The roadmap gives an overview of the research directions. The research topics in the group may appear to be unrelated at first glance, however, there is a focus. The problems of interest in biology today are often very complex. We are interested in designing new ways to look at biomolecules using optical spectroscopy. In order to use vibrational spectroscopy to advantage it is important to assign the molecular vibrations (Vibrational Spectroscopy). In order to design functional tests for molecules it is important to understand their dynamics (Protein dynamics). In order to utilize microscopy it is convenient to immobilize molecules on a surface and interrogate their interaction with other molecules (Nanoscale materials). Thus, we see the focus as a new way to look at molecules using spectroscopy. We fix a molecule in a location so that we can probe it with a laser or single-bounce attenuated total reflection infrared spectroscopy. Then we examine that molecules interaction with other molecules. Our goal is to examine protein binding to DNA, proteins and small molecule targets in this way and to apply infrared and Raman spectroscopies to the study of the processes and in the cell cycle.
Assignment of vibrational spectra is an important problem. The information content of vibrational spectroscopy is difficult to unravel because the normal modes of vibration often contain contributions from many nuclei simultaneously. Moreover, the normal mode picture itself is a simplification and the reality is that molecules are constantly executing complicated Lissajou patterns of motion due to anharmonic coupling of one mode to another. However, there is hope! Density functional theory (DFT) calculations provide an excellent starting point for analysis of the components of biological molecules (amino acids and the nucleobases, A, T(U), G and C) and even more complicated structures (polypeptides and base pairs as well as hydrogen bonded structures. We are implementing new approaches to the analysis of vibrational spectra to explain some of the spectral anomalies. These studies have already revealed that assignments of spectra in double-stranded DNA can be made. This has been done in part by understanding the anharmonicity of the amino group in the nucleobase adenine (A) and partly using comparisons of isotopomers of DNA with vibrational calculations within the harmonic approximation.
There are other important aspects of vibrational spectroscopy of interest such as vibronic coupling mechanisms that affect optical transitions and the vibrational Stark effect. We have made theoretical contributions in these areas relevant to an understanding of protein dynamics and electrostatics, respectively.
Protein dynamics is important for understanding protein function. What does an enzyme do after it binds the substrate? This question addresses mechanism in molecular terms. We have studied the classic biophysical example of myoglobin to obtain a baseline for understanding these issues in more complicated systems. The next logical step is to study a protein that looks like myoglobin, but functions as an enzyme. Dehaloperoxidase (DHP) from the marine worm Amphitrite ornata is such an enzyme. DHP has high structural homology, but low sequence with globins. provides an interesting comparison between a peroxidase enzyme and oxygen carriers. The enzyme is capable oxidizing p-bromophenol and other halophenols to quinone in the presence of peroxide. This model system is a small enzyme that can be exploited using surface attachment strategies. Spectroscopy can both characterize and detect binding in sensor applications using the p-bromophenol ligand binding site in the heme pocket as a probe.
Most surface work has used gold-thiol chemistry to create self-assembled monolayers. We use gold-thiol chemistry as a breadboard, but the most useful surfaces are solids that can be used for total internal reflection spectroscopy. In the infrared these are ZnSe, Ge and Si. The oxides of Ge and Si can be functionalized. However, there is a great deal of chemistry that must be done to exploit these surfaces for biological applications. Any surface will have a tendency to interact with a biomolecule. Proteins can denature on surfaces or bind non-specifically. We avoid this by placing a passivating layer on the surface. Detection of a monolayer is difficult at best and so we are working to create organized multilayers in the volume sampled by an optical beam or attenuated wave.
We have also begun to explore the functionalization of metal oxide conductors such as indium tin oxide and fluorine-doped tin oxide. These studies have revealed interesting spectroscopic applications. Metal oxide conductors reflect in the infrared and transmit in the visible. This means that IR reflection-absorption spectroscopy can be used to characterize surface adlayers. In addition, this means that the plasma frequency of the metal oxide is the near infrared (typically near 1 micron wavelength). Plasma resonance frequency shifts can be used to detect binding events. Thus, these materials can also be used to make sensors.
