Biomolecular interaction analysis (BIA) techniques

Biomolecular Interaction Analysis

(BIA) Techniques

By: Naghmeh Poorinmohammad

University of Tehran

Microbial Technology and Products (MTP)

Research Center

November 2014

• Introduction

• BIA techniques

1. Biochemistry & biophysics methods

2. Molecular Methods

3. Computer-aided techniques

4. Novel creative approaches

• Summary/Conclusion

• References

2

Outline

3

• Introduction

• BIA techniques






• References

Outline

Biomolecular Interaction Analysis (BIA)

• Almost all biological processes can be described as a sequence of

interaction events between macromolecules/small molecules.

• The study of such interactions (BIA) is key for:

1. Understanding of biology at the molecular level

2. Drug discovery

3. Development of diagnostic tests

• It is thus crucial to develop techniques that can:

1. Probe

2. characterize

3. Discriminate

4

• Introduction

• BIA techniques:

1. Biochemistry and biophysics methods





• References

Outline

5

1. Biochemistry and biophysics methods

Optical

Fluorescence

SPR

MST

ATR-FTIR

Raman

MS

NMR

Electrochemistry

Equilibrium dialysis

Chromatography

ITC

DSC

Electrophoresis

UV

Non-optical

CD Pull-down

6

OPTICAL METHODS

Fluorescence spectroscopy

• Fluorescence originates from electronic transitions in molecules with

conjugated bonds.

• When a fluorophore, which is excited by a photon, returns from an excited

singlet electronic state to the ground singlet electronic state, it may emit light

in the form of fluorescence.

• The observable fluorescence intensity, If , is a wavelength dependent quantity

and is related to the quantum yield and optical density quantitative

treatment of fluorescence binding data.

• In general, the optimal spectroscopic conditions should be selected to exploit

the properties of the excitation and emission scans of the complex and its

constituents.

Mocz, Gabor, and Justin A. Ross. "Fluorescence Techniques in Analysis of Protein–Ligand Interactions." Protein-Ligand

Interactions. Humana Press, 2013. 169-210.

8

Fluorescence Resonance Energy Transfer (FRET)

Song, Yang, et al. "Protein interaction affinity determination by quantitative FRET technology." Biotechnology and

bioengineering 109.11 (2012): 2875-2883.

From: Broussard et al. 2013; nature protocols

9

• The donor absorption and emission

spectra should have a minimal

overlap to reduce self-transfer.

• The absorption spectrum of the

acceptor must overlap the

fluorescence emission spectrum of

the donor.

• Donor and acceptor molecules must

be in close proximity (10–100 Å).


From: http://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html

10


• Intensity ≈ [sample]; when OD< 0.05

Song, Yang, et al. "Protein interaction affinity determination by quantitative FRET technology." Biotechnology and

bioengineering 109.11 (2012): 2875-2883.

11

• Binding free energy can be derived from: ΔG= RTlnKd

• In greater OD values the relation is non-linear, thus complicated for calculations.

Advantages Drawbacks

Allows the study in living cells High background noise

Short time of trial (<90s) Donor reabsorbs emission

Quantitative Natural fluorescence of cells

Sensitive Oxygen quenching effect

Needs low concentration of sampleExternal light source required : Here

comes BRET


12

• Absorption spectroscopy in the visible and ultraviolet spectral regions is a

powerful technique by which ligand binding equilibria can be studied.

Th

ree

dif

fere

nt

inte

rna

l

chro

mo

ph

ore

sin

Bio

mo

lecu

le

The peptide bond (absorbs weakly 220 nm)

Aromatic amino acids F, Y, W, H (230–300 nm)

Many biological molecules show strong absorbance in the visible region of the spectrum as a result of the presence of metal ions and prosthetic groups with extended π-electron systems, such as chlorophyll, carotinoid, flavin, heme.

Ultra Violet-visible (UV-Vis) spectroscopy

Nienhaus, Karin, and G. Ulrich Nienhaus. "Probing heme protein-ligand interactions by UV/visible absorption

spectroscopy." Protein-Ligand Interactions. Humana Press, 2005. 215-241.

