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Introduction
to UV Raman Studies of Protein Folding
The recent completion
of the human genome project elucidated to the primary sequence of
all human proteins. The primary sequence encodes both the native
structure as well as the protein folding mechanism. The arguably
most important problem in enzymology is to translate the primary
sequence into the encoded protein folding mechanism(s), and to use
this information to predict the ultimate native structure from the
primary sequence information.
Solving this problem
will also reveal the mechanisms of many diseases of both humans
and animals. For example, mutations in the secondary structure of
proteins lead to diseases such as Huntington’s, Parkinson’s, cystic
fibrosis, as well as Alzheimer’s and prion diseases. In addition,
solving the protein folding problem will open new opportunities
in drug design and drug delivery.
Scientists use many
experimental techniques to elucidate the protein folding mechanisms:
x-ray diffraction, nuclear magnetic resonance (NMR), and various
optical spectroscopic methods. X-ray diffraction is certainly a
gold standard for solid state structural studies of proteins, while
the NMR is a gold standard for studying protein solutions at high
concentrations of proteins if dynamical processes of interest are
not too much faster than the msec time scale.
In contrast, optical
spectroscopic techniques, which monitor the characteristic light
absorption, emission, or scattering, are the only ones at the moment,
which allow to study proteins at low concentration as well as to
study fast dynamic processes. These techniques include the ultraviolet
(UV), visible (VIS) and infrared (IR) absorption; circular dichroism
(CD); vibrational circular dichroism (VCD); fluorescence; visible
and near-infrared Raman scattering (normal Raman); Raman optical
activity (ROA), and UV resonance Raman (UV Raman) scattering techniques.
Recent developments
in UV Raman spectroscopy makes this technique especially attractive
for protein conformational studies. The ability of UV Raman to selectively
monitor the vibrations of chosen chromophore group(s) without an
overlap from the other groups dramatically simplifies the spectral
analysis, comparing to normal Raman. In addition, UV Raman is able
to monitor both very high and very low concentrations of proteins
in aqueous solutions even down to 0.2 mg/ml, able to study turbid
and even not transparent samples, has no significant interference
from water. Moreover, it can quantitatively monitor the protein
energy landscape and protein folding coordinates.
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UV
Resonance Raman Spectroscopy
Resonance
Raman Spectroscopy (RRS) is a very powerful tool which is used to
study molecular structure and dynamics. Resonance Raman scattering
requires excitation within an electronic absorption band and results
in a large increase of scattering (up to 108 fold when compared
to "normal" Raman scattering). Few molecules have visible absorption
bands; however everything absorbs in the deep UV. So, by using UV
light we are able to study a wide variety of colorless chromophores,
and we have the additional benefit of avoiding interference from
fluorescence. (Typically, condensed phase species show no fluorescence
below 260nm.) Furthermore, we can selectively
excite electrons of different functional groups with different
excitation wavelengths. This approach allows us to investigate specific
parts of macromolecules by using different excitation wavelengths.
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Experiments
With the development
of cutting edge technology, the powerful UV lasers and specialized
spectrometers required for UVRR experiments are available. Almost
totally tunable pulse lasers provide excitation from 184 nm to near
IR, while intracavity
doubled Ar and Kr ion lasers provide continuous wave excitation
at discrete lines from 206.5 to 257 nm. The UV scattering, obtained
from the laser light interacting with a sample, is collected by
a modified Triplemate spectrometer equipped with novel high efficiency
mirrors. While this type of instrument is quite efficient and provides
Rayleigh rejection, a single stage spectrometer containing mirrors
with high reflectance in the UV region and Rayleigh rejection filters
was also developed to further improve our signal to noise ratio.
The combination of specialized lasers and spectrometers enables
us to obtain high signal to noise Raman spectra for nearly any molecule.
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Proteins and
Peptides - Steady State Studies
Steady-State Studies
Research is being done on the fundamental properties of the peptide
bond. A new method was developed to measure ground state activation
barriers for trans-cis isomerization in secondary amides and studies
are being done to determine the orientation of the transition dipole
moment in dipeptides. Furthermore, we discovered a
new charge transfer band in glycyl-glycine.
Insight into the
nature of the peptide bond has permitted us to develop new methodology
for determining protein secondary structure. Protein spectra obtained
using excitation at 206.5nm are dominated by amide bands, and we
were able to correlate the position of these amide I, II, III and
alpha C-H amide bending vibrations to secondary structure. This
correlation was accomplished by empirically determining the average
pure a-helix,
b-sheet and random coil spectra from the spectra of thirteen
proteins with known crystal structures, and the average spectra
are used as standards to directly determine protein secondary structure.
We utilized this new method in combination with 229 nm excited protein
spectra to monitor acid unfolding of horse
heart myoglobin to the intermediate state. Since 229 nm excitation
almost exclusively enhances tryptophan and tyrosine side chain vibrations,
we were able to correlate secondary structure changes with alterations
in side-chain environment. This approach proved to be powerful for
monitoring protein
folding and unfolding. Currently, we are working on the development
of time-resolved deep UVRR technique and its application in protein
folding/ unfolding studies.
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Proteins and
Peptides - Kinetic Studies
Kinetic Studies
in aqueous solution, many native proteins unfold (denature) when
the solution temperature is raised or lowered. Using the pump-probe
technique described earlier, we are able to initiate folding and
unfolding events in proteins and polypeptides and to monitor the
time scale in which they occur. UVRR spectra are obtained at intervals
ranging from ten nanoseconds to several hundred nanoseconds following
the heat-producing pump pulse. Our first success using this technique
was the denaturation
of the alpha-helical polypeptide, A5(A3RA)3A. A pump pulse that
induced a temperature jump of 54 degrees Celsius was used. Structural
changes were observed after 22 ns, and a random coil structure was
observed after 95 ns. Studies on bioactive proteins are underway
to answer questions surrounding the protein folding problem.
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Meteorites
Another area of
research being conducted within the group involves meteorites which
contain a rich varieties of complex minerals and inorganics. Their
detailed examination yields information on temperature history and
processing, while compositional anomalies can suggest origin. Our
micro UV Raman measurements enable us to not only characterize the
type and distribution of these minerals within meteorites, but also
monitor perturbations of their characteristic phonon frequencies
indicative, for example, of crystal structure, cation substitution,
hydration and percentile composition in solid solutions. In addition
to characterizing the predominant inorganic matrices of meteorites,
our UV Raman measurements enable the identification and characterization
of various solid state carbon material inclusions such as diamond,
graphite (ordered and disordered), various amorphous carbon species
and silicon carbide. In the cases of the hard amorphous carbon species,
UV Raman can readily probe the tetrahedrally bonded carbon sites
within these materials, enabling their rapid characterization. Furthermore,
we have demonstrated that UV Raman enables us to probe the PAH and
amino acid content of these systems. Even in solid form, the high
sensitivity of UV Raman measurements enables the characterization
of these photolabile materials without observable photo or thermal
degradation.
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Some Spectroscopy
Group Pictures
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