gfp for exploring protein-protein interactions - nelson giovanny rincon silva

GFP FOR EXPLORING

PROTEIN-PROTEIN-INTERACTIONS

By: Giovanny Rincon-Silva1

2

CONTENT

Introduction

Protein–Protein Interactions

Types of protein–protein interactions

Methods to investigate protein-protein interactions

Green fluorescent Protein as a signal for protein-protein

interactions.

Detecting Protein-Protein Interactions with a Green

Fluorescent Protein Fragment Reassembly Trap.

Introduction3

PPIs refer to intentional physical contacts established between two or

more proteins as a result of biochemical events and/or electrostatic forces.

In fact, proteins are vital macromolecules, at both cellular and systemic levels, but

they rarely act alone.

Aberrant PPIs are the basis of multiple diseases, such as Creutzfeld-

Jacob, Alzheimer's disease, and cancer.

4

Introduction

Casado-Vela J, Matthiesen R, Sellés S, Naranjo, JR Protein-Protein Interactions: Gene Acronym Redundancies and Current Limitations

Precluding Automated Data Integration Proteomes 2013, 1(1), 3-24.

PPIs have been studied from different perspectives:

Biochemistry

Quantum Chemistry

Molecular Dynamics

Signal Transduction

This information enables the creation of large protein interaction networks

similar to metabolic or genetic/epigenetic networks.

5

Protein–protein interactions



6

Protein–protein interactions



PPIs have been studied from different perspectives:

Biochemistry

Quantum Chemistry

Molecular Dynamics

Signal Transduction

This information enables the creation of large protein interaction networks

similar to metabolic or genetic/epigenetic networks.

Cell metabolism

In many biosynthetic

processes enzymes interact with each

other to produce small compounds or

other macromolecules.

Muscle contraction

Myosin filaments act as molecular

motors and by binding

to actin enables filament sliding.

Examples of protein–protein interactions

8 Casado-Vela J, Matthiesen R, Sellés S, Naranjo, JR Protein-Protein Interactions: Gene Acronym Redundancies and Current Limitations


Signal transduction

The activity of the cell is regulated by extracellular signals.

Transport across membranes

A protein may be carrying another protein.

TYPES OF PROTEIN–PROTEIN

INTERACTIONS

9

Bacillus amyloliquefaciens

proteins in a complex

Homo-oligomers are macromolecular complexes constituted by only one type of protein

subunit.

Disruption of homo-oligomers in order to return to the initial individual monomers often

requires denaturation of the complex.

homo-oligomers complex

Several enzymes, carrier proteins and transcriptional regulatory factors carry out their

functions as homo-oligomers.

Homo-oligomers vs. hetero-oligomers

10 Joel Edt, Shoshana Wodak..Protein Modules and Protein-Protein Interaction 2002..16, 232-245

Distinct protein subunits interact in hetero-oligomers, which are essential to

control several cellular functions.

Homo-oligomers vs. hetero-oligomers

11

Hemoglobin Hb or HgbEdit by: Giovanny Rincon

Joel Edt, Shoshana Wodak..Protein Modules and Protein-Protein Interaction 2002..16, 232-245

S.I: These are usually the case of homo-

oligomers (e.g. cytochrome c).

T.I.: A protein may interact briefly and in a

reversible manner with other proteins in only

certain cellular contexts – cell type, cell cycle

stage. (biochemical cascades)

Stable interactions vs. transient interactions

12

Example, some G protein-coupled

Activation cycle of a G-protein (purple) by a G-protein-

coupled receptor (light blue) receiving a ligand (red).

Joel Edt, Shoshana Wodak..Protein Modules and Protein-Protein Interaction 2002..16, 232-245

C: Are those with the strongest association and are formed by disulphide

bonds or electron sharing.

Although being rare, these interactions are determinant in some post

translational modifications, as ubiquitination and SUMOylation

(Small Ubiquitin-like Modifier (or SUMO)).

Non-covalent bonds are usually established during transient interactions by the

combination of weaker bonds:

Hydrogen bonds

Ionic interactions

Van der Waals forces

Hydrophobic bonds

Covalent vs. non-covalent


Protein concentration, which in turn are affected by expression levels and

degradation rates.

