molecular interactions 1 lecture 10.pdf

7/27/2019 molecular interactions 1 lecture 10.pdf

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BS204: Exp. Molecular and Cell Biology

-

Prof. Surajit Bhattacharyya


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Lecture 1

Basic Concepts of Molecular Interactions

Methods: Physical methods and Spectroscopic methods


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y u y o ecu ar n erac ons

RNA

Lipids

PROTEINS MasterInteractors

LifeisSustainedbyInteractions


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Proteins:DiversityinBinding

Enzyme-substrate Antibody-Antigen DNA-Protein Interactions

RNA-Protein InteractionsMembrane-Protein InteractionsCarbohydrate-protein

Interactions


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MultiproteinComplexes

Transcriptioncomplex

Signalingcomplex

Transcription complex: informationprocessed local

Signaling complex: information

processed distal


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WhydoProteinsabletoInteractwith

LargeDiverseMolecules?

vo u on

Proteinstructures:

Shapecomplementarities

Toolarge

to

fit

Toosmalltofit

Goodfit


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CharacteristicsofProteinBindingSurface

BindingSurfaceisExposed(beforebinding)

B n ngIn uce Con ormat ona C anges

BindingSurface

has

hot

spot

BindingAffinityishighlyDiverse:VeryweaktoVerystrong

Bindingsarenoncovalentandreversible


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CharacteristicsofProteinBindingSurface

What are the forces that stabil ize Interactions?

Hydrogenbonds:mainlysidechainmediated

(polaramino

acids:

Ser,

Thr,

Asn,

Gln)

Ionic Interactions Glu As L s Ar His

2

NH2Ser

Asn

CH2CO

O+NH3 (CH2)4

L s

vanderWaalspacking:closecontactamongatoms(nonpolar

residues)


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Molecular Interactions

+

ProteinA ProteinB ComplexofAandB

,

Sizeexclusionchromatography,DynamicLightScattering(DLS)


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Howdowedetectbinding?

Native(Nondenaturing)Gel

+++

++

+++

++

3negativecharge

2positivecharge

20KD 10KD 30KD

(MW)(size)and/orcharge

ComplexformationincreasesMWandchangecharge

SDSPAGEGel:ProteinsaretreatedwithSDS,separatedbysize,

Complexcannotbedetected,denatured


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Native(Nondenaturing)Gel

AllSamplesareloadedattop

Sametime

_ca o e

p

Protein

A

ComplexA+B

+

rote n

B

anode

ForNativegelsproteinsamplesarepreparedusingcommonbuffers:phosphatebuffers,

Trisbufferetc.Gelisstainedbyabluedye(CoomassieBrilliantBlue)


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Originalmixtureoflarge

andsmallproteinsinbufferHowdowedetectbinding?

SizeExclusionChromatographySmall protein

Large protein

Loadintocolumn

Porous beads

based on their sizes

Proteins mixture loaded

beadsinbufferM grat on

Contains porous beads

Lar e roteins come out of

Large proteinsmall protein

the column faster than

small proteins

ein

centration

does not interact wi th

small protein, since they

come out at different time

Time(min)

Pro

con

20min 40min


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Howdowedetectbinding?

S ectrosco ic Methods

UV-Vis spectroscopy

Fluorescence spectroscopy

NMR (Yr 3, Elective)

ElectromagneticMolecules absorb electromagnetic

radiationra a on

Molecules emit electromagnetic

radiation after absorpt ion


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Electromagnetic Radiation

E = h = hc/ c= 3 x 108 m/s=Frequency of light or electromagnetic radiation h = Plancks constant


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Absorption of EMR and Electronic Transition

Molecules will change energy: when it

absorbs or emits l ight

E3E4E5E6

-1.5

-0.37

)E4E5E6-0.37

E21st excited state

exc e s a e

-4.0

ne

rgy(eV

E2-4.0

- .

ergy(eV)

Ground stateE1-14.0 E1-14.0

E

Absorption of EMREmission of EMR

E2: 1st excited state, E3: 2ndexcited state


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Absorption of EMR and Electronic Transition

