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BIOS E-170 Term Paper

Jessica Nguyen ID# 1125075
Protein Crystallization Characterization through Raman Spectroscopy
Raman scattering is a vibrational imaging technique that was founded in India, due to
seeing sunlight effects on tetrachloride which resulted in a visible of excitation while light is
being annihilated and a single light of photo is created. This research resulted in C.V Raman
winning the Novel Prize in 1930. Raman is the usage of light that has been inelastically scattered
to give us a label. Because of the potential to use scattered light, Raman has the potential to even
replace fluorescence. This is because the vibrations are used to pick up molecules and identify
the chemistry of the functional groups. The spectrum results in bands with "line intensities [that
are directly a] function of intrinsic polarization o the signal, polarization conditions of the
excitation and signal collection, concentration, Raman scattering cross-section and molecular
interactions[1]... " Because Raman is so versatile as a technique it can be used as Raman
scattering, stimulated Raman Scattering, confocal Raman imaging and many more techniques.
The more Raman is used, the more molecular maps of molecules can be determined. If one can
spot out a specific functional group on the Raman spectra you can map out a chemical functional
groups of a sample.
Raman spectroscopy uses the scattering phenomenon to map out surface interfaces of
samples with little to no sample preparation, resulting in the authenticity in the spectrums.
Raman spectra are used to identify certain characteristics of the molecules of the sample. In this
paper, protein identification through Raman will be explained. "The Raman spectrum of a protein
or nucleic acid consists of numerous discrete bands [that] represent molecular normal modes of

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vibration and serves as a sensitive and selective fingerprint of three-dimensional structure,

intermolecular interactions and dynamics[2]."
When using Raman, the tagging of small molecules for the identification of the sample's
chemistry is required, which means overcoming certain challenges such as the behavior of the
sample changing dependent on labels added to it. For example, glucose is often times tagged as
small molecules tend to take up more glucose quickly. Through addition of an alkyne moiety,
which have a triple bond which has a higher vibrational frequency, to the glucose which small
molecules are taking up more rapidly, we have the potential to see where these molecules are
going and traveling to. This means we have the capability of tracking where small molecules are
going without any side effects of the small molecules because cells can metabolize glucose with
the alkyne moiety with little to no complications. Using a vibrational imaging technique such as
Raman can help identify important information in the identification of proteins such as the
"...conformation or orientation of constituent molecules and sub-molecular groups, local
hydrogen-bonding interactions and time dependence of structural or organizational properties[2]."
Raman data collection is often compared to FTIR. Even though some challenges of
Raman may be the signal-to-noise levels are considered inferior as it is difficult to compare
scattering sensations and quantifying those sensation. However, because FTIR lacks the
capabilities to produce a spectrum as it collects information through the use of an interferograms
(which then undergoes a Fourier Transform to produce a spectrum), Raman spectrums are
preferred due to the information that it can provide about localized molecular vibrations. These
vibrations between the atoms, creates the signal to generate a Raman band, usually associating
both polarity as well as dipole moments or bias towards a more electronegative atom.

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One Raman mode known as the normal mode takes a sample with "...N atoms [and] has
3N+6 independent modes of vibration[3]" to form a spectrum based on the vibrations that happen.
Water has 3 normal modes which include a symmetric, asymmetric and a bending mode. These
modes correlate at certain wavenumbers on the spectrum. The symmetric stretching for water is
around 3200 cm-1, asymmetric cm-1 and bending is around 1600 cm-1. Water has a large band due
to the bias of the dipole moment of the O-H sharing resulting in an overly broad band.
For protein identification through the Raman spectroscopy, protein crystallization and
nucleic acid assemblies must be observed to thoroughly map out functional group characteristics
to determine what kind of protein the subjected sample is. The Raman spectrum uses the energy
between bonds and the atoms of those bonds to create a spectrum of functional groups. Using a
polyethylene chain, which is found in many proteins, for example, is "an infinite trans zigzag
chain, is constructed by repeating -CH2- units which have nine proper vibrations: three atoms
with three degree of freedom (x, y and z)[4]." When the atoms are moving towards a certain atom
with bias, the energy whether the atoms are symmetric, asymmetric or are in a bending motion,
these three types of atom interactions have different stretching values that help correlate to the
orientations of the atoms that are the molecular makeup of the protein.
Because Raman is an imaging technique that models the atoms of a molecule as a system
of balls and springs with resonant energy interactions, the resonances result in a frequency range
of energy which has a large correlation to larger displacements to higher energy vibrations of
those atoms of the molecule. The increase of displacement occurs with the localized vibrations of
multiple bonded electron rich groups adjacent to each other. C=O, C=N, C=C, S-S, S-C, S-H are
some of the most common and most electron rich types of bonds that result in large and intense
signal peaks due to the vibrations of the oscillating dipole moment. For example, Sulfur is one

