http://teachline.ls.huji.ac.il/~92984/

From Transgene to Protein, Station

IVb

T. Bruck, Dr. E.R. Bennett, E. Podoly

Introduction

Methods• Experimental

•

Computational

• Interactome• Driving

Forces• Energy

Proteins

Proteins rarely function in isolation. Protein-Protein interactions

(PPIs) are an integral part of cellular processes and functions.

Understanding and characterization

of data coming from proteomic

experiments is one of the major

challenges of the post-genomic era:

This is the time of the InteractomeInteractome.Genomics > Proteomics > Interactomics

The Post-Genomic Era

Interactomics is a study of interaction

network, aimed at mapping the molecular

interactions in cells using computational

and experimental methods.

Interactome – The Protein’s Social Utility

proteins

The Driving Forces of Interactions

· Disulfide bonds

· Hydrophobic

· Hydrogen bonds

· Electrostatic

I. Covalent Interactions

II. Non-Covalent Interactions

Covalent bonds result from sharing pairs of electrons between atoms.

Disulfide bonds are formed

between the thiol groups of cysteine

residues.

The Driving Forces of Interactions

Non-covalent bonds involve dispersed variations of electromagnetic interactions:

Hydrophobic interactions are formed between non-polar

atoms.

Hydrogen bonds are formed between an electronegative atom

and a hydrogen atom bonded to another electronegative atom.

Electrostatic interactions are formed between charged atoms.

Bond Strength (kcal/mole)

Hydrophobic interactions 1-2

H-bonds 3-7

Ionic bonds 10

Disulfide bridge 51

Energy of Interactions

Noncovalent interactions are weak as

compared with a covalent bond, but:

1. They work jointly to hold two

interacting surfaces together.

2. A surface topography enables

substantial areas of two interacting

surfaces to approach each other closely.

Methods to Study PPIs

Tracking PPIs and delineating their detailed information are both

interest for researchers in many fields. Multitude of methods detect

PPIs, each has its own strengths and weaknesses.

Specificity: most of the interactions

detected occur in reality.

Sensitivity: many interactions that

occur in reality are detected.

Two-Hybrid System (Y2H)

A genetic method based on transcription factor structure in yeast.

A functional transcription factor consists of two separable domains:

DNA binding domain (BD)

Activation domain (AD)

In Y2H system, these two

domains are separated and each is

fused to the protein of interest (X

and Y, respectively).

Physical interaction between BD-X and AD-Y reconstitutes the

transcription factor that activates the transcription of reporter gene.

Monitoring interactions of two proteins, one of them is labeled with

a donor and the other with an acceptor, by following the energy

transfer between them.

Fluorescence Resonance Energy Transfer*

* Termed also: "Förster Resonance Energy Transfer“, after the German scientist Theodor Förster

Protein A is labeled with CFP

while protein B is labeled with

YFP.

If the A and B are adjacent to each

other, an energy transfer happens.

Co - ImmunoPrecipitation

An antibody specific to protein X is added to a cell lysate.

Identification of proteins in the pellet can be determined by western

blot/sequencing a purified protein band/antibody against protein Y.

The antibody-protein complex is pelleted with protein-G sepharose.

Unbound protein are washed.

Size Exclusion Chromatography*

* Termed also: Gel Filtration Chromatography

Porous beads

separate different

sizes of

molecules.Small proteins

enter the pores

and have a

longer path and

longer transist

time than bigger

proteins.

Size Exclusion Chromatography

Protein Complexes will

have even shorter path and

transist time compared to

the its protein components.

Affinity Chromatography

A tagged protein of interest

binds to beads coated with

antibodies against its tag.

A cell lysate is passed

through the affinity

column.

Beads coated with

antibodies that

bind their antigen.

Affinity Chromatography

Binding partners are not eluted.

Tag can be broken by an

enzyme, following by proteins’

identification as before.

SPR reflectivity measures the changes in the local index of

refraction upon adsorption of the target molecule to the metal

surface.

Surface Plasmon Resonance*

* Termed also: Dual Polarisation Interferometry

Comparison of Methods

Y2H

FRET

Co-IP

Size Ch.

Affinity Ch.

Fluorescence

SPR

Crosslinking

Sensitivity Specificity StrengthsTag/Ig

High Low

Immobilization dependent

C/E/P

+/-/-

+/+/+

+/+/+

+/+/+

-/-/-

+/-

+/+/+

-/+

-/-

+/+/+ +/-Kinetics

Equilibrium

Simplicity

Preparative

Screening+/+

High

High

High

Med.