How much energy to give to make a transition?? (Absorption)

h = E = Ehigh- ElowE6-0.37

E2

E3E4

st

2ndexcited state

-4.0

-1.5

(e

V)

E2-1 = E2- E1 = hE5-1 = E5- E1 = h

Energ

High frequency light to move molecule

Ground stateE1-14.0 From E1 to E5 in comparison to E1 to E2


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Aromatic Amino Acids of Proteins and Nucleic Acid bases (A, T, G, C, U)

absorb UV l i ht

Proteins and Bases in Nucleic Acids Absorb UV Light

Absorb UV lights: Trp; 280 nm, Tyr; 278 nm, Phe; 270 nm

Nucleic Acid bases (A, T, G, C, U) absorb UV light at 260 nm

Trp absorbs more UV light than Tyr and Phe


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UV-Vis and Fluorescence Experiments are done in Spectrophotometer

Cuvette contains samplesSpectrophotometer:

UV l i ht source

A to D conver ter

UV lamp Monochromator Sample photodetector

Intens

it

wavelength


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Enzyme SubstrateInteractions

NAD+ NADH

Substrate(glyceraldehyde

3Phosphate)

Enzyme (Glyceraldehyde3Phosphate

dehydrogenase)withNAD+

EnzymewithNADH

Substrateconcentration

increasesfrom

Bottomspectrumtotopspectrun

(0M,2M,3M,5Mand10M)

Enzymecatalysesconversionofsubstratetoproductwithreductionof

NADtoNADH,thatcanbemonitoredbytakingabsorbanceat340nm,

SinceNADHabsorbsat340nm,noabsorptionfromNADorthesubstrate


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o ac or-con a n ng ro e ns sor g o eren ave eng

HemeProtein (Myoglobin,hemoglobin)

Fe

HemeGroup

HemeGrouphasdifferentabsorptionintheabsenceofproteinsandwhen

boundtoproteins


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FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions

A: Absorption, F: Fluorescence

ity

Intens

Wavelength (nm)

270 290 310 330 350 370 390

max

max

max: wavelength at which intensity is maximum.


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How much energy will be emitted from a transit ion?? (Emission)

E2 Excited state

Emit lightAbsorptionof light

E1

m e g s

detected as Fluorescence

The emitted light is low in energy as compared to energy of the light

.

(solvents)

E= hhc , ,


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Fluorophores in Proteins

m no c -------- sorpt on max ---- m ss on emPhe---------------260------------ 282

Tyr---------------275------------- 304

Trp--------------280--------- ---- 353


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Fluorophores in Proteins

In protein Trp contributes more for the fluorescence spectra.

Trp f luorescence is very sensit ive to conformational change, ligand

binding association/aggregation.

Contribution from Tyr is quenched by peptide groups or

energy transfer with Trp.


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Protein-Peptide interactions: Calmodulin/Kinase Derived Peptide interaction

Case Study

Ca+2

Release in Muscle

Activates Calmodulin

Activate M osin Li ht Chain Kinase MLCK

Phosphorylates Myosin Light Chain

Phosphorylated Myosin Hydrolyze ATP

Activation of Myosin Light Chain Kinase by Calmodulin


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Protein Kinases are Multi-domain Proteins Containing Kinase domain,

Inhibitory Domain and Other Regulatory Domains

Re ulator Domain

Kinase domain

Occupies the Active site of Kinase

Binds to Calmodul in

Inhibitory Domain

A Repressed State of Calmodulin dependent Kinase(e.g. Myosin Light Chain Kinase, MLCK)

was sufficient to bind to Calmodulin


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: n o ecu ar n erac ons

How do we record Trp fluorescence

Spectrum?

Light at290 nm

Sample in

Collect si nal

from 310-425 nm


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max of Trp of RS20 ina sence o a mo u n:

355 nm

max presence of Calmodulin:

323 nm

of RS 20is high in presence

of calmodulin

Fluorescence spectra of RS20 peptide in the absence of calmodulin

2 , .

No Trp amino acid in Calmodulin


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molecular interactions 1 lecture 10.pdf

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