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