Raman Spectroscopy For Proteins - Horiba

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2012 HORIBA Scientific. All rights reserved. 2012 HORIBA Scientific. All rights reserved.

Raman Spectroscopy for proteinsCatalina DAVID Ph.D.– application scientist 2012 HORIBA Scientific. All rights reserved.

OutlineRaman spectroscopy in few wordsWhat is Raman spectroscopy ?What is the information we can get?Basics of Raman analysis of proteinsRaman spectrum of proteinsEnvironmental effects on the protein Raman spectrumContributions to the protein Raman spectrumUV Resonances Raman for proteinsPolarization measurements for proteinsLow-frequency measurements for proteinsConclusions 2012 HORIBA Scientific. All rights reserved.

Raman Spectroscopy in few words 2012 HORIBA Scientific. All rights reserved.

What is Raman SpectroscopyRaman effect Inelastic Light Scatteringλdiff λlaseranti-Stokes Ramanmoleculeor crystalλlaserExcitationλdiff λlaserRayleigh Diffusionλdiff λlaserStokes RamanThe frequency (ν 1/λ) difference between the incident and the scattered lightcharacterises the molecule vibration.νscattered νlaser νvibration 2012 HORIBA Scientific. All rights reserved.

What is the information we can getBand position:Chemical species, crystal phases,alloy compositionsFrequency Shift:Strain, ural disorderA Raman spectrum provides a fingerprint which represents the set of bonds present in thematerial: vibrational frequencies are characteristic of chemical bonds or groups of bondsVibrational frequencies are sensitive to details of the structure and local environment of amolecule, such as symmetry, crystal phase, polymer morphology, solvents, interactions, Relative intensities corresponds principally to the species concentration but it can be relatedto the orientation of the material or molecule with respect to the incoming laser polarization. 2012 HORIBA Scientific. All rights reserved.

Basics of Raman analysis of proteinsRaman spectrum of proteins 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteins 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsSecondary structure of the proteinSpatial arrangement of bonds (C O)Coupling between individual vibrations(C O)Amide I Raman bandThe precise positions of bands depend on inter andintra molecular effects, including peptide-bond anglesand hydrogen-bonding patterns 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsSecondary structure analysis Nine normal modes are allowed for theamide band of proteins.These are called A, B, and I-VII in order ofdecreasing frequencyAmide Raman bandsAmide I band80% C O stretch, near 1650cm-1Amide II band 60% N–H bend and 40% C–N stretch, near 1550 cm-1Amide III band 40% C–N stretch, 30% N–H bend, near 1300 cm-1 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsAmide I bandsThe different types of secondary structuresare characterized by amide I bands slightlydifferent in position and shape 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsAmide II band Parallel / antiparallelβ- sheet structure 1550 cm-1 It is a weak band It can not be observed in the absence ofresonance excitation It is hardly affected by the side-chain vibrations but thecorrelation between secondary structure and frequency is lessstraightforward than for the amide I vibration. It can be sensitive to H/D exchange 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsAmide III bands Assignmentα-helix : 1270-1300 cm-1Random coil : 1243-1253 cm-1β-sheet : 1229-1235 cm-1 The structure of amide III band can becorrelated to the amide I band complementarystructural information on the protein structureand in this way it is possible to get someadditional details to the amide I 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsOther important spectral features in proteins spectra Disulphide Bridges (S-S bonds) Aromatic aminoacids (Phenylalanine - Phe, tryptophan - Trp, tyrosine - Tyr,hystidin - His) 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsS-S bond stretching Experimental studies, shows that forthe proteins whose structure containsS-S bridges, the S-S Raman bandsare located in the spectral range 500550 cm-1. The factors affecting the frequencyof vibration are: the relativeconformation of atoms Cα-CβS-S'C'βC'α around Cβ-S and Cβ-S’ bonds,the mode coupling and the hydrogenbondsThe analysis of the lysozyme Raman spectrum in the 450–600 cm-1spectral range using Lorentzian functions. Experimental spectrum inblack and simulated spectrum in red (band decomposition in blue)David et al, PCCP, 2009 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsAromatic aminoacids Some of the vibrational bands of tyrosine (Tyr), tryptophan (Trp) or phenylalanine(Phe) are sensitive to the microenvironment Theirs band positions may vary up to 5 cm-1 in the Raman spectra of proteins.Important Raman modes of aromatic aminoacids withinthe protein structure 2012 HORIBA Scientific. All rights reserved.