13

These bands are sensitive to the surrounding polypeptide environment and

reflect structural changes, oxidation states, and the binding of ligands.

Ultra Violet-visible (UV-Vis) spectroscopy


Simple (more than with

fluorescence)Less sensitive than fluorescnece

Inexpensive Less specific than fluorescence

Quantitative Overlap bands

16

Circular Dichroism (CD) Spectroscopy

• CD is the difference in absorption of left

and right circularly polarized light.

• Proteins and DNA and many ligands are

chiral.

• Molecular interactions between chiral

and achiral compounds can give rise to

induced circular dichroism (ICD) of the

achiral counterpart.

• If it is chiral then its ICD is the

difference between its own CD spectrum

and the spectrum in the presence of the

protein.

Rodger, Alison, et al. "Circular dichroism spectroscopy for the study of protein-ligand interactions." Protein-Ligand

Interactions. Humana Press, 2005. 343-363.

17


Daviter, Tina, Nikola Chmel, and Alison Rodger. "Circular and linear dichroism spectroscopy for the study of protein–

ligand interactions." Protein-Ligand Interactions. Humana Press, 2013. 211-241.

18


Gives structural information Machines are more expensive than UV

SensitiveMeasures binding strength of the order

of μM

Relatively rapid (20 min) -

Quantitative -

No extensive sample preparation -


19

Nuclear Magnetic Resonance (NMR) Spectroscopy

• A physical phenomenon in which nuclei in a magnetic field absorb and re-

emit electromagnetic radiation.

• NMR detects ligand binding through changes in the resonant frequencies

(chemical shifts) of NMR-active nuclei.

Mittermaier, Anthony, and Erick Meneses. "Analyzing Protein–Ligand Interactions by Dynamic NMR

Spectroscopy." Protein-Ligand Interactions. Humana Press, 2013. 243-266.

20

• Larger molecules (>25 kDa), additional enrichment with 2H.

• NMR spectroscopy detects and reveals protein ligand interactions with a large

range of affinities (10 9–10-3 M).

• Protein samples need to be isotopically enriched (15N and/or 13C).

• Isotopically labeled protein over-express in bacteria grown in minimal media

containing 15NH4Cl and/or 13C glucose as the sole sources of nitrogen and/or

carbon.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Advantage Drawback

Very sensitive to weak interactionsNeeds concentrated isotopically lablled

sample 50μM- 2mM)

Reveals the portion of molecule

involved in interactionNot suitable for >100 KDa

Accurate kinetics even for short

lifetime bounds (< 1ms)Needs high purity sample

Assay in equilibrium solutionRequires ligand-receptor buffer

harmony

Quantitative (large range of affinities)Strong magnetic fields needed for high

quality expensive

- Long assay time

21

Mass Spectroscopy

• Using ESI-MS, it is possible to transfer weakly associated complexes from

solution into the gas phase inside the mass spectrometer source.

• ESI-MS not only provides a direct readout of binding stoichiometry but can

also be used to determine dissociation constants ranging from nM to mM.

• The number of ligands bound for a given protein–ligand system can be

determined directly from the spectrum based on the mass difference between

free protein and its ligated complexes.

• In addition to exploiting the ‘x axis’ of the mass spectrum (that is, the mass-

tocharge ratio, m/z), the ‘y axis’ of the mass spectrum (that is,

abundance/intensity) provides important information about affinity and

specificity.

- Pacholarz, Kamila J., et al. "Mass spectrometry based tools to investigate protein–ligand interactions for drug discovery." Chemical

Society Reviews41.11 (2012): 4335-4355.

- Hofstadler, Steven A., and Kristin A. Sannes-Lowery. "Applications of ESI-MS in drug discovery: interrogation of noncovalent

complexes." Nature Reviews Drug Discovery 5.7 (2006): 585-595.

22

Mass Spectroscopy

Pacholarz, Kamila J., et al. "Mass spectrometry based tools to investigate protein–ligand interactions for drug

discovery." Chemical Society Reviews41.11 (2012): 4335-4355.

23

Surface-enhanced laser desorption/ionization (SELDI)

From: Tang et al. 2004; Mass spectrometry reviews

• SELDI-TOF MS, combines two powerful techniques: chromatography and

mass spectrometry.

