proteins by salman ul islam
TRANSCRIPT
DetectingProtein-Protein
interactions
Salman Ul Islam (MS)Cellular Bio Chemistry Lab
CONTENTS
Introduction
Types of protein-protein interactions
Methods of detection
INTRODUCTION
Importance for cell biology and biochemistry Localization and trafficking posttranslational modifications signaling networks Essential in viral replication Difficult to predict two main patterns: ■ domain-domain interactions ■domain-peptide interactions
CHARACTERISTICS OF PROTEINS
• Nitrogenous compounds, contain carbon, hydrogen, oxygen, nitrogen, and sulfur
• Basic building block is the amino acid• Serve as structural components of animals• Serve as control molecules (enzymes)• Serve as transport and messenger molecules
AMINO ACID
FORMATION OF A DIPEPTIDE
THE MECHANISM OF INTERACTION
Non-covalent so reversible Van del waals forces Hydrophobic interactions Electrostatic bonds Hydrogen bonds For strong couplings very accurate
force field potentials are needed
POLYPEPTIDE CHAIN STRUCTURE
WHY ARE PROTEIN-PROTEIN INTERACTIONS SO IMPORTANT?
The binding of one signaling protein to another can have a number of consequences:
• Such binding can serve to recruit a signaling protein to a location where it is activated and/or where it is needed to carry out its function.
• The binding of one protein to another can induce conformational changes that affect activity or accessibility of additional binding domains, permitting additional protein interactions.
IMPACT ON OTHER FIELDS
• Cancer Biology The study of protein-protein interactions has
provided important insights into the functions of many of the known oncogenes, tumor suppressors, and DNA repair proteins.
• Pharmacogenetics Pharmacogenetic research has expanded to include
the study of drug transporters, drug receptors, and drug targets.
THE TYPES OF PROTEIN INTERACTIONS
• Binary protein-protein interactions
• Scaffolding proteins
THE TYPES OF PROTEIN INTERACTIONS-ANOTHER CLASSIFICATION
• Metabolic and signaling (genetic)pathways
• Morphogenic pathways in which groups of proteins participate in the same cellular function during a developmental process
• Structural complexes and molecular machines in which numerous macromolecules are brought together
MORPHOGENIC PATHWAYS
HOW TO STUDY PROTEIN PROTEIN INTERACTION?
OVERVIEW OF TECHNIQUES
• Gel filtration• Far western blot• Affinity
chromatography• Co-
immunopercipitation
• Capillary electrophoresis
• Biosensor
• FRET microscopy• Confocal
microscopy• 2 hybrid assay• Protein microarry• Maspec• NMR• Co-crystallization
for crystallography
GEL FILTRATION CHROMATOGRAPHY
Also called ”Size exclusion”
Porous made up of cross-linked polymers
Small molecules are trapped by the beads
For self assembling proteins monomers come later
FAR WESTERN BLOT
Also called ”Blot overlay”
Fractionating proteins on SDS-PAGE
Blotting to nitocellulose or PVDF membrane
Overlaying with a solution of the protein of interest
Binding the added protein to an immobilized protein on the membrane
Detection with antibody against the overlaying protein
CO-IMMUNOPRECIPITATION
Protein A binds to antibodies
Sepharose beads coated with protein A
Specific antibody binds to the protein of interest
The complex is precipitated by binding to the beads via protein A
Proteins are released from beads by boiling
Western blot
AFFINITY CHROMATOGRAPHY
In the case of His- tagged proteins
The His-tagged protein binds to nickel or cobalt column
His-tagged protein and it’s associated protein are eluted from the column by adding imidazole
FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)
FRET CONT
Cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP) are spectral variants of GFP
Plasmid constructs to fuse the proteins of interest to CFP and YFP
Co-transfection of plasmids to the cells Fixation of the cells and view by confocal microscopy Disadvantage:False negative results: If the fluorophores are over 200Ǻ apart while the
proteins interact with each other, no signal will be observed
FRET USING CFP & YFP
YEAST TWO HYBRID ASSAY
Transcription factor, Gal4p, has DNA binding (BD)(aa1-147) and transcriptional activator(AD)(aa768-881) domains
Stimulates transcription at a promoter reconized by Gal4p (upstream activating sequence,UAS)
Lac Z reporter gene encodes beta-galactosidase which produces blue pigment when the colony is grown in a media containing X-Gal
Disadvantage: time consuming!
2 HYBRID SYSTEM
MAMALIAN TWO-HYBRID ASSAY
Is analogous to Y2H assay Plasmids: 1)Gal4pBD-fusion vector 2)VP16AD-fusion vector(viral activator) 3)luciferase reporter plasmid contaning multiple copies of Gal4p binding
sites(UAS)
Co-transfection: in the case of interaction, luciferase activity will be detected
Advantage: good for studying mammalian proteins: they may not fold correctly in yeast or they may require post-tranlational modifications for protein interaction
WHAT ARE BIOSENSORS?
• Transducer converts physical change(heat, change in charge, light absorbance, mass) into an electrical signal
CONFOCAL MICROSCOPY
A good technique to detect intracellular co-localization of proteins
Point scan laser system minimizes overlaps in image (perfect for imaging Co-localization of proteins)
CONFOCAL MICROSCOPY CONT.
Thanks