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of the atoms of usually found in the amino acids methionine, cysteine, homocysteine and taurine.
The polarization between the S-H atoms create a stretch that occurs at a given 2500-2600 cm-1.
Common electron rich atoms next to each other give off wavenumbers that are recorded in
database due to the common electron behaviors which result in similar Raman signal
intensities[5]. The vibrational spectrums of "large biological macromolecules can produce large
shifts in Raman band positions, often referred to as frequency shifts, empowering the technique
in the diagnosis of protein secondary structure, determination of side-chain configurations, and
detection of interacting side-chain groups...[6]."
Raman Spectroscopy is a non-invasive form of spectroscopy that does not disrupt the
sample that is of interest. Because of this characteristic, the viewing of samples in an undisrupted
environments allows for observations of the natural interaction of amino acids and protein
building in a specific environment free of distractive variables. When you a running a protein
subject to Raman spectroscopy conditions, there are several signals that are going to peak which
can be due to noise as well as the molecular interactions between adjacent molecules. However,
different types of bands that are associated with peptide bonds (-CONH) are usually indicative of
amide stretching. The wave number stretching patterns for both the acidic and basic side of the
amino acids have different signals usually stretching from 3500 cm-1 (amide A with CONH) and
3100 cm-1 (NH)[7]. From these wave numbers the skeletal structure of the protein can be
identified as the three main signals of interest will a C=O stretching, C-N stretching and N-H
stretching. These bands around 600 cm-1 - 1600 cm-1 are usually found between a pair of 3000+
cm-1 wavenumbers for the end carbons, which allows for the identification of amino acid resides
for each protein signal.

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Due to the special identification of the amide bands in Raman, this feature allows the spectra of
amino acids to be extremely useful in determining the amino acid side chains and their signals
for example a tryptophan doublet at 1360/1340cm-1, a tyrosine double 860/833 cm-1 or even C-S
stretching where the carbon atom is in a trans position from the S atom 740-760 cm-1 [7]. These
different major standards in vibrational bond identification play a major role in the secondary
structures of proteins as well. Through the vibrational modes of amides in position I and modes
of amides in position III, the determination of the orientation of the proteins unfolding allows for
the identification of the a-helix as well as the B-sheet structures.
Raman does have challenging limitations such as autofluorescence and getting over
chemically complex molecules, however the foundation for the Raman spectra allows for
analysis to be done on biological molecule giving users information about conformation,
orientation and intermolecular interactions making it a vital and important imaging technique
because it does not physically cause changes to the sample and requires little to no sample prep.

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References
1. Dubessy, J., Caumon, M.C., Rull, F., Sharma, S. "Instrumentation in Raman Spectroscopy:
Elementary Theory and Practice" EMU-CNRS International School: Applications of Raman
Spectroscopy to Earth Sciences and Cultural Heritage. 1-59 (2012).
2. Thomas, George J., "Raman Spectroscopy of Protein and Nucleic Acid Assemblies" Annual
Review of Biophysics and Biomolecular Structure. 25:1-27 (1999).
3. McClelland, Arthur. "Introduction to Vibrational Spectroscopy" BIOS E-170: Introduction to
Microscopy. HES. (2016).
4. Motoyama, Michiyo. :"Structure and Phase Characterization of Triacylglycerols by Raman
Spectroscopy" Bull Naro Institute of Livestock and Grassland Science. 12:19-68 (2012).
5. Tuma, Roman. "Raman Spectroscopy of Proteins from Peptides to Large Assemblies" Journal
of Raman Spectroscopy. 34: 4-11 (2005).
6. Miura, Takashi., Thomas, George J. Raman Spectroscopy of Proteins and their Assemblies"
Subcellular Biochemistry: Proteins Structure, Function and Engineering. 25:55-99 (1995)
7. Rygula, A., Majzner, K., Marzec, K.M., Kaczor, A., Pilarczyk, M., Baranska, M. "Raman
Spectroscopy of Proteins: A Review" Journal of Raman Spectroscopy. 44: 1061-1076 (2013)