• Different SELDI plates are available: CM10 (ion-exchange), H50 (like C6

to C12 reverse phase)

24

Advantage Drawback

Binding stochiometry and kineticsDoes not discriminate between specific

and non-specific binding

Sensitive/specific lower sample

consumption

Does not provide information

regarding the binding site of the ligand

and structure changes

High throughput capability (nano-

ESI-MS)Instrumental appropriate tuning

Label free -

No stabilization needed -

Mass Spectroscopy

25

Fourier transform infrared (FTIR) spectroscopy

• Interaction of infrared light with the vibrational modes of the molecules being

interrogated.

• High IR absorptivity of water IR samples are very thin, the relative water .

26

Attenuated Total Reflectance (ATR)-FTIR

• ATR Molecule being studied may be in contact to water above, which is not

sensed by the IR beam.

• Its characteristics allow ligands to be detected only when they are bound to

receptors closely linked to the sensor surface.

Kazarian, Sergei G., and KL Andrew Chan. "ATR-FTIR spectroscopic imaging: recent advances and applications to

biological systems." Analyst 138.7 (2013): 1940-1951.

From:

27

ATR-FTIR spectroscopy

Advantage Drawback

Rapid assay (10ms) Needs concentrated sample (2-10mM)

label free Needs ATR correction software

low-cost Stabilization process (in some cases)

Low sample volume requirement (1/3

NMR)Quantitative determination

Suitable for large/membrane proteins Low Sensitivity

28

Surface Enhanced Raman Spectroscopy

• It can be dramatically enhanced (102–1014) by adsorption of molecules on a

surface of noble metal, transition metal, or semiconductor substrates (SERS).

Siddhanta, Soumik, and Chandrabhas Narayana. "Surface Enhanced Raman Spectroscopy of Proteins: Implications in

Drug Designing." Nanomaterials and Nanotechnology 2.1 (2012): 1-13.

From: Siddhanta et al. 2012; Nanomaterials and

Nanotechnology

From: www.semrock.com

29

Raman IR

Due to the scattering of light by the vibrating

molecules.

Result of absorption/reflectance of light by

vibrating molecules.

The vibration is Raman active if it causes a

change in polarisability.

The vibration is IR active if there is a

change in dipole moment during the

vibration.

Many distinguishable peaks with high

intensities

Few distinguishable peaks with weak

intensities

(even in ATR-FTIR)

Water can be used as a solvent.Water cannot be used due to its intense

absorption (not for ATR).

Sample preparation is not very elaborate

sample can be almost in any state.

Sample preparation is elaborate

Gaseous samples can rarely be used.

Cost of instrumentation is very high Comparatively inexpensive.

30

Surface Plasmon Resonance (SPR)

• SPR is the resonant oscillation of conduction electrons at the interface which

will affect with polirized light.

Stahelin, Robert V. "Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular

interactions." Molecular biology of the cell 24.7 (2013): 883-886.

From:

31

Advantage Drawback

Label freeTethering of molecules to surfaces may

affect the binding constants measured

Enables quantitative determinationAny artifactual RI change other than

from the interaction can also give

signal

Low sample volume requirementStabilization process (in some cases)

Real time assay

SensitiveCannot verify the stability of the

complex formed

Surface Plasmon Resonance (SPR)

32

Micro-scale thermophoresis (MST)

• It measures the motion of molecules along microscopic temperature

gradients and detects changes in their hydration shell, charge or size.

• An infrared-laser is used to generate precise microscopic temperature

gradients within thin glass capillaries that are filled with a sample in a

buffer or bioliquid of choice.

• Thermophoresis, is very sensitive to changes in size, charge, and

solvation shell of a molecule and thus suited for bioanalytics.

• The fluorescence of molecules is used to monitor the motion of

molecules along these temperature gradients. The fluorescence can be

either intrinsic (e.g. tryptophan) or of an attached dye or fluorescent

protein (e.g. GFP).

Jerabek-Willemsen, Moran, et al. "Molecular interaction studies using microscale thermophoresis." Assay and drug

development technologies 9.4 (2011): 342-353.

