Transcript Montse Fabrega`s presentation

Stepping into the world
of proteins
INTRODUCTION TO STRUCTURAL BIOLOGY
AND X-RAY CRYSTALLOGRAPHY
Montserrat Fàbrega Ferrer
Miquel Coll’s lab – Structural biology of protein & nucleic acid complexes and molecular machines
Outline
A. Structural biology
1. Definition
2. Proteins and its structure
3. Main techniques
B. X-ray crystallography
1. General scheme of work
2. From the gene to the protein
3. From the protein to the crystal
4. From the crystal to the structure
C. Biomedical applications: structure-based drug design
D. Practical session
A. Structural biology
Definition of structural biology
WHAT?
Structural biology is a branch of molecular biology, biochemistry and biophysics that studies:
-The molecular structure of biological macromolecules (proteins and nucleic acids)
-The acquisition of the structures
-Structure and function relationships
WHY?
Macromolecules are only able to carry out most of the their functions when they are coiled into specific
three-dimensional shapes.
Definition of structural biology
HOW?
1.
Experimental techniques
2.
Bioinformatic predictions
Proteins
Proteins are large biological molecules consisting of one or more chains of amino acids.
Proteins perform many different functions within living organisms:
-Metabolic reactions
-Replication of DNA
-Response to stimuli
-Transport of molecules
-Maintenance of cell’s shape
-Cell signaling
-Immune responses
-Cell adhesion
-Cell cycle
Protein structure: primary structure
Protein structure: secondary structure
RANDOM COIL
Unfolded polypeptide chain lacking any fixed three-dimensional structure.
Protein structure: tertiary structure
Myoglobin
Protein structure: quaternary structure
Hemoglobin
Basic vocabulary about protein structure
STRUCTURAL DOMAIN
Self-stabilizing element that folds independently.
STRUCTURAL MOTIF
Short segment of protein three-dimensional structure found in a large number of different proteins.
Basic vocabulary about protein structure
SUPERSECONDARY STRUCTURE
Specific combination of secondary structure elements.
PROTEIN FOLD
General protein architecture.
Techniques
Indirect methods  No atomic data
Proteolysis assays
Spectroscopic methods  Atomic data
Techniques
Spectroscopic methods
X-ray crystallography
3·1016 – 3·1019 Hz
Electrons
Scattering
Nuclear magnetic resonance (NMR)
6·104 – 1·106 Hz
Atomic nucleus
Absorbance and re-emission of electromagnetic radiation
B. X-ray crystallography
General scheme of work
Gene
Structure
Protein
Crystals
From the gene to the protein
Gene
HETEROLOGOUS
EXPRESSION
RECOMBINANT DNA
TECHNOLOGY
Protein
Express a protein codified by a certain gene in a host
organism which does not naturally have this gene
Laboratory methods to bring together
genetic material from multiple sources,
creating sequences that would not otherwise
be found in biological organisms
Escherichia coli
From the gene to the protein
Escherichia coli
-Gram-negative, rod-shaped bacterium
-Most widely studied prokaryotic organism
-Can grow easily and inexpensively in a laboratory setting
-Host organism for the majority of work with recombinant DNA
From the gene to the protein
Cloning the gene
Polymerase chain reaction (PCR)
CLONING = Making several copies!
From the gene to the protein
Polymerase chain reaction (PCR)
We need:
-DNA from the interest organism
-Thermo resistant DNA polymerase
-Magnesium (cofactor)
-Primers
-Deoxyribonucleotides (dNTPs)
-Thermociclator
http://www.youtube.com/watch?v=HMC7c2T8fVk
From the gene to the protein
What happens in one cycle of PCR reaction?
From the gene to the protein
Progression of the PCR reaction
From the gene to the protein
Cloning the gene
Expression vector: Plasmid or virus designed for protein expression in cells. The vector is
used to introduce a specific gene into a target cell and take advantage of the cell’s
mechanism for protein synthesis to produce the protein encoded by the gene.
Your gene: codifies for the interest protein
Promoter: drives expression in the host organism
Ori: allows replication of the vector
Antibiotic resistance gene: avoids contaminations and
the loss of the plasmid
From the gene to the protein
Transforming Escherichia coli
Transformation: Genetic alteration of a cell resulting from the direct uptake of exogenous
DNA from its surroundings.
From the gene to the protein
Producing the protein
Colony
Small preculture
Big culture
Protein production is induced with IPTG (isopropyl β-D-1-thiogalactopyranoside)
Antibiotic present in all the steps to avoid contaminations!
From the gene to the protein
Purifying the protein
Centrifuge at high speed to separate proteins from the other cellular
components (membranes and DNA)
Soluble protein
Chromatography purification steps to separate the interest protein from the
others
Pure protein
High concentration
Homogeneous and stable
Considerable amount (mg)
From the protein to the crystal
Gene
Structure
Protein
Crystals
From the protein to the crystal
Crystallization principles
INCREASE IN THE PROTEIN CONCENTRATION
Vapor diffusion
REDUCING THE PROTEIN SOLUBILITY
Salt type and concentration
Temperature
pH
Organic additives or polymeric precipitants
Starting protein concentration
From the protein to the crystal
Main crystallization techniques: vapor diffusion technique
Hanging drop
Sitting drop
From the protein to the crystal
How to find the correct condition?
96 well plates
Sitting drops (0.1 μL + 0.1 μL)
TRYING LOTS OF CONDITIONS!!
High-throughput screening
From the protein to the crystal
Reproduction of the conditions at larger scale and optimization of the crystals
24 well plates
Hanging or sitting drops (1 μL + 1 μL)
From the crystal to the structure
Gene
Structure
Protein
Crystals
From the crystal to the structure
Fishing and freezing the crystals
From the crystal to the structure
X-ray source
X-rays
Crystal
Diffraction pattern
Structure
Data processing
C. Biomedical application: structurebased drug design
Drug discovery
DRUG DISCOVERY: Process by which new candidate medications are discovered.
RATIONAL DRUG DESIGN: Process of finding new medications based on the
knowledge of a biological target.
Rational drug design
LIGAND-BASED DRUG DESIGN: The structural details of the target are not known.
Based on information about a know subset of ligands.
STRUCTURE-BASED DRUG DESIGN: The target structure is known. The main
technique used in this method is molecular docking.
PDB file with the
structure of the
target protein
X-ray crystallography
NMR
The first success: a peptide-based HIV protease
inhibitor (1990)
D. Practical session
Experiment 1. Production of protein
Small preculture
1. Follow the growth of the cells
2. Induce expression
Big culture
Experiment 1. Production of protein
3. Collect and break the cells
4. Analyze expression and solubility of the protein (SDS-PAGE)
Experiment 2. Crystallization techniques
Starting with a sample of pure protein and a known condition of crystallization
A. Hanging drops
B. Sitting drops
24 well plates
Thank you!
Miquel Coll’s lab
Structural biology of protein & nucleic acid complexes and molecular machines