Raman spectrum of proteinsAromatic aminoacids - Phenylalanine1000 Phe shows very intense band around 1000 cm-1 This band is not sensitive to conformational changes of protein and therefore canbe used for normalization of the Raman spectra of proteinBreathing mode800 2012 HORIBA Scientific. All rights reserved.1 0001 2001 400Raman shift (cm ¹)1 600

1340Raman spectrum of proteins1361Aromatic aminoacids - TryptophanThe components of the Fermi doubletof Trp : 1340 and 1360 cm-1.1 200 1 3001 400Raman shift (cm - ¹)I1360/I1340 serves as a hydrophobicity marker.The 1360 cm-1 band is strong in hydrophobic solvents (I1360/I1340 1.1)The 1340 cm-1 band is stronger in hydrophilic environment (I1360/I1340 0.9 )The 1010 cm-1 band is sensitive to the strength of van der Waals interactions ofthe Trp ring with surrounding residues ν near or below 1010 cm-1 indicates weak or no van der Waals interactionsν near 1012 cm-1 or higher reflect stronger van der Waals interactions 2012 HORIBA Scientific. All rights reserved.1 500

Raman spectrum of proteinsAromatic aminoacids - Tyrosine Tyrosine doublet Raman bands near 830 and 850 cm-1. They are caused by Fermi resonance between the in-plane breathing mode of thephenol ring and an overtone of out-of-plane deformation mode The intensities of these two bands depend on the hydrogen bonding condition ofthe phenol side chain. The ratio I850/I830 is often analyzed- 6.7 corresponds to non-hydrogen bonded Tyr- 2.5 the OH group of tyrosine is a strong hydrogen bond acceptor,- 0.3 corresponds to tyrosine as a donor of a strong hydrogen bond- 1.25 shows that the OH group serves both as an acceptor and a donor of ahydrogen bond. 2012 HORIBA Scientific. All rights reserved.

Raman analysis of proteinsSize of proteins makes spectrum complexPolypeptide backbone– Secondary structure – amides bands– Tertiary structure – background, aromatic aminoacidsAminoacids in side chains– H-bonding– Environment– Intermolecular interactions– Aromatic aminoacids are sensitive to micro-environment 2012 HORIBA Scientific. All rights reserved.

Raman analysis of proteinsbackbone 2012 HORIBA Scientific. All rights reserved.amino acidsS-S

Basics of Raman analysis of proteinsEnvironmental effects on the proteinRaman spectrum 2012 HORIBA Scientific. All rights reserved.

Environmental effects on the proteins Raman spectrumFour major types of interactions stabilize the native structure of proteins : The hydrogen bonds The covalent disulphide bonds The ionic bonds The hydrophobic effectThese interactions can be disturbed by reactionswith external agents, solvents, pH, temperature,salt concentration thus the protein can suffersome conformational changesThe conformational changes can be often observed and followed on the RamanspectrumRaman spectroscopy allows the study of folding / unfolding processes inproteins 2012 HORIBA Scientific. All rights reserved.

Environmental effects on the protein Raman spectrumpH effectchanges in the ionization of proteins sidechainshydrogen bonds can be strongly disturbedThe native structure of the protein can beaffectedRepresentative example is the study doneby SW Ellepola et al. on rice globulin protein,for different pH valuesThey found a slightly shift of amide I andamide III bands indicating a transition from αhelical structure to β-sheets and disorderedstructures under different pH conditions 2012 HORIBA Scientific. All rights reserved.pH11pH9pH7pH5pH3Raman spectra of rice globulin under differentpH conditions.

Environmental effects on the protein Raman spectrumTemperature effectsVery high or very low temperatures can lead to the weakness or even broking ofbonds inside the protein structure – Secondary structure is destabilized - Tertiarystructure will be affectedExample below : Study of the thermal denaturation of human haptoglobin (aglycoprotein from a blood serum which plays an important role in immune systemand in response to shock conditions)T. Pazderka et all., Proceeding 2012 HORIBA Scientific. All rights reserved.

Environmental effects on the protein Raman spectrumSolvent effects15711612154414421675Lysozyme 2596975250052253957013271652The interaction of the protein with the solvent often induces broadening of bandsand shifts of their maxima1 5001 200Raman shift (cm-¹) 2012 HORIBA Scientific. All rights reserved.1 4001 6001 6001 650Raman shift (cm-¹)Lysozyme inpowder1 0001 5501 8002 0001 7001 750

Environmental effects on the protein Raman spectrumChemical reactions effects(chemical unfolding) α-synuclein unfoldingcaused by reactionwith methanolthe analysis wasperformed in theamide I band regionMaiti et al, J.Am.Chem.Soc., 2004, 126, 2399 2012 HORIBA Scientific. All rights reserved.