33


From: http://www.nanotemper-technologies.com/uploads/pics/MST-Techo-Artikel.jpg

34


From: Jerabek-Willemsen et al. 2011; Assay and drug development technologies.

35


36


37


38


Advantage Drawback

Sample concentration (pM/nM) and

small volume (< 4 µl)

Buffer condition must be absolutely

stable

Quantitative (K: pM/nM to mM range

and n)

Conformational changes induced by

IR-Laser heating may be problematic

measures interactions with essentially

no limitation on molecule size or

molecular weight.

-

Immobilization free -

Free in choosing buffer type-

39

NON-OPTICAL METHODS

Isothermal Titration Calorimetry (ITC)

• All chemical, physical and biologic processes are performed along with heat

exchange criteria.

• When a protein interacts with a ligand, heat is either released or absorbed.

• ITC relies only on the detection of a heat effect upon binding not relies on

the presence of chromophores or fluorophores.

• Can be used to measure the binding constant, the enthalpy of binding, and

the stoichiometry.

• Modern instruments, like the MicroCal and Calorimetry Sciences

Corporation ITCs, make it possible to measure heat effects as small as 0.4 μJ

(0.1 μcal) allowing the determination of binding constants, K’s, as large as

108 to 109 M–1.

Lewis, Edwin A., and Kenneth P. Murphy. "Isothermal titration calorimetry."Protein-Ligand Interactions. Humana

Press, 2005. 1-15.

41

• The linear parameters ΔH and n will be better determined than the nonlinear

parameter K.

• The best results will be obtained at 10/K <[M] <100/K, and [L] 20 to 50 ×

[M], subject to solubility and heat signal considerations.

• Titrant and titrate concentrations must be accurately known.

• (Nonintegral values for n are often the result of concentration errors. Errors

in titrate concentration contribute directly to a similar systematic error in n.

Errors in titrant concentration or titrant delivery contribute directly to errors

in K and ΔH.)


Lewis, Edwin A., and Kenneth P. Murphy. "Isothermal titration calorimetry."Protein-Ligand Interactions. Humana

Press, 2005. 1-15.

42


43


From: http://www.endocytosis.org/techniqs/ITC.htmlLewis, Edwin A., and Kenneth P. Murphy. "Isothermal titration calorimetry."Protein-Ligand Interactions. Humana

Press, 2005. 1-15.

44

Advantage Drawback

Label free Large sample volumes required

Enables quantitative determination (K

and n)High ligand concentrations

Can be done on solutions that are

either homogeneous or heterogenous

Presence of impurities or inactive

protein will have a direct impact on the

stoichiometry

Universal -


45

Differential Scanning Calorimetry (DSC)

• Measures heat capacity in a range of temperatures.

• If a ligand binds preferentially to the native state of the protein, the

temperature at which the protein-ligand complex denatures will be higher

compared to the temperature at which the free protein unfolds.

• Since the degree of stabilization or destabilization of the native protein

depends on the magnitude of the binding energy, comparison of the stability

of the complex with the stability of the ligand-free protein allows the binding

energy to be estimated.

• DSC thus provides a direct measure of whether ligand binding to a protein is

stabilizing or destabilizing, and so can complement studies of binding

equilibria obtained by isothermal titration calorimetry (ITC).

Chiu, Michael H., and Elmar J. Prenner. "Differential scanning calorimetry: an invaluable tool for a detailed

thermodynamic characterization of macromolecules and their interactions." Journal of Pharmacy And Bioallied

Sciences 3.1 (2011): 39.

46


http://lnbio.cnpem.br/spectroscopyandcalorimetry/files/2012/08/DSCnote1.pdf

47


Advantage Drawback

Label freeSensitivity depends on many

parameters

Quantitative (relatively)useful in characterizing very tight

binding interactions which equilibrate

very slowly (mins to hrs)

Gives information on the nature of

binding event-

48

Equilibrium Dialysis

• The molecular weight cut off (MWCO) is chosen such that it will retain the

receptor component.

• A known concentration and volume of ligand is placed into one of the

chambers. The ligand is small enough to pass freely through the membrane.

• A known concentration of receptor is then placed in the remaining chamber in

an equivalent volume to that placed in the first chamber.

