
 Ab Initio–Based Methods

• It measures the relative propensity(Natural

tendency) of each amino acid belonging to a
certain secondary structure element.
• The propensity scores are derived from
known crystal structures.
• Examples of ab initio prediction are the
Chou–Fasman and Garnier, Osguthorpe,
Robson (GOR) algorithms
 The Chou–Fasman algorithm
• ( http://fasta.bioch.virginia.edu/fasta/chofas.htm )
• determines the propensity or intrinsic tendency of
each residue to be in the helix, strand, and β-turn
conformation using observed frequencies found in
protein crystal structures.
 Homology-Based Methods
• This type of method combines the ab initio
secondary structure prediction of individual
sequences and alignment information from
multiple similar sequences (>35% identity).
• The idea behind this approach is that close
protein homologs should adopt the same
secondary and tertiary structure.
• This homology based method has helped
improve the prediction accuracy by another
10% over the second-generation methods.
• Eg.,- PHD
 PHD – neural network algorithm
for secondary structure prediction
• PHD are neural network systems (a
sequence-to-structure level and a structure-
structure level)to predict secondary structure
(PHDsec)
• PHDsec focuses on predicting hydrogen
bonds. The use of the evolutionary
information held by a multiple sequence
alignment increases the prediction accuracy.
• PHD uses two levels of Neural Networks •
trained networks
 How good are the methods?
• Single sequence, single residue methods
Chou & Fasman -50%
• Single sequence, multiple residues methods
GOR IV -65%
• Multiple sequence methods
PHD 71%
THANKYOU