Horiba, Ltd. · TSE:6856

Horiba — Technical Presentation (2012)

53 slides from Horiba's technical presentation, 2012

Slide 3: Outline
Outline 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 Raman for proteins Po
Slide 5: What is Raman Spectroscopy
What is Raman Spectroscopy Raman effect = Inelastic Light Scattering λdiff < λlaser anti-Stokes Raman molecule or crystal λdiff = λlaser Rayleigh Diffusion λlaser Excitation λdiff > λlaser Stokes Raman The frequency (ν = 1/λ) difference between the incident and the scattered light characterises the
Slide 6: What is the information we can get
What is the information we can get Band position: Chemical species, crystal phases, alloy compositions Frequency Shift: Strain, temperature Intensity: Concentration Linewidth: Structural disorder A Raman spectrum provides a fingerprint which represents the set of bonds present in the material: vibra
Slide 9: Raman spectrum of proteins
Raman spectrum of proteins Secondary structure of the protein Spatial arrangement of bonds (C=O) Coupling between individual vibrations (C=O) Amide I Raman band The precise positions of bands depend on inter and intra molecular effects, including peptide-bond angles and hydrogen-bonding patterns © 2
Slide 10: Raman spectrum of proteins: secondary structure analysis
Raman spectrum of proteins Secondary structure analysis • Nine normal modes are allowed for the amide band of proteins. • These are called A, B, and I-VII in order of decreasing frequency Amide Raman bands Amide I band 80% C=O stretch, near 1650cm-1 Amide II band 60% N–H bend and 40% C–N stretch, ne
Slide 11: Amide I bands
Raman spectrum of proteins Amide I bands The different types of secondary structures are characterized by amide I bands slightly different in position and shape © 2012 HORIBA Scientific. All rights reserved.
Slide 12: Amide II band
Raman spectrum of proteins Amide II band • Parallel / antiparallelβ- sheet structure ~ 1550 cm-1 • It is a weak band It can not be observed in the absence of resonance excitation • It is hardly affected by the side-chain vibrations but the correlation between secondary structure and frequency is les
Slide 13: Amide III bands
Raman spectrum of proteins Amide III bands • Assignment α-helix : 1270-1300 cm-1 Random coil : 1243-1253 cm-1 β-sheet : 1229-1235 cm-1 • The structure of amide III band can be correlated to the amide I band = complementary structural information on the protein structure and in this way it is possibl
Slide 14: Other important spectral features in proteins spectra
Raman spectrum of proteins Other 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.
Slide 15: S-S bond stretching
Raman spectrum of proteins S-S bond stretching • Experimental studies, shows that for the proteins whose structure contains S-S bridges, the S-S Raman bands are located in the spectral range 500- 550 cm-1. • The factors affecting the frequency of vibration are: the relative conformation of atoms Cα-
Slide 16: Aromatic aminoacids
Raman spectrum of proteins Aromatic 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
Slide 17: Aromatic aminoacids - Phenylalanine
Raman spectrum of proteins Aromatic aminoacids - Phenylalanine • Phe shows very intense band around 1000 cm-1 • This band is not sensitive to conformational changes of protein and therefore can be used for normalization of the Raman spectra of protein 1000 Breathing mode 800 1 000 1 200 1 400 1 600
Slide 18: Aromatic aminoacids - Tryptophan
Raman spectrum of proteins 1340 Aromatic aminoacids - Tryptophan 1361 The components of the Fermi doublet of Trp : 1340 and 1360 cm-1. 1 200 1 300 1 400 1 500 Raman shift (cm - ¹) • I1360/I1340 serves as a hydrophobicity marker. • The 1360 cm-1 band is strong in hydrophobic solvents (I1360/I1340 > 1
Slide 19: Aromatic aminoacids - Tyrosine
Raman spectrum of proteins Aromatic 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 the phenol ring and an overtone of out-of-plane deformation mode • The intensities of these two bands depend on
Slide 20: Raman analysis of proteins
Raman analysis of proteins Size of proteins makes spectrum complex Polypeptide backbone – Secondary structure – amides bands – Tertiary structure – background, aromatic aminoacids Aminoacids in side chains – H-bonding – Environment – Intermolecular interactions – Aromatic aminoacids are sensitive to
Slide 23: Environmental effects on the proteins Raman spectrum
Environmental effects on the proteins Raman spectrum Four major types of interactions stabilize the native structure of proteins : • The hydrogen bonds • The covalent disulphide bonds • The ionic bonds • The hydrophobic effect These interactions can be disturbed by reactions with external agents, so
Slide 24: pH effect
Environmental effects on the protein Raman spectrum pH effect changes in the ionization of proteins side pH11 chains hydrogen bonds can be strongly disturbed pH9 The native structure of the protein can be affected pH7 Representative example is the study done pH5 by SW Ellepola et al. on rice globuli
Slide 25: Temperature effects
Environmental effects on the protein Raman spectrum Temperature effects Very high or very low temperatures can lead to the weakness or even broking of bonds inside the protein structure – Secondary structure is destabilized - Tertiary structure will be affected Example below : Study of the thermal d
Slide 26: Solvent effects
Environmental effects on the protein Raman spectrum Solvent effects The interaction of the protein with the solvent often induces broadening of bands and shifts of their maxima 1652 1327 1000 1442 1544 752 1249 1354 1612 868 892 925 1096 500 1119 1571 969 1189 1026 1069 849 522 828 539 Lysozyme in 5
Slide 27: Chemical reactions effects (chemical unfolding)
Environmental effects on the protein Raman spectrum Chemical reactions effects (chemical unfolding) • α-synuclein unfolding caused by reaction with methanol • the analysis was performed in the amide I band region Maiti et al, J.Am.Chem.Soc., 2004, 126, 2399 © 2012 HORIBA Scientific. All rights reser
Slide 28: Chemical reactions effects (reductive unfolding)