• A complete binding curve is generated by measuring Y at different ligand

concentrations.

• The relationship between binding and ligand concentration is then used to

determine the number of binding sites, the ligand affinity, kd. Because this kind

of experimental data used to be analyzed with (Scatchard plots)

Hatakeyama, Tomomitsu. "Equilibrium Dialysis Using Chromophoric Sugar Derivatives." Lectins. Springer New York,

2014. 165-171.

49


www.nestgrp.com/pdf/Ap1/EqDialManual.pdf

50

Advantage Drawback

Truely label-free Not very rapid

Quantitative Large sample volumes



51

Affinity Capillary Electrophoresis (ACE)

• The technique uses the resolving power of CE to distinguish between free and bound

forms of a receptor as a function of the concentration of free ligand.

• ACE experiments are most commonly performed in fused silica capillaries by injecting a

receptor and neutral marker with increasing concentrations of ligand in the separation

buffer.

• By studying the mobility change of a certain molecule when it interacts with another

molecule of different mobility it is possible to determine the binding constant between

the two compounds.

• The binding of the ligand to the receptor produces a migration time shift in the effective

mobility due to a change in the charge:size ratio of the complex.

• Scatchard analysis of the effective mobilities measured as a function of ligand

concentration provides the binding affinity of the receptor–ligand complex.

- Dinges, Meredith M., Kemal Solakyildirim, and Cynthia K. Larive. "Affinity capillary electrophoresis for the determination

of binding affinities for low molecular weight heparins and antithrombin‐III." Electrophoresis 35.10 (2014): 1469-1477.

- Chen, Zhi, and Stephen G. Weber. "Determination of binding constants by affinity capillary electrophoresis, electrospray

ionization mass spectrometry and phase-distribution methods." TrAC Trends in Analytical Chemistry 27.9 (2008): 738-748.

52

From: Galievsky et al. 2014; Analytical chemistry

53

Affinity Capillary Electrophoresis (ACE)

Advantage Drawback

Truely label-freelittle information about the

experimental conditions

Quantitative (k)Does not give stoichiometry

information


Low sample and ligand consumption -

relatively short analysis time -

high and weak affinity interactions -

Can be performed in solution under

physiological buffer conditions-

Impure samples can be used -

54

Co-Immunopercipitation (Co-IP)

• It can only be applied if there is a suitable antibody against the target.

From: http://www.tankonyvtar.hu/

55

Pull-down assay

• Can be regarded to as

Affinity Chromatography.

From: Rigaut et al., 1999; nature

56

Electrochemical methods

• Typically in (bio-)electrochemistry, the reaction under investigation:

• Generate current (amperometric)

• Generate potential or charge accumulation (potentiometric)

• Alter the conductive properties of a medium (conductometric)

between electrodes

• Alter impedance

• NANO The higher surface-to-volume ratio of nano-objects makes

their electrical properties increasingly susceptible to external

influences.

Grieshaber, Dorothee, et al. "Electrochemical biosensors-Sensor principles and architectures." Sensors 8.3 (2008): 1400-

1458.

57

From: Grieshaber et al. 2008; Sensors

58

Electrochemical Impedance Spectroscopy (EIS)

• Allows analysis of

interfacial changes

originating from bio-

recognition events at

electrode surfaces.

• Formation of the complex

on a conductive or semi-

conductive surface alters

the capacitance and the

resistance at the surface

electrolyte interface.

Hu, Yaofang, Peng Zuo, and Bang-Ce Ye. "Label-free electrochemical impedance spectroscopy biosensor for direct detection

of cancer cells based on the interaction between carbohydrate and lectin." Biosensors and Bioelectronics 43 (2013): 79-83.

59

Chromatography

• A novel technique: kinetic size-exclusion chromatography with mass

spectrometry detection (KSEC-MS).

• Size-exclusion chromatography (SEC) allows generic separation of SM from

P−SM without the immobilization of SM or P. Mass spectrometry (MS), in turn,

allows the generic detection of SM without labeling it.

• Affinity chromatography was described under the title of pull-down assay.

• You may think of other chromatography techniques such as HIC which can

discriminate binding events.

