Asher Research Group

Department of Chemistry University of Pittsburgh

 

Spectroscopy Group

 

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The spectroscopy group was recently covered in a C&E News cover story. Click here to read more.

 

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 Huntingtonís, Parkinsonís, cystic fibrosis, as well as Alzheimerís 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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