Optical Spectroscopic and Microscopic Techniques: Analysis of Biological Molecules PDF
Preview Optical Spectroscopic and Microscopic Techniques: Analysis of Biological Molecules
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
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