Bao, Jiayin, et al. "Kinetic Size-Exclusion Chromatography with Mass Spectrometry Detection (KSEC-MS): an Approach

for Solution-Based Label-Free Kinetic Analysis of Protein-Small Molecule Interactions." Analytical chemistry (2014).

61

KSEC-MS

From: Bao et al. 2014; Analytical chemistry

62

KSEC-MS

From: Bao et al. 2014; Analytical chemistry

63

64

Outline

• Introduction

• BIA techniques:






• References

2. Molecular techniques

Y2H

Phage Display

TAP

Microarrays

65

Yeast two-hybrid (Y2H) system

• Testing for physical interactions between two proteins or protein/DNA.

• Is based on the properties of the yeast GAL4 protein, which consists of

separable domains responsible for DNA-binding and transcriptional

activation.

• Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-

binding domain fused to protein X and the other consisting of the GAL4

activation domain fused to protein Y, are constructed and introduced into

yeast.

• Interaction between proteins X and Y leads to the transcriptional activation

of a reporter gene containing a binding site for GAL4.

Caufield, J. H., Neha Sakhawalkar, and Peter Uetz. "A comparison and optimization of yeast two-hybrid

systems." Methods 58.4 (2012): 317-324.

66

• AD: activation Domain

• DBD: DNA Binding

Domain

• Reporter gene:LacZ reporter - Blue/White

Screening


67

http://2010.igem.org/File:TH.jpg

http://2010.igem.org/File:TH.jpg


Advantage Drawback

in vivoAn interaction may not happen in

yeast

SensitiveMammalian protein is sometimes not

correctly modified in yeast

-

If test proteins are not localized to the

nucleus two interacting proteins may

be found to be non-interacting

-

Some proteins might interact when

they are co-expressed in the yeast, (in

reality they are never present in the

same cell at the same time)

68


Y2H

Phage Display

TAP

Microarrays

69

• For the study of protein–protein, protein–peptide, and protein DNA interactions.

• A gene encoding a protein of interest is inserted into a phage coat protein gene,

causing the phage to "display”.

• These displaying phages can then be screened against other proteins, peptides or

DNA sequences to detect interaction.

• The most common bacteriophages used in phage display

are M13 and filamentous phage, though T4, T7, and λ phage have also been

used.

Phage Display

Bratkovič, Tomaž. "Progress in phage display: evolution of the technique and its applications." Cellular and molecular

life sciences 67.5 (2010): 749-767.

70

Phage Display

From: Wikipedia Phage display

71

Advantage Drawback

Libraries commercially available Complicated structures cannot be

incorporated

Screening can be done on whole cellsTwo hosts required (phage,bacterium)

Phages are amenable to many

conditionsQualitative only

Phage Display

72


Y2H

Phage Display

TAP

Microarrays

73

Tandem Affinity Purification (TAP)

From: Huber, 2003; Nature Reviews Molecular Cell Biology

74

Advantage Drawback

Significantly reduces non-specific

background

Not suitable for screening transient

protein interactions

More sensitive that Y2H Qualitative

Capable of disclosing multicomponent

interactions

possibility of a cleavage of the

proteins by the TEV protease

Tandem Affinity Purification (TAP)

75


Y2H

Phage Display

TAP

Microarrays

76

Microarrays: 1. Protein Chip

From: Barillot et al. 2012; Mathematical & Computational biology

77

Microarrays: 2. Nucleic acid Chip

From: Wikipedia DNA chip

78

79

Outline

• Introduction

• BIA techniques:






• References


Molecular docking

Virtual Screening

Molecular dynmic

simulation

Visualization and

Databses

80

Molecular docking technique

• A method which predicts the preferred orientation and energitics of one

molecule to a second when bound to each other to form a stable complex.

• strength of the predicted bound can be reported as different scoring types.

81

1. Ligand 3D structure

2. Receptor 3D structure

.

3. An appropriate docking algorithm

Molecular Docking Requirements

82

• Can be fetched from various databases for ligands:

ChemDB

Zinc

ChEMBL

PubChem

KEGG DRUG and ...

• Can be generated using cheminformatic tools:

eDesign

HyperChem

Marvin Suite

CORONA and ...