Protein affinity for proteins or other binding ligands.

Presence of other proteins, nucleic acids, and ions.

Ligands concentrations (substrates, ions, etc.).

Electric fields around proteins.

Factors that regulate protein–protein

interactions


The molecular structures of many protein complexes have been unlocked by the

technique of X-ray crystallography.

The first structure to be solved by this method by Sir John Cowdery Kendrew.

Techniques to study the molecular structure of

protein complexes

15

Crystal structure of modified Gramicidin S

horizontally determined by X-ray

crystallography

Stephen W Michnick. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments.

Prorein Science, Cambridge University Press. 2011, 8: 1256 -1265

NMR also started to be applied with the aim of unravelling the molecular

structure of protein complexes.

Techniques to study the molecular structure of

protein complexes

16

NMR structure of cytochrome C

illustrating its dynamics in solution

Stephen W Michnick. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments.


Yeast two-hybrid screening

Investigating interactions of proteins within the yeast nucleus. (transfected with two

plasmids; Bait and Prey).

Tandem affinity purification (TAP):

Detects cell interactions in the real environment (eg in the cytosol of a mammalian cell).

Methods to investigate protein-protein

interactions

17 Stephen W Michnick. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments.


Coimmunoprecipitation:

Is considered the best assay

for detecting PPIs.

Endogenous proteins

Edit: by Giovanny Rincon

Quantitative immunoprecipitation combined with knock-out:

(QUICK is based on the co-immunoprecipitation, quantitative mass

spectrometry (SILAC) and RNA interference (RNA interference - RNAi).

Dual Polarization Interferometry (DPI):

Provides measurements of molecular size, density and mass, in real time

and with high resolution.

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

Based on the migration of the protein complexes in polyacrylamide gels

according to their molecular weight.

18 Stephen W Michnick. Exploring protein interactions by interaction-induced folding of proteins from complementary peptide fragments.



interactions

Green fluorescent Protein as a signal for

protein-protein interactions.

19

From the jelly fish Aequorea victoria has exceptional physical and

chemical properties:

Spontaneous fluorescence

High thermal stability

Resistance to detergents

Organic solvents and proteases


interactions

20 Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science,

Cambridge University Press. 1997, 6: 2344 &2349.

Use of S65T GFP as the basis of two methods for exploring PPIs

21

1.) Fluorescence gel retardation assay: Based on

the electrophoretic mobility of a protein-DNA

complex being less than that of either molecule

alone.

Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science,



interactions

2.) Fluorescence polarization assay. A complex between two molecules

rotates more slowly than do the free molecules.

22 Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science,



interactions

MATERIALS AND METHODS

23

Monitoring protein-protein interactions

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System characterized interaction of the S-peptide and S-protein fragments

of bovine pancreatic ribonuclease (RNase) A

S-peptide (residues 1-20) and S-protein (residues 21-124)

Structure of RNase A

S15 was used in this study

Specifically, the generated fusion proteins in

which S15 is fused to the N or C terminus of

S65T GFP.



25

The His6 tag and S65T mutation were introduced simultaneously into the cDNA

that codes for wild-type GFP by PCR mutagenesis using three primers:

P39

GGCATATGCACCACCACCACCACCACGGCGGTAGCAAAGGAGAAGAAC

for the His6 tag and an Nde I site

M5 CCATGGCCAACACTGGTCACCACTTTCACCTATGGTGTTCAATGCTT

for the S65T change

P36

GTGAATTCTTGTATAGTTCATCCATGCCA for an EcoRI site



His6-GFP(S65T)-S15 construction

His6-GFP(S65T)-S15 construction

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The resulting PCR fragment was digested with EcoR I and Nde I and inserted into an

EcoR I/Nde I site of PET-29 ª.

The crystallographic structure of restriction

Endonuclease EcoRI

The DNA fragment encoding SI5 was generated from PET-29a by PCR using P37:

GGAATTCCGGCGGCAAAGAAACCGCTGCTGCTAAA with an EcoR I site)

and P38 (TGGTCGACTTAGCTGTCCATGTGCTGG CGTTCGA with a Sal I site) and

inserted into EcoR I/Sal I site of the above plasmid to give pSH24.



