PROTEOMICS & PROTEIN ENGINEERING
UNIT_1
• Protein Introduction
Proteins are essential large, complex molecules made up of amino acids
that perform a wide range of functions in the body
Protein structure refers to the three-dimensional arrangement of atoms in a
protein molecule.
They are the building blocks of cells, tissues, and organs, and are crucial
for growth, repair, and overall bodily function.
Proteins act as enzymes, hormones, antibodies, and structural
components, playing vital roles in virtually every cellular process
• Protein Structure
It's crucial for a protein's function and is typically described in four levels:
primary, secondary, tertiary, and quaternary.
Primary Structure: This is the linear sequence of amino acids in a
polypeptide chain, held together by peptide bonds.
Secondary Structure: This involves local folding patterns of the
polypeptide chain, like alpha-helices and beta-sheets, stabilized by
hydrogen bonds between amino acids.
Tertiary Structure: This describes the overall three-dimensional shape of
a single polypeptide chain, including the folding of secondary structures
and interactions between side chains.
Quaternary Structure: This applies to proteins composed of multiple
polypeptide chains (subunits), and it describes the spatial arrangement of
these subunits and how they interact.
Proteins are built from 20 different amino acids, each with a unique side
chain that influences the protein's shape and function. The specific
sequence of amino acids (primary structure) determines how the protein
folds into its secondary, tertiary, and quaternary structures. These
structures are essential for a protein's biological activity, such as acting as
enzymes, structural components, or transport molecules.
PROTEOMICS
The word “proteome” represents the complete protein pool of an
organism encoded by the genome.
In broader term, Proteomics, is defined as the total protein content of a
cell or that of an organism.
Proteomics helps in understanding of alteration in protein expression
during different stages of life cycle or under stress condition. Likewise,
Proteomics helps in understanding the structure and function of different
proteins as well as protein-protein interactions of an organism.
A minor defect in either protein structure, its function or alternation in
expression pattern can be easily detected using proteomics studies. This
is important with regards to drug development and understanding various
biological processes, as proteins are the most favorable targets for
various drugs.
Classification of Proteomics:
Proteomics can be broadly classified into structural, functional, and
expression proteomics. Structural proteomics focuses on the 3D structure
of proteins, functional proteomics investigates protein functions and
interactions, while expression proteomics analyzes protein levels and
changes under different conditions.
1. Structural Proteomics:
• Focus:
Determining the three-dimensional structures of proteins and protein
complexes.
• Goal:
Understanding how protein structure relates to its function. This can be
achieved through techniques like X-ray crystallography and NMR
spectroscopy.
• Significance:
Helps in understanding protein function, identifying potential drug
targets, and understanding protein-protein interactions.
2. Functional Proteomics:
• Focus:
Studying protein functions, interactions, and mechanisms within a
biological system.
• Goal:
Understanding how proteins interact with each other and with other
biomolecules, and how these interactions contribute to cellular
processes.
• Significance:
Crucial for understanding cellular pathways, identifying protein targets
for drug development, and understanding disease mechanisms.
• Methods:
Affinity purification, mass spectrometry, yeast two-hybrid system, and
protein microarrays.
3. Expression Proteomics:
• Focus:
Studying the qualitative and quantitative expression levels of proteins
under different conditions.
• Goal:
Identifying proteins that are differentially expressed in various
conditions, such as healthy versus diseased tissues.
• Significance:
Helps in identifying potential biomarkers for diseases, understanding
disease progression, and developing diagnostic tools.
• Methods:
2D gel electrophoresis, mass spectrometry, and protein microarrays
Steps in Proteomic Analysis
The following steps are involved in analysis of proteome of an organism
1. Purification of proteins: This step involves extraction of protein
samples from whole cell, tissue or sub cellular organelles followed by
purification using density gradient centrifugation, chromatographic
techniques (exclusion, affinity etc.)
2. Separation of proteins: 2D gel electrophoresis is applied for
separation of proteins on the basis of their isoelectric points in one
dimension and molecular weight on the other. Spots are detected using
fluorescent dyes or radioactive probes.
3. Identification of proteins: The separated protein spots on gel are
excised and digested in gel by a protease (e.g. trypsin). The eluted
peptides are identified using mass spectrometry. Analysis of protein
molecules is usually carried out by MALDI-TOF (Matrix Assisted Laser
Desorption Ionization-Time of Flight) based peptide mass
fingerprinting.
Determined amino acid sequence is finally compared with available
database to validate the proteins.
Several online tools are available for proteomic analysis such as
Mascot, Aldente, Popitam, Quickmod, Peptide cutter etc.