Ligand 3D structure

83

• Can be fetched from Protein Data Bank or other structural databases.

• If not available (for proteins), it can be modelled:

1. Homology modeling

SwissModel

M4T

Modeller

2. De novo modeling

I-TASSER

Phyre2

3. Threading

NovaFold

I-TASSER

Receptor 3D structure

84

• Software:

AutoDock

Molegro

ArgusLab

Hex

• Web-based server:

Cluspro

SwissDock

PatchDock

Docking algorithm

85


Molecular docking

Virtual Screening

Molecular dynmic

simulation

Visualization and

Databses

86

Virtual Screening

• In drug discovery, search libraries of small molecules in order to identify those

structures which are most likely to bind to a drug target, typically

a protein receptor or enzyme.

• Success of a virtual screen is defined in terms of finding interesting new

scaffolds rather than the total number of hits.

• Interpretations of virtual screening accuracy should therefore be considered

with caution. Low hit rates of interesting scaffolds are clearly preferable over

high hit rates of already known scaffolds.

• Follow the same rules as mentioned.

87


Molecular docking

Virtual Screening

Molecular dynmic

simulation

Visualization and

Databses

88

Molecular Dynamics (MD) Simulation

“If we were to name the most powerful

assumption of all, which leads one on and

on in an attempt to understand life, it is

that all things are made of atoms, and that

everything that living things do can be

understood in terms of the jigglings and

wigglings of atoms.”

____Richard Feynman, 1965 Nobel Prize winner in Physics

Durrant, Jacob D., and J. Andrew McCammon. "Molecular dynamics simulations and drug discovery." BMC

biology 9.1 (2011): 71.

89


• This computational method

calculates the time dependent

behavior of a molecular system.

• MD simulations have provided

detailed information on the

fluctuations and conformational

changes of proteins and nucleic

acids.

• These methods are now routinely

used to investigate the structure,

dynamics and thermodynamics of

biological molecules and their

complexes.

90


• MD simulation Soft-wares:

GROMACS

AMBER

CHARMM

GROMOS

NAMD

90


Molecular docking

Virtual Screening

Molecular dynmic

simulation

Visualization and

Databses

91

Interaction network visualization and databases

• Softwares of available to visualize PPI networks: eg. Cytoscape.

• Many Databases of experimental and predicted interactions are

also available:

- STRING

- BIND: the Biomolecular Interaction Network Database

- IntAct

- MINT: Molecular INTeraction database

- iRefIndex

92

http://mint.bio.uniroma2.it/mint/search

93

Outline

94

• Introduction

• BIA techniques:






• References

Helper-interaction FRET (hiFRET)

Jalink, Kees. "hiFRET: some tailwind for FRET resolves weak protein interactions." Nature methods 10.10 (2013):

947-948.

95

Liposome microarray (LiMA)

Saliba, Antoine-Emmanuel, et al. "A quantitative liposome microarray to systematically characterize protein-lipid

interactions." Nature methods (2013).

96

Micro/nano needle biosensor

Esfandyarpour, Rahim, et al. "Microneedle biosensor: A method for direct label-free real time protein

detection." Sensors and Actuators B: Chemical 177 (2013): 848-855.

97

Outline

98

• Introduction

• BIA techniques:






• References

Summary

• Study the interaction of biomolecules with each other and with other

molecules (usually drugs) are of utmost importance.

• There are various types of techniques with different origins and nature.

• We must choose the best techniques which can satisfy our research aim

needs.

• One technique cannot solely be conclusive.

• Optimization of the mentioned techniques are still being done.

• We are not yet able to analyze biomolecular interactions with just one

flawless technique.

99

Outline

100

• Introduction

• BIA techniques:






• References

Some key references

• Williams, Mark A., and Tina Daviter, eds. Protein-ligand Interactions: Methods

and Applications. Humana Press, 2013.

• Nienhaus, G. Ulrich. "Protein-Ligand Interactions." Totowa, NJ: Humana (2005).

• Wetie, Armand G. Ngounou, et al. "Protein–protein interactions: switch from

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Biomolecular interaction analysis (BIA) techniques

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