Research

Calmodulin, or calcium modulated protein (CaM), is a hinge protein that takes on several different structures, which makes it a good choice to measure THz TDS measurements of internal structural vibrations. The main role of the protein is to regulate biological processes like metabolism and nerve growth. The protein itself has 3 distinct shapes. The first structure is the natural structure that has no bonding with other substances. The second occurs when four calcium atoms bond to the calmodulin. This causes the original structure to bend to a dumbbell shape. From here, the third shape is achieved by a bond with a substrate, or polypeptide. A polypeptide is a chain of amino acids bound with peptide bonds. They can bind together to form proteins or can be broken up to create shorter polypeptide chains. With the substrate bonded, the calmodulin chain wraps around the substrate to break it apart. All proteins can also be denatured, in which the chain of the protein stays intact but looses its structure. These multiple structures makes calmodulin an ideal protein for THz TDS measurements.

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In terahertz time-domain spectroscopy (THz-TDS), short pulses of terahertz radiation are used to probe properties of matter. The Markelz Research Group has developed new THz-TDS techniques to study conformational changes in biomolecules. The state of the art developed at University at Buffalo is Stationary Sample Anisotropic Terahertz Microscopy (SSATM) in which the interrogated sample remains stationary while the linear state of THz polarization is rotated through 360° to identify the orientation of intramolecular vibrations in the THz frequency range.

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We use THz Time-Domain Spectroscopy (THz-TDS) and Microscopy to measure correlated vibrational modes in proteins. Our technique, Crystal Anisotropy THz Microscopy (CATM), has proven to be successful in measuring vibrational modes in molecular crystals. For the first time, this technique enables benchtop measurement and identification of large scale protein vibrational modes widely accepted to be related to protein function and binding. Using an optical technique we select only those modes with a high dipole coupling. Measurements are made at room temperature, with full hydration, on a table top with small sample requirements. Measurements are done in the near field to overcome the THz diffraction limit due to the small size of protein crystals (~300 μm).

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Through temperature dependent measurements we examine how protein dynamics move from harmonic to anharmonic response. We are examining how this transition is related to protein stability, often defined through the denaturing temperature.

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Protein vibrations may enable protein structure to efficiently access structural changes necessary for function. In addition, the motions may effect biochemistry by bringing catalytic partners together. Anisotropic THz spectroscopy provides a spectrum of vibrational excitations, but to understand what the spectra mean we need to compare these to calculations. Calculations enable us to more readily see trends with changes in temperature, ligand binding and mutation. However, calculations may be inaccurate for such complex systems. By combining calculations and measurements we can both improve the computational accuracy and analyze what the data mean. We use a number of different software packages to perform molecular dynamics simulations (mainly charmm and NAMD), and analyze the results using different visualization software (VMD and PyMOL). We have developed some python/TCL scripts to more readily visualize the data within VMD.

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In many THz microspectroscopy configurations, detection of a transmitted THz beam is performed in the far-field. This is often done to facilitate environmental control of the sample, such as the humidity needed for protein crystals or vacuum conditions needed to isolate properties of semiconductor materials. Sample sizes of only 100-300 μm are typically used to measure the structural dynamics in fingerprinting protein dynamics, for example. This presents a need to use a subwavelength aperture to filter out the components of the beam that do not pass through the sample is paramount. Post-measurement corrections of artifacts arising in the detected absorbance spectrum account for known mechanisms such as etalon or signal drift. However, a loss of spectral fidelity is observed in the far field whose mechanisms remain largely unknown.

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Structure and dynamics of proteins play a crucial role in their function. THz time domain spectroscopy has been employed to study proteins dynamics as their vibrational responses fall in the THz range. But the response from the structural motions of proteins in the solution state, which is closest to in vivo states as opposed to powders and crystals, are often masked by the relaxational contributions by water and protein side chains that also fall in the same frequency range.

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