BS204: Exp.

Molecular and Cell Biology

Molecular Interactions in Biological Systems-Part I Molecular Interactions in Biological Systems-Part I
Prof. Surajit Bhattacharyya
Lecture 1
Basic Concepts of Molecular Interactions
Methods: Physical methods and Spectroscopic methods
Wh St d M l l I t ti ? Why Study Molecular Interactions?
DNA DNA
RNA
Lipids
PROTEINS MasterInteractors
LifeisSustainedbyInteractions
Proteins:DiversityinBinding
Enzyme-substrate
Antibody-Antigen DNA-Protein Interactions
RNA-Protein Interactions Membrane-Protein Interactions
Carbohydrate-protein
Interactions
Proteins:DiversityinBinding
MultiproteinComplexes
Transcriptioncomplex
Signalingcomplex
Transcription complex: information
processed local
Signaling complex: information g g p
processed distal
Proteins:DiversityinBinding
WhydoProteinsabletoInteractwith
LargeDiverseMolecules?
E l ti ? Evolution?
Proteinstructures:
Shapecomplementarities
Toolargetofit
Toosmalltofit
Goodfit
Proteins:DiversityinBinding
CharacteristicsofProteinBindingSurface
BindingSurfaceisExposed(beforebinding)
Bi di I d d C f i l Ch BindingInducedConformationalChanges
BindingSurfacehashotspot
BindingAffinityishighlyDiverse:VeryweaktoVerystrong
Bindingsarenoncovalentandreversible
Proteins:DiversityinBinding
CharacteristicsofProteinBindingSurface
What are the forces that stabilize Interactions?
Hydrogenbonds:mainlysidechainmediated
(polaraminoacids:Ser,Thr,Asn,Gln)
CH O H O=C C
Ionic Interactions (Glu, Asp, Lys, Arg, His)
CH
2
OHO=CC
NH
2
Ser
Asn
IonicInteractions(Glu,Asp,Lys,Arg,His)
CH
2
C
O
O
+NH
3
(CH
2
)
4

Asp
Lys
vanderWaalspacking:closecontactamongatoms(nonpolar
residues)
Asp
Lys
Molecular Interactions
How do we detect binding? How do we quantify binding? Howdowedetectbinding? Howdowequantifybinding?
+
20 KD 10 KD 30 KD 20KD 10KD 30KD
ProteinA ProteinB ComplexofAandB
Physical methods to detect binding: Gel (Native) electrophoresis Physicalmethodstodetectbinding:Gel(Native)electrophoresis,
Sizeexclusionchromatography,DynamicLightScattering(DLS)
Howdowedetectbinding?
Native(Nondenaturing)Gel
5 positive charge
+
+ +
+
+

+
+ +
+
+

3negativecharge
5positivecharge
2positivecharge
The mobility of a molecule in a gel is dependent on: molecule weight
20KD 10KD
30KD
Themobilityofamoleculeinagelisdependenton:moleculeweight
(MW)(size)and/orcharge
ComplexformationincreasesMWandchangecharge
SDSPAGEGel:ProteinsaretreatedwithSDS,separatedbysize,
Complexcannotbedetected,denatured
Native(Nondenaturing)Gel
A Case Study ACaseStudy
AllSamplesareloadedattop
Sametime
th d
_
cathode
p
Protein
A
P t i
Complex
A+B
+
Protein
B
anode
ForNativegelsproteinsamplesarepreparedusingcommonbuffers:phosphatebuffers,
Trisbufferetc.Gelisstainedbyabluedye(CoomassieBrilliantBlue)
Originalmixtureoflarge
andsmallproteinsinbuffer
Howdowedetectbinding?
SizeExclusionChromatography
Small protein
Large protein
Loadintocolumn
Proteins can be separated
Gel filtration
Porous beads
Mi i
Proteins can be separated
based on their sizes
Proteins mixture loaded
into gel filtration column: Gelfiltration
beadsinbuffer
Elution from column
Migration
into gel filtration column:
Contains porous beads
Large proteins come out of
Elutionfromcolumn
Large protein
small protein
g p
the column faster than
small proteins
In this example: large protein
t
e
i
n
c
e
n
t
r
a
t
i
o
n
In this example: large protein
does not interact with
small protein, since they
come out at different time
Time(min)
P
r
o
t
c
o
n
20min 40min
Size Exclusion Chromatography
Howdowedetectbinding?
The complex has larger
Si
SizeExclusionChromatography
Size
ProteinA ProteinB ComplexofA&B
Elute from the column
Earlier than two components
o
n
20 min 30 min
P
r
o
t
e
i
n
c
o
n
c
e
n
t
r
a
t
i
o
Method is well suited
to purify complex for
structural studies
20min 30min
10min
Pc
Howdowedetectbinding?
Spectroscopic Methods p p
UV-Vis spectroscopy
Fluorescence spectroscopy
NMR (Yr 3, Elective)
Electromagnetic
di ti
Molecules absorb electromagnetic
radiation
radiation
Molecules emit electromagnetic
radiation after absorption
Electromagnetic Radiation
E = hv = hc/ [ c= 3 x 10
8
m/s) [ )
v
=Frequency of light or electromagnetic radiation