Environmental effects on the protein Raman spectrum Chemical 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-S will be cleaved. Thus the stru
Slide 29: Chemical reactions effects (reductive unfolding)
Environmental effects on the protein Raman spectrum Chemical reactions effects (reductive unfolding) S-S band before/after reaction Amide I band before/after reaction Contributions of the different peaks are calculated after deconvolution Physical parameters, such as reaction rate, free enthalpy, ac
Slide 31: The solvent
Contributions to the protein Raman spectrum The solvent The reactions in which are involved the proteins occur mostly in solution : water or buffers The buffers as well as the water have their own Raman signature composed of one or more bands. Theirs bands may overlap to the protein ones – the Raman
Slide 32: The solvent - example
Contributions to the protein Raman spectrum The solvent - example 18 000 18 000 Raman spectrum of aqueous solution of lysozyme 16 000 16 000 Subtraction of the water contribution Intensity (counts) 1 500 Intensity (counts) 14 000 14 000 Blue spectrum12–000Green spectrum Intensity (counts) 12 000 10
Slide 33: The fluorescence
Contributions to the protein Raman spectrum The fluorescence The fluorescence can strongly affect the Raman spectrum - it can cause significant background noise • Intrinsic fluorescence (caused by the presence of some aromatics aminoacids) - it is possible to remove the fluorescence by selecting a s
Slide 34: The fluorescence - example
Spectra analysis and the contributions to the baseline The fluorescence - example Pure Raman 1 200 spectrum of lysozyme Red curve = Polynomial After baseline (fluorescence) subtraction function with a fluorescence background for the baseline 1 000 Intensity (counts) 1 500 1 500 800 Intensity (counts
Slide 35: The signal-to-noise ratio
Contributions to the protein Raman spectrum The signal-to-noise ratio A quantitative spectral analysis based on Raman spectroscopy requires a Raman spectrum with minimal signal-to-noise ratio. Longer acquisitions in optimal conditions could be requested The signal averaging is also a way to improve
Slide 36: The signal-to-noise ratio - Example
Contributions to the protein Raman spectrum The signal-to-noise ratio - Example 1 second 8 000 20 seconds 400 1 accumulations 1 accumulations 6 000 Intensity (counts) Intensity (counts) 300 4 000 200 2 000 100 0 0 1 500 1 600 1 700 1 800 1 500 1 600 1 700 1 800 Raman shift (cm-¹) Raman shift (cm-¹)
Slide 38: UV Resonances Raman for proteins
UV Resonances Raman for proteins Deep UV resonance Raman spectroscopy (DUVRRS) usually employs excitation wavelengths in the 190nm to 300nm range The peptide bond strongly absorb at 190 nm – strong resonances are created and Raman signal coming from the peptide chains (proteins) can be efficiently a
Slide 39: Advantages of deep UV Raman Resonance
UV Resonances Raman for proteins Advantages of deep UV Raman Resonance ☺ Raman signal enhancement : Rayleigh law enhancement (1/λ4) and resonance enhancement (~σs2 – Raman cross section) ☺ Raman Spectra Simplification : resonance enhancement occurs only within the Raman bands associated with the ele
Slide 40: Disadvantages of deep UV Raman Resonance
UV Resonances Raman for proteins Disadvantages of deep UV Raman Resonance Thermal damage of the sample : usually pulsed lasers are used Photochemical damage : depends on total photon dose and wavelength (photon/illuminated spot) DUVRRS systems require a high level of operator skill, require regular
Slide 42: Polarization measurements for proteins
Polarization measurements for proteins In addition to the “normal” Raman, polarised Raman provides information about molecular orientation and symmetry of the bond vibrations All molecules can be more or less symmetric. Thus, the positioning of these molecules (or of the sample) with respect to the
Slide 43: Polarization measurements for proteins
Polarization measurements for proteins Proteins 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 the totally symmetric modes i
Slide 44: Polarization effect on the Raman spectrum of Lysozyme
Polarization measurements for proteins Example : Polarization effect on the Raman spectrum of Lysozyme Experimental configuration • Z is the direction of the incident radiation propagation • Y is the direction of observation of the scattered light • X is the direction of polarization of the electric
Slide 45: Polarization effect on the Raman spectrum of Lysozyme
Polarization measurements for proteins Example : Polarization effect on the Raman spectrum of Lysozyme • I|| is the intensity of scattered radiation with its electric vector polarized along the X axis I|| • I⊥ is the radiation whose polarization is directed along the Z axis I⊥ • Depolarization ratio
Slide 47: Low-frequency measurements for proteins
Low-frequency measurements for proteins The bio-macromolecules exhibit low-frequency motions These motions are collective motions of all atoms in a protein These vibrations are very sensitive to the microenvironment variations due to the : temperature, pressure, solvation, pH or ionic strength The R
Slide 50: Conclusions
Conclusions Raman spectroscopy can be successfully used as a method for probing the structure and conformation of native proteins Important structural information can be deduced from specific Raman vibrational bands as: amide I, amide II and amide III bands. The influence of chemical reactions mecha
Slide 51: Conclusions
Conclusions The experimental results are usually submitted to complex processing which may offer the access to important physical and chemical parameters for the understanding of studied mechanisms Thus, it is possible to determine the reaction free enthalpy and free enthalpies for the unfolding of
Slide 52: Conclusions
Conclusions Widely used in Raman spectroscopy method, the polarization measurements and more precisely the depolarization ratio can facilitate the assignment of unknown bands to molecular vibrations Deep ultraviolet resonance Raman spectroscopy as well as the low- frequencies measurements are powerf