Med.

Weaknesses

False pos.

Apparatus

High

High

High

Kinetics Equilibrium

Kinetics Equilibrium

Crosslinker dependent

Med.

Med. High

Screening In vivo

High

T.C Apparatus

Time Consuming

Time Consuming

Time Consuming

Resource Comments

BIND Biomolecular Interaction Network Database at the University of Toronto, Canada.

CYGD PPI section of the Comprehensive Yeast Genome Database.

DIP Database of Interacting Proteins at UCLA. No species restriction.

GRID General Repository for Interaction Datasets. Mount Sinai Hospital, Toronto, Canada

HPRD The Human Protein Reference Database. Institute of Bioinformatics, Bangalore, India & Johns Hopkins University, Baltimore, MD, USA.

HPID Human Protein Interaction Database. Department of computer Science and Information Engineering Inha University, Inchon, Korea

iHOP iHOP (Information Hyperlinked over Proteins). Protein association network built by literature mining

IntAct Protein interaction database at EBI. No species restriction.

InterDom Database of putative interacting protein domains. Institute for InfoComm Research, Singapore.

JCB PPI site at the Jena Centre for Bioinformatics, Germany

MetaCore Commercial software suite and database. Manually curated human PPIs

MINT Molecular INTeraction database at the Centro di Bioinformatica Moleculare, Universita di Roma, Italy.

MRC PPI links

Commented list of links to PPI databases and resources maintained at the MRC Rosalind Franklin Centre, Cambridge, UK

OPHID The Online Predicted Human Interaction Database. Ontario Cancer Institute and University of Toronto, Canada.

PDZbase Database of PDZ mediated protein-protein interactions.

Predictome Predicted functional associations and interactions. Boston University.

PathCalling Proteomics and PPI tool/database. CuraGen Corporation.

PIM Hybrigenics PPI data and tool, H. pylori. Free academic license available

RIKEN Experimental and literature PPIs in mouse.

STRING Protein networks based on experimental data and predictions at EMBL.

YPD "BioKnowledge Library" at Incyte Corporation.

Bioinformatic Approaches

http://dip.doe-mbi.ucla.edu/

Bioinformatic Approaches

http://160.80.34.4/mint/Welcome.do

Bioinformatic Approaches

http://mips.gsf.de/genre/proj/mpact/yeast/query/interaction

Bioinformatic Approaches

http://www.thebiogrid.org/

Bioinformatic Approaches

http://www.himap.org/main/main.jsp

Bioinformatic Approaches

http://www.ihop-net.org/UniPub/iHOP/

Quanternary Organization: A Case Study of PPIs

1º 2º 3º 4º

The workshop focuses not in the proteins themselves but in their

homo-oligomerization state as a tool to study the field of PPI and a

new methodology to approach this field.

15-Lipoxygenase

A 94-kDa protein with iron binding site. Three alternatively spliced transcript variants encoding two distinct isoforms have been described.

Activity: Arachidonate + O2 → 15S-hydroperoxyeicosatetraenoic acid

Function: Arachidonic acid metabolism & linoleic acid metabolism.

Interactions: Unknown.

Oligomerization: Monomer.

Beta-Enolase

A 47-kDa protein with magnesium binding site (required for catalysis and dimerization). Three isozyme subunits (alpha, beta, gamma) are known.

Activity: 2-phospho-D-glycerate → 2-phosphoenolpyruvate + H2O

Oligomerization: Homodimers or heterodimers.

Function: Glycolysis, Muscle development and regeneration.

Interactions: AChE.

Pyruvate Kinase Muscle Isozyme

A 57-kDa protein with four metal binding sites (magnesium and potassium). Three alternatively spliced transcript variants encoding two distinct isoforms have been reported and four mammalian isozymes: L (liver), R (red cells), M1 (muscle, heart and brain) and M2 (early fetal tissues).

Activity: ADP + 2-phosphoenolpyruvate → ATP + pyruvate

Oligomerization: Homotetramer; On subunit dissociation, a dimeric intermediate is formed.

Function: Glycolytic pathway.

Interactions: Thyroid hormone & Opa protein.

PK Oligomerization

The tetramer:dimer ratio of PK is not constant, and determines whether glucose carbons are converted to energy (tetrameric) or into synthetic processes (dimeric).

Oxygen starvation or highly accumulated glycolytic intermediates, such as fructose 1,6-bisphosphate or the amino acid serine, induce the reassociation of the dimeric form to the tetrameric form.

PEP

pyruvate

ADP

ATP

Ser/ fructose 1,6-P2