= Wavelength of light

= Wavelength of light
h = Plancks constant
Absorption of EMR and Electronic Transition
Molecules will change energy: when it
absorbs or emits light
E3
E4
E5
2
nd
it d t t
E6
-1.5
-0.37
V
)
E3
E4
E5
E6
1 5
-0.37
E2
1
st
excited state
2
nd
excited state
-4.0
E
n
e
r
g
y

(
e
V
E2
E3
-4.0
-1.5
n
e
r
g
y

(
e
V
)
Ground state
E1
-14.0
E
Ground state
E1
-14.0
E
n
Absorption of EMR
Ground state
Emission of EMR
E2: 1st excited state, E3: 2ndexcited state
Absorption of EMR and Electronic Transition
How much energy to give to make a transition?? (Absorption)
hv = AE = E
high
- E
low
E5
E6
-0.37
E2
E3
E4
E5
1
st
excited state
2
nd
excited state
-4.0
-1.5
y

(
e
V
)
AE
2-1
= E
2
- E
1
= hv1
AE
5-1
= E
5
- E
1
= hv4
1
st
excited state
E
n
e
r
g
y
v4 >v1
High frequency light to move molecule
Ground state
E1
-14.0
From E1 to E5 in comparison to E1 to E2
Aromatic Amino Acids of Proteins and Nucleic Acid bases (A, T, G, C, U)
absorb UV light
Proteins and Bases in Nucleic Acids Absorb UV Light
g
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
UV-Vis and Fluorescence Experiments are done in Spectrophotometer
Cuvette contains samples Spectrophotometer:
UV light source g
A to D converter
UV lamp
Monochromator Sample photodetector
y
I
n
t
e
n
s
i
t
y
wavelength
How do we detect binding?
Cofactor-containing Proteins Absorb Light at Specific Wavelength
H H
Enzyme -Substrate Interactions
H H
Glyceraldehyde3Phosphate
dehydrogenase dehydrogenase
NAD+ (oxidized)
(Nicotinamide adenine
dinucleotide)
NADH (reduced)
(Nicotinamide adenine
dinucleotide)
[absorbat340nm] [noabsorptionat340nm]
EnzymeSubstrateInteractions
NAD+
NADH
Substrate
(glyceraldehyde
3Phosphate)
Enzyme(Glyceraldehyde3Phosphate
dehydrogenase)withNAD+
EnzymewithNADH
Substrateconcentration
increasesfrom
300 350 400 Wavelength (nm)
Bottomspectrumtotopspectrun
(0M,2M,3M,5Mand10M)
300 350 400 Wavelength(nm)
Enzymecatalysesconversionofsubstratetoproductwithreductionof
NADtoNADH,thatcanbemonitoredbytakingabsorbanceat340nm,
SinceNADHabsorbsat340nm,noabsorptionfromNADorthesubstrate
C f t t i i P t i Ab b Li ht f Diff t W l th Cofactor-containing Proteins Absorb Light of Different Wavelength
HemeProtein(Myoglobin,hemoglobin)
Fe
HemeGroup
HemeGrouphasdifferentabsorptionintheabsenceofproteinsandwhen
boundtoproteins
H ( l ) b ti
Cofactor-containing Proteins Absorb Light of Different Wavelength
3
c
e
Heme+Protein
Heme (alone) absorption
spectrum shows a peak 355 nm
Heme in complex with
2
A
b
s
o
r
b
a
n
c
OnlyHeme
Heme in complex with
Protein shows an absorption
spectrum with a peak 420 nm
0
1
This spectral change occurs
due to new bond formation of
Fe atom in Heme with proteins:
causing structural change
400 350 300 450 500
Wavelength
0
g g
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
A: Absorption, F: Fluorescence
i
t
y
I
n
t
e
n
s
(Fluorescence) > (Absorption)
Wavelength (nm)
270 290 310 330 350 370 390

max
(Fluorescence) >
max
(Absorption)