Environmental effects on the protein Raman spectrumChemical reactions effects (reductive unfolding)The tertiary structure of the BSA is very well stabilized by 17 disulphide bridges(S-S).When the protein will react with a reducing agent, as DTT for example, the S-Swill be cleaved. Thus the structure will be perturbed thus the protein unfold occursDTTThe kinetic of the reaction is monitored by measuring Raman spectra over timeTwo spectral regions can be analyzed- the S-S Raman band – cleavage of the bond- the amide I Raman band 2012 HORIBA Scientific. All rights reserved.

Environmental effects on the protein Raman spectrumChemical reactions effects (reductive unfolding)S-S band before/after reactionAmide I band before/after reactionContributions of the different peaks are calculated after deconvolutionPhysical parameters, such as reaction rate, free enthalpy, activation energycan be calculated – quantitative resultsC. David et al. 2008, Biopolymers 2012 HORIBA Scientific. All rights reserved.

Basics of Raman analysis of proteinsContributions to the protein Raman spectrum 2012 HORIBA Scientific. All rights reserved.

Contributions to the protein Raman spectrumThe solventThe reactions in which are involved the proteins occur mostly in solution : water orbuffersThe buffers as well as the water have their own Raman signature composed of oneor more bands.Theirs bands may overlap to the protein ones – the Raman quantitative analysisbecome difficultRaman spectra of buffers should be measured separately and used as referencesThe most convenient solvent in terms of spectral contribution is water. Water it is aweak Raman scatterer, leading to little or no interference from water in Ramanspectra. 2012 HORIBA Scientific. All rights reserved.

Contributions to the protein Raman spectrumThe solvent - example18 00018 000Raman spectrum of aqueoussolution of lysozymeIntensity (counts)12 00010 0008 0006 00050016 000Subtraction of thewater contribution14 000Blue spectrum12–000Green spectrum1 50014 000Intensity (counts)Intensity (counts)16 0001 00010 0008 000Raman spectrum of water6 00015000001 500Raman shift (cm ¹)2 0005001 0001 500Raman shift (cm-¹)Pure Raman spectrum of lysozyme5001 0001 500Raman shift (cm-¹) 2012 HORIBA Scientific. All rights reserved.2 0002 000

Contributions to the protein Raman spectrumThe fluorescenceThe fluorescence can strongly affect the Raman spectrum - it can causesignificant background noise Intrinsic fluorescence (caused by the presence of some aromatics aminoacids)- it is possible to remove the fluorescence by selecting a suitablewavelength Extrinsic fluorescence (caused by impurities, solvents, buffers )- biological solutions should be pure as much as possible- photobleaching can be used to decrease the fluorescence background(pay attention to the exposure time and the subsequently the thermaldegradation of molecules within the sample solution) 2012 HORIBA Scientific. All rights reserved.

Spectra analysis and the contributions to the baselineThe fluorescence - exampleIntensity (counts)1 5001 0005001 0001 500800Intensity (counts)Intensity (counts)Red curve PolynomialPure Raman1 200 spectrum of lysozymeAfter baseline (fluorescence)subtraction functionfor the baselinewith a fluorescence background6004001 0005002005001 0001 500Raman shift (cm ¹)2 0005001 0000500Raman shift (cm-¹)1 0001 500Raman shift (cm-¹) 2012 HORIBA Scientific. All rights reserved.1 5002 0002 000

Contributions to the protein Raman spectrumThe signal-to-noise ratioA quantitative spectral analysis based on Raman spectroscopy requires a Ramanspectrum with minimal signal-to-noise ratio.Longer acquisitions in optimal conditions could be requestedThe signal averaging is also a way to improve signal-to-noise ratio.For the reproducible measurements, several spectra accumulations can beperformed, thus improving the signal-to-noise ratio.for n accumulations, the signal-to-noise ratio will be improved by a factor n 2012 HORIBA Scientific. All rights reserved.

Contributions to the protein Raman spectrumThe signal-to-noise ratio - ExampleIntensity (counts)400Intensity (counts)8 0001 second1 accumulations30020020 seconds1 accumulations6 0004 0002 00010001 50001 5001 6001 700Raman shift (cm-¹)Intensity (counts)3002502001501 6001 700Raman shift (cm-¹)8 0001 second100 accumulations350Intensity (counts)1 8001 80020 seconds4 accumulations6 0004 0002 0001005001 6001 700Raman shift (cm-¹) 2012 HORIBA Scientific. All rights reserved.1 80001 5001 6001 700Raman shift (cm-¹)1 800

Basics of Raman analysis of proteinsUV Resonances Raman for proteins 2012 HORIBA Scientific. All rights reserved.

