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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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Proteins:DiversityinBinding
MultiproteinComplexes
Transcriptioncomplex
Signalingcomplex
Transcription complex: informationprocessed local
Signaling complex: information
processed distal
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Proteins:DiversityinBinding
WhydoProteinsabletoInteractwith
LargeDiverseMolecules?
vo u on
Proteinstructures:
Shapecomplementarities
Toolarge
to
fit
Toosmalltofit
Goodfit
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Proteins:DiversityinBinding
CharacteristicsofProteinBindingSurface
BindingSurfaceisExposed(beforebinding)
B n ngIn uce Con ormat ona C anges
BindingSurface
has
hot
spot
BindingAffinityishighlyDiverse:VeryweaktoVerystrong
Bindingsarenoncovalentandreversible
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Proteins:DiversityinBinding
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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FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
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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FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
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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FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
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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FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
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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