S15-GFP(S65T)-His6 Construction

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The coding region of GFP(S65T) was amplified from pSH24:

P53 (TCAAGATCTTAGCAAAGGAGAAGAACTT with a Bgl I1 site)

P54 (GCCCTCGAGCTTGTATAGTTCATCCATGC with an Xho I site).

The PCR fragment was digested with Bgl I1 and Xho I and inserted into Bgl II/Xho I

site of PET-29 b to give pSH41.



Gel Retardation Assay

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Purified fusion proteins were quantified

Є= 39.2 mM-1 cm-1 at 490 nm of S65T GFPS-protein Є= 9.56 mM-1 cm-1 at 280 nm

S15-GFP(S65T)-His6 was incubated with varying

amounts of S-protein.

20´ the mixtures were loaded onto a native

polyacrylamide gel, and was subjected to

electrophoresis at 4 °C at 10 V/cm.

After the gel was scanned by a Fluorimager SI

System (490 nm for excitation and 2515 nm for

emission).

The fluorescence intensities of bound and free

S15- GFP(S65T)-His6 were quantified by using

the program Image QuaNT 4.1.

Values of R (= fluorescence intensity of bound S15-GFP(S65T)-His6/ total fluorescence

intensity) were determined from the fluorescence intensities and Kd was determined:

)6

)65(15(1

totalHisTSGFTSRtotalproteinS

RR

dK

Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science, Cambridge University Press.

1997, 6: 2344 &2349.

Polarization Assay

29

Measured with a Beacon Fluorescence Polarization System

Purified S15-GFP(S65T)-His6 was incubated with various concentrations of S-protein

in Tris-HCI buffer, pH 7.5, 8.0, or 8.5, containing NaCl (0 or 0.10 M).

Polarization measurements were made at each

S-protein concentration.

Values of Kd were determined by using the

program DeltaGraph 4.0 to fit the data with:

In Eq. 2, P is the measured polarization:

(ΔP =Pmax- Pmin). F is the concentration of free

S-protein. The fraction of bound S-protein (ƒB)

was obtained from the eq: (3)

)2(min

PF

dK

PFP

)3(min

P

PP

Bf

Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science, Cambridge University

Press. 1997, 6: 2344 &2349.

Results

30

Purification and detection of S65T GFP Fusion

Proteins

31

DNA encoding SI5 and six histidine residue (His6) was added to 5' and 3' ends

of the cDNA encoding S65T GFP.

The two resulting proteins, His6-GFP-(S65T)-S15 and SI5-GFP-(S65T)-His6,

were produced in Escherichia coli strain BL21(DE3)

Escherichia coli strain BL21(DE3)


Press. 1997, 6: 2344 &2349.

Purification and detection of S65T GFP fusion

proteins

32

Purified by affinity chromatography using a Ni2+ -NTA column:

SDS-PAGE analysis of purified GFP fusion proteins.


Press. 1997, 6: 2344 &2349.

Purification and detection of S65T GFP fusion

proteins

33

Zymogram electorphoresis analysis of purified GFP fusion proteins.

Lane 1. Sl5-GFP(S65T)-His6. Lane 2. His6-GFP(S65T)-S15.


Press. 1997, 6: 2344 &2349.

Fluorescence gel retardation assay

34

The slower migrating isoform of His6-GFP(S65T)-

S15 was shifted upon binding to S-protein during

native PAGE, indicating that only this species has

an accessible S15.


Press. 1997, 6: 2344 &2349.

Fluorescence gel retardation assay

35

Fluorescence intensities of bound and free S15-GFP (S65T)-His6 were

quantified.

From the relative fluorescence intensities of the bound and free

S15-GFP-(S65 T)-His6, the binding ratio, at each concentration was obtained:

The dissociation constant (Kd) is (6 ± 3) * 10-8 M. (Average)


Press. 1997, 6: 2344 &2349.

36

Effect of pH.

The Kd values obtained were

1.4* 10-8 M, 1.1*10-8 M and

1.0*10-8 M.

Polarization assay


Press. 1997, 6: 2344 &2349.