Horiba — Technical Presentation (2012) on 6ix

Horiba — Technical Presentation (2012)

Slide 1: © 2012 HORIBA Scientific. All rights reserved.

Slide 2: Raman Spectroscopy for proteins

Slide 3: Outline

Slide 4: Raman Spectroscopy in few words

Slide 5: What is Raman Spectroscopy

Slide 6: What is the information we can get

Slide 7: Basics of Raman analysis of proteins

Slide 8: Raman spectrum of proteins

Slide 9: Raman spectrum of proteins

Slide 10: Raman spectrum of proteins: secondary structure analysis

Slide 11: Amide I bands

Slide 12: Amide II band

Slide 13: Amide III bands

Slide 14: Other important spectral features in proteins spectra

Slide 15: S-S bond stretching

Slide 16: Aromatic aminoacids

Slide 17: Aromatic aminoacids - Phenylalanine

Slide 18: Aromatic aminoacids - Tryptophan

Slide 19: Aromatic aminoacids - Tyrosine

Slide 20: Raman analysis of proteins

Slide 21: Raman analysis of proteins

Slide 22: Environmental effects on the protein Raman spectrum

Slide 23: Environmental effects on the proteins Raman spectrum

Slide 24: pH effect

Slide 25: Temperature effects

Slide 26: Solvent effects

Slide 27: Chemical reactions effects (chemical unfolding)

Slide 28: Chemical reactions effects (reductive unfolding)

Slide 29: Chemical reactions effects (reductive unfolding)

Slide 30: Contributions to the protein Raman spectrum

Slide 31: The solvent

Slide 32: The solvent - example

Slide 33: The fluorescence

Slide 34: The fluorescence - example

Slide 35: The signal-to-noise ratio

Slide 36: The signal-to-noise ratio - Example

Slide 37: UV Resonances Raman for proteins

Slide 38: UV Resonances Raman for proteins

Slide 39: Advantages of deep UV Raman Resonance

Slide 40: Disadvantages of deep UV Raman Resonance

Slide 41: Polarization measurements for proteins

Slide 42: Polarization measurements for proteins

Slide 43: Polarization measurements for proteins

Slide 44: Polarization effect on the Raman spectrum of Lysozyme

Slide 45: Polarization effect on the Raman spectrum of Lysozyme

Slide 46: Low-frequency measurements for proteins

Slide 47: Low-frequency measurements for proteins

Slide 48: Low-frequencies modes of chymotrypsin

Slide 49: Conclusions

Slide 50: Conclusions

Slide 51: Conclusions

Slide 52: Conclusions

Slide 53: Thank you