UV Resonances Raman for proteinsDeep UV resonance Raman spectroscopy (DUVRRS) usually employs excitationwavelengths in the 190nm to 300nm rangeThe peptide bond strongly absorb at 190 nm – strong resonances are created andRaman signal coming from the peptide chains (proteins) can be efficiently andselectively enhanced Excitation at 415 nm results in an intense RRspectra which contains heme ring vibrations Excitation at 229 nm shows RR spectradominated by Tyr and Trp aromatic ring sidechain vibrations Excitation at 206.5 nm, shows RRspectra dominated by the peptide bond amidevibrationsAsher et al., J. Phys. Chem. Lett., 2011 2012 HORIBA Scientific. All rights reserved.

UV Resonances Raman for proteinsAdvantages of deep UV Raman Resonance Raman signal enhancement : Rayleigh law enhancement (1/λ4) and resonanceenhancement ( σs2 – Raman cross section) Raman Spectra Simplification : resonance enhancement occurs only within theRaman bands associated with the electronic transition – few bands Ambient Background Elimination : measurements in outdoor environmentsduring full sunlight 2012 HORIBA Scientific. All rights reserved.

UV Resonances Raman for proteinsDisadvantages of deep UV Raman ResonanceThermal damage of the sample : usually pulsed lasers are usedPhotochemical damage : depends on total photon dose and wavelength(photon/illuminated spot)DUVRRS systems require a high level of operator skill, require regularalignment and servicing of the systems and the laser - the cost can be high 2012 HORIBA Scientific. All rights reserved.

Basics of Raman analysis of proteinsPolarization measurements for proteins 2012 HORIBA Scientific. All rights reserved.

Polarization measurements for proteinsIn addition to the “normal” Raman, polarised Raman provides information aboutmolecular orientation and symmetry of the bond vibrationsAll molecules can be more or less symmetric. Thus, the positioning of thesemolecules (or of the sample) with respect to the laser beam, can greatly influencethe intensity of Raman bandsThe excitation of the molecule on one or another symmetry axes can lead to adifferent Raman scattering 2012 HORIBA Scientific. All rights reserved.

Polarization measurements for proteinsProteins have no only one symmetry but several (cyclic, dihedral, cubic, helical.)and these symmetries are local and not global (as for smaller molecules)Polarization measurements are possible – it is a practical way to identify thetotally symmetric modes in the Raman spectra of molecules possessing a certaindegree of symmetryTherefore, the interpretation of spectra is not an easy task to do due to the strongvibration modes coupling 2012 HORIBA Scientific. All rights reserved.

Polarization measurements for proteinsExample : Polarization effect on the Raman spectrum ofLysozymeExperimental configuration Z is the direction of the incident radiationpropagation Y is the direction of observation of thescattered light X is the direction of polarization of theelectric vector of the incident beam 2012 HORIBA Scientific. All rights reserved.

Polarization measurements for proteinsExample : Polarization effect on the Raman spectrum ofLysozyme I is the intensity of scatteredradiation with its electric vectorpolarized along the X axisI I Depolarization ratio (ρ) can facilitate theassignment of unknown bands to molecularvibrations 2012 HORIBA Scientific. All rights reserved. I is the radiation whosepolarization is directed along theZ axisI ρ I ΙΙ

Basics of Raman analysis of proteinsLow-frequency measurements for proteins 2012 HORIBA Scientific. All rights reserved.

Low-frequency measurements for proteinsThe bio-macromolecules exhibit low-frequency motionsThese motions are collective motions of all atoms in a proteinThese vibrations are very sensitive to the microenvironment variations due tothe : temperature, pressure, solvation, pH or ionic strengthThe Raman bands associated to these vibrations are very broad and difficult toassign. Thus, the experimental results are very often compared with thetheoretical analysis (Normal Mode Analysis and Calculations) 2012 HORIBA Scientific. All rights reserved.

Low-frequency measurements for proteinsExample : Low-frequencies modes of chymotrypsin 2012 HORIBA Scientific. All rights reserved.

Conclusions 2012 HORIBA Scientific. All rights reserved.

ConclusionsRaman spectroscopy can be successfully used as a method for probing thestructure and conformation of native proteinsImportant structural information can be deduced from specific Raman vibrationalbands as: amide I, amide II and amide III bands.The influence of chemical reactions mechanism involving proteins(folding/unfolding, oxidation, reduction, phosphorylation, and polymerization) canbe monitored by following the evolution over the time of Raman bands: thedisulphide bridges stretching, aromatics ring vibrations, protein side chaindeformation etc 2012 HORIBA Scientific. All rights reserved.

ConclusionsThe experimental results are usually submitted to complex processing whichmay offer the access to important physical and chemical parameters for theunderstanding of studied mechanismsThus, it is possi

Raman spectroscopy in few words What is Raman spectroscopy ? What is the information we can get? Basics of Raman analysis of proteins Raman spectrum of proteins Environmental effects on the protein Raman spectrum Contributions to the protein Raman spectrum UV Resonances

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