Optical Spectroscopic and Microscopic Techniques: Analysis of Biological Molecules

260 Pages·2022·8.215 MB·English

by  Harekrushna Sahoo

Harekrushna Sahoo   Editor
Optical
Spectroscopic
and Microscopic
Techniques
Analysis of Biological Molecules
Optical Spectroscopic and Microscopic
Techniques
Harekrushna Sahoo
Editor
Optical Spectroscopic
and Microscopic
Techniques
Analysis of Biological Molecules
Editor
HarekrushnaSahoo
BiophysicalandProteinChemistry
Laboratory,DepartmentofChemistry
NationalInstituteofTechnology(NIT)
Rourkela
Rourkela,Odisha,India
CenterofNanomaterials
NationalInstituteofTechnology(NIT)
Rourkela
Rourkela,Odisha,India
ISBN978-981-16-4549-5 ISBN978-981-16-4550-1 (eBook)
https://doi.org/10.1007/978-981-16-4550-1
#TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerNatureSingapore
PteLtd.2022
Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whether
thewholeorpartofthematerialisconcerned,specificallytherightsoftranslation,reprinting,reuseof
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation, computer software, or by
similarordissimilarmethodologynowknownorhereafterdeveloped.
Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublication
doesnotimply,evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevant
protectivelawsandregulationsandthereforefreeforgeneraluse.
The publisher, the authors, and the editorsare safeto assume that the adviceand informationin this
bookarebelievedtobetrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsor
theeditorsgiveawarranty,expressedorimplied,withrespecttothematerialcontainedhereinorforany
errorsoromissionsthatmayhavebeenmade.Thepublisherremainsneutralwithregardtojurisdictional
claimsinpublishedmapsandinstitutionalaffiliations.
ThisSpringerimprintispublishedbytheregisteredcompanySpringerNatureSingaporePteLtd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Contents
1 AbsorptionSpectroscopy:WhatCanWeLearnAbout
ConformationalChangesofBiomolecules?. . . . . . . . . . . . . . . . . . . 1
ManaliBasuandPadmajaPrasadMishra
2 CircularDichroismSpectroscopy:PrincipleandApplication. . . . . 19
SuchismitaSubadini,PratyushRanjanHota,DeviPrasannaBehera,
andHarekrushnaSahoo
3 Steady-StateFluorescenceSpectroscopyasaTooltoMonitor
Protein/LigandInteractions. .. . . . . . . . .. . . . . . . . .. . . . . . . . .. . 35
RoopaKenoth,BalamuraliM.M.,andRaviKanthKamlekar
4 FluorescenceAnisotropy:ProbingRotationalDynamics
ofBiomolecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
GourabPrasadPattnaikandHirakChakraborty
5 FluorescenceLifetime:AMultifacetedToolforExploring
BiologicalSystems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
SubhrajitMohantyandUsharaniSubuddhi
6 FromEnsembleFRETtoSingle-MoleculeImaging:Monitoring
IndividualCellularMachineryinAction. . . . . . . . . . . . . . . . . . . . . 113
FarhanaIslam,ManaliBasu,andPadmajaPrasadMishra
7 NanosecondTime-ResolvedFluorescenceAssays. . . . . . . . . . . . . . 143
Yan-CenLiuandAndreasHennig
8 FluorescenceCorrelationSpectroscopy:AHighlySensitiveTool
forProbingIntracellularMolecularDynamicsandDisease
Diagnosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
BikashChandraSwain,AnandKantDas,JanmejayaRout,
ShrutidharaBiswas,andUmakantaTripathy
9 PrinciplesandApplicationsofFluorescenceMicroscopy. . . . . . . . . 197
BibhuRanjanSarangi
vv
vi Contents
10 AnalysisofBiomolecularDynamicsUnderFourierTransform
InfraredSpectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
SanjeevKumarPaikraandMonalisaMishra
11 RamanSpectroscopyinBiology:PerspectivesandEmerging
Frontiers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
PradeepK.Sengupta
About the Editor
HarekrushnaSahoo iscurrentlyworkingasanAssociateProfessorofChemistry
at the NIT (National Institute of Technology) in Rourkela, India. He previously
served as a guest scientist at the Max-Bergmann Center (Dresden, Germany). He
completedhisPh.D.atJacobsUniversityBremen(Bremen,Germany)in2006,prior
to engaging in postdoctoral research at the University of Massachusetts (Amherst,
USA)andTechnicalUniversityDresden(Dresden,Germany).Hisresearchchiefly
focusesonusingbiophysicalchemistrytounderstandthekineticsanddynamicsof
various types of proteins, along with the impacts of environmental stress. He has
served as a reviewer for a number of international peer-reviewed and reputed
journals,includingJournalofPhysicalChemistryB,ACSOmega,ACSSustainable
Chemistry and Engineering, Journal of Photochemistry and Photobiology, and
International Journal of Biological Macromolecules. He has more than 9 years of
teaching experience in General Chemistry, Physical Chemistry, Supramolecular
Chemistry, Structure and Function of Biomolecules, Biophysical Chemistry, and
OpticalSpectroscopy.
He has also published more than 50 research articles in peer-reviewed interna-
tionaljournalsandauthoredorcoauthoredseveralbookchapters.Heisamemberof
many international scientific societies and organizations, e.g., American Chemical
Society, Biophysical Society, Indian Photobiology Society, and Orissa Chemical