max
: wavelength at which intensity is maximum.
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
How much energy will be emitted from a transition?? (Emission)
Emission
E2
E itt d li ht i
Excited state
Emit light
Absorption
of light
Emission
E1
Emitted light is
detected as Fluorescence
Ground state Ground state
The emitted light is low in energy as compared to energy of the light
absorbed Some energy is lost with interactions with environments absorbed. Some energy is lost with interactions with environments
(solvents)
E= hv= h(c/)
(v: frequency c: speed of light = wavelength) (v: frequency, c: speed of light, = wavelength)
Fluorophores in Proteins
A i A id Ab i ( ) E i i ( ) Amino Acid--------Absorption (
max
)----Emission(
em
)
Phe---------------260------------ 282
Tyr---------------275------------- 304
Trp--------------280--------- ---- 353
Fluorophores in Proteins
In protein Trp contributes more for the fluorescence spectra.
Trp fluorescence is very sensitive to conformational change, ligand
binding association/aggregation.
Contribution from Tyr is quenched by peptide groups or
energy transfer with Trp.
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
Protein-Peptide interactions: Calmodulin/Kinase Derived Peptide interaction
Case Study
Ca
+2
Release in Muscle
Activates Calmodulin
Activate Myosin Light Chain Kinase (MLCK)
Phosphorylates Myosin Light Chain
y g ( )
Phosphorylated Myosin Hydrolyze ATP
Activation of Myosin Light Chain Kinase by Calmodulin
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
Protein Kinases are Multi-domain Proteins Containing Kinase domain,
Inhibitory Domain and Other Regulatory Domains
Regulatory Domain
Kinase domain
g y
Occupies the Active site of Kinase
Binds to Calmodulin
p
Inhibitory Domain
A Repressed State of Calmodulin dependent Kinase
(e.g. Myosin Light Chain Kinase, MLCK)
Only 20 Amino acid long peptide (called RS20) of Inhibitory domain Only 20 Amino acid long peptide (called RS20) of Inhibitory domain
was sufficient to bind to Calmodulin
FLUORESCENCE SEPCTROSCOPY I M l l I t ti FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
How do we record Trp fluorescence p
Spectrum?
Light at
290 nm
Sample in
cuvette cuvette
Collect signal g
from 310-425 nm
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions

max
of Trp of RS20 in
b f C l d li absence of Calmodulin:
355 nm
of Trp of RS20 in
max
of Trp of RS20 in
presence of Calmodulin:
323 nm
The fluorescence intensity The fluorescence intensity
of RS 20is high in presence
of calmodulin
Fluorescence spectra of RS20 peptide in the absence of calmodulin
(in H O purple line) and in the presence of Calmodulin (red line) (in H
2
O, purple line) and in the presence of Calmodulin (red line).
No Trp amino acid in Calmodulin
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
Trp fluorescence spectrum is sensitive to binding
Binding causes a change in the environment and hence change
in spectrum
t water
Exposed
Free peptide
Bound peptide with
Calmodulin, not exposed to water,
Free peptide
, p ,
Binding pocket of calmodulin is hydrophobic
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
Energy level diagram of Trp fluorescence in polar (or water)
and non-polar environment
E2
Excited state (water)
E it li ht
Emission
E2
Excited state (non-polar)
Emit light
Absorption
of light
Emission
Emit light
E1
E1
Ground state
The emitted light is low in energy in water as compared to energy of the
Emitted light in non-polar environment.
E1
Ground state
Emitted light in non polar environment.

max
in water >
max
in non-polar environment
Fluorescence intensity is lower in polar environment due Fluorescence intensity is lower in polar environment due
to loss in energy
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
Binding Surface of the Peptide (RS16) in Complex with Calmodulin
RS16: RKWQKTGHAVRAIGRLS
: RKFWKTGHAVRAIGRLS
: RKFQWTGHAVRAIGRLS
Several peptides were made with
T i diff t l ti
: RKFQWTGHAVRAIGRLS
Trp occupying different location
Fluorescence spectra were recorded Fluorescence spectra were recorded
with calmodulin
Insertion of Trp into Insertion of Trp into
Calmodulin
FLUORESCENCE SEPCTROSCOPY: In Molecular Interactions
345
8
15
335
340
1
2
5
8
9
11
14
15
Trp in these location
(1, 5, 8, 14, 15)
are exposed to water
325
330
2
4
6 7
9
10
11
12 16
Trp in these location (3, 6, 7, 10, 13)
315
320
0 2 4 6 8 10 12 14 16
3
10
13
p ( , , , , )
are buried into non-polar pocket of
calmodulin
Trp-position