In this assay, the formation of a complex is deduced from an increase in

fluorescence polarization.

37

Effect of salt concentration on complex formation. The value of Kd increased by

3.8-fold when NaCl was added to a final concentration of 0.10 M.

Polarization assay

At pH 8.0. Values of Kd in the presence

of 0 and 0.10 M NaCl were 1.1*10-8 M

and 4.2 *10-8 M, respectively.

Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science, Cambridge University Press. 1997, 6:

2344 &2349.

38

GENERAL IDEAS

In fluorescence gel retardation method the interaction between the two

proteins is evident by a decrease in the mobility of the fluorescent fusion

protein that results from complex formation.

The fluorescence polarization assay, provides a more accurate

assessment of the value of Kd.

Park, Sang-Hyun And Raines, Ronald T. Green fluorescent protein as a signal for protein-protein interactions. Prorein Science, Cambridge University Press. 1997, 6:

2344 &2349.

39

Detecting Protein-Protein Interactions with a Green

Fluorescent Protein Fragment Reassembly Trap

40

Fusing strongly interacting antiparallel

leucine zippers to the C- and N-termini of the

N-terminal and C-terminal fragments of GFP

Magliery, Thomas J. et al. Detecting Protein-Protein Interactions with a Green Fluorescent Protein Fragment Reassembly Trap: Scope and

Mechanism. CHEM. SOC. 2005, 127, 146-157

Leucine zippers

41

Compatible plasmids (e.g., pMRBAD-Z-CGFP and pET11a-Z-NGFP) were either

cotransformed or sequentially transformed into BL21(DE3) Escherichia coli by

electroporation.

Cells were screened on LB agar supplemented with anamycin, ampicillin and arabinose.

Cells were either grown for 3 days at room temperature 22 °C.

Fluorescence was observed under a hand-held long-wave UV lamp 365nm.

Screening



42

To improve the utility of the GFP-based fragment complementation

Assay; two compatible vectors that can be comaintained in E. coli

Compatible Vectors for Comaintenance of

GFP Fusions.



43

If the Z-NGFP or Z-CGFP plasmids are replaced with link-NGFP or link-CGFP

plasmids, respectively, then no cellular fluorescence is observed



Compatible Vectors for Comaintenance of

GFP Fusions

(Middle row) GFP reassembly occurs when the

interacting peptides are fused to the GFP

fragments, with the original fusion architecture

To confirm the visual phenotypes, it also harvested cells

from the screening agar plates and examined the

fluorescence in cleared lysates for equal numbers of cells

44

Parallel and antiparallel coiled coils

associate by the interaction of

hydrophobic residues at the peptide-

peptide interface.

As well as charge-charge interactions

between “edge” positions.

Antiparallel Leucine Zipper Libraries for

Determining Interaction Requirements

Magliery, Thomas J. et al. Detecting Protein-Protein Interactions with a Green Fluorescent Protein Fragment Reassembly Trap: Scope

and Mechanism. CHEM. SOC. 2005, 127, 146-157

Helical wheel diagram

A portion of the antiparallel leucine zipper from

T. thermophilus SerRS

45

General Applicability of the Screen:

Protein-Peptide Interactions



Quantified from lysates

Interaction of TPR1, TPR2A, and TPR2B on

NGFP with the Z peptide or C-terminal peptides

from Hsc70 or Hsp90 in CGFP

46

It have engineered a pair of compatible plasmids that greatly facilitate the use

of GFP fragment reassembly as a screen for protein-protein interactions in

bacteria.

The vectors allow facile subcloning of the genes of interest as fusions to the

GFP fragments, and their compatibility and independent transcriptional control

afford faithful reporting of interactions.

The screen can detect weak (KD = 1 mM) and probably transient interactions

due to irreversibility of the reassembly reaction,



GENERAL IDEAS

GFP FOR EXPLORING

PROTEIN-PROTEIN-INTERACTIONS

By: Giovanny Rincon-Silva48

gfp for exploring protein-protein interactions - nelson giovanny rincon silva

Science

membranesa protein

current limitations

gene acronym redundancies

current knowledge

matthiesen r

geneticepigenetic networks

introductioncasadovela

biochemical cascades