Society.
vviiii
1
Absorption Spectroscopy: What Can We
Learn About Conformational Changes
of Biomolecules?
Manali Basu and Padmaja Prasad Mishra
Abstract
The quantification of interaction between electromagnetic radiation and matter
servesas aneffective toolinthe characterizationof materials inorder toidentify
andquantifyspecificsubstances.Absorptionspectroscopyisbasedonthephenom-
enon of wavelength-dependent absorption, that is, the attenuation of radiation
intensitywhenpassedthroughsolutioncontainingsampleinturnhelpstoquantify
theconcentrationaswellasthenatureofsubstancespresentwithinthesample.
1.1 Introduction
Sincetheadventofresearchesonatomsandmolecules,therehasbeenanenormous
curiosity among chemists, physicists, and other scientists about the structure of
molecules;nonetheless,theinfinitesimaldimensionofmoleculeshasposedtobea
major setback in visualizing their structure. However, the initial comprehension of
molecular structure could be indirectlyderivedfrom thetechniqueknown asspec-
troscopy. Spectroscopy deals with the precise measurement of the molecular
interactions with electromagnetic waves leading to the transitions between energy
levels upon absorption of suitable radiations dictated by the quantum mechanical
selectionrules[1].Theenergy(frequencytobeprecise)ofelectromagneticradiation
is used to promote electrons to an excited state from the ground state when the
energy of photons is comparable to the difference in energy between the two
molecularenergy levels.Aspectrum thus obtained ismeasuredasafunctionofits
M.Basu·P.P.Mishra(*)
ChemicalSciencesDivision,SahaInstituteofNuclearPhysics,Kolkata,WestBengal,India
HBNI,Mumbai,India
e-mail:[email protected]
#TheAuthor(s),underexclusivelicensetoSpringerNatureSingaporePte 1
Ltd.2022
H.Sahoo(ed.),OpticalSpectroscopicandMicroscopicTechniques,
https://doi.org/10.1007/978-981-16-4550-1_1
2 M.BasuandP.P.Mishra
wavelength or frequency and is termed as absorption spectrum. The origin of
spectrallinesinmolecularspectroscopynotonlyarisesduetoabsorptionofphotons
(e.g.UV-Vis,IR,NMR),emission(luminescenceandfluorescence),andscattering
ofphotons(Ramanspectroscopy)butalsoactivelycontributedduetochangesinthe
energy level of molecules. However, in this chapter, we will be focusing on
absorption,namelyUV-Visspectroscopyinparticular.
1.2 Origin of UV/Vis Spectrum
Visiblelightformsasmallpartofelectromagneticradiation(Fig.1.1)comprisingof
oscillatingelectricandmagneticfieldsthataremutuallyperpendiculartoeachother,
whoseenergy“E”isgivenby:
E ¼hϑ¼hc=λ ð1:1Þ
whereh,c,λ,andϑarePlanck’sconstant,speed,wavelength,andfrequencyofthe
light.Iftheenergyofalightphotonisthesameasthedifferencebetweentheenergy
of the ground and the excited state, then the molecule absorbs the photon. The
redundant energy may be released in the form of a photon with lower energy
(fluorescence or phosphorescence) or as radiation of heat. Occasionally, it ends up
inducingchemicalchangesintheabsorbingmolecule.Thesymmetryofmolecules
andtheselectionruletunestheprobabilityoftheabsorptionofaphotonofagiven
energy.Thisfurtherdependsonthedifferencebetweentheenergyconfigurationof
themolecularorbitalsandthenexthighestenergeticallyallowedelectronicconfigu-
ration. The commonly observed low-energy electronic transitions of molecules are
n!π* and π!π* type (where* represents an excitedstate),although σ ! σ(cid:2) and
n!σ(cid:2)transitionoccurashigher-energytransitionsfallinginthelowerwavelength
regionofspectra(Fig.1.2.).
Fig.1.1 Comparisonofenergy,frequency,andwavelengthoftheelectromagneticspectrum
1 AbsorptionSpectroscopy:WhatCanWeLearnAboutConformationalChangesof... 3
Fig.1.2 Schematic
representationofallthe
possibleelectronictransition
ofanymolecules
Fig.1.3 Possibleelectronic
transitionsincase
formaldehyde
Ingeneral,aromaticsystemswithdelocalizedelectronsabsorblightinthevisible
(400–800nm)orthenear-UV(150–400nm)region.Iftheelectronicconfigurationsof
molecules contain bonding and nonbonding electrons, they are readily excitable at
optical wavelengths. For instance, heteroatomic molecular systems like carbonyls
have both π bonding electrons (C¼O) and nonbonding (n) electrons at the O atom
(Fig. 1.3.). Although both of these types of electrons can be excited, they certainly
absorb at different wavelengths that could be used to distinguish the nature of
transitions.
However, the extent of conjugation is one of the pivotal factors affecting the
wavelength of absorption by a molecule. Molecules having extended conjugation
aredefinedasasystemhavingconnectedporbitalwithdelocalizedπ electrons,i.e.,
containing alternating double and single bonds, which are stable compared to their
nonconjugated counterparts. The bonding orbitals of organic molecules are almost
always filled (HOMO), and the antibonding orbitals are usually always empty
(LUMO). Greater is the extent of conjugation of molecules, less energy is required
totransfertheelectrons,andthustheenergydifferencebetweenHOMOandLUMO