Prof. Martin Zanni at the University of Wisconsin in Madison, and collaborators James Skinner (UW-Madison), Daniel Raleigh (SUNY at Stony Brook), and Sean Decatur (Oberlin College) are supported by the Collaborative Research in Chemistry Program to devise and test methods based on two-dimensional infrared (2D IR) spectroscopy for characterization of protein structure and function. The techniques are particularly important for studying systems for which NMR and x-ray crystallography are difficult to apply, such as proteins in cell membranes and protein aggregates. The work brings together a diverse and complementary team of researchers to develop a rigorous protocol for calculating 2D IR spectra from atomistic structural models through a novel combination of theory, experiment, and synthesis. To develop the theoretical methodology, well-characterized proteins are either isotope-labeled or mutated at individual residues so that the linewidths and/or couplings of specific residues can be measured with 2D IR spectroscopy.
While the work is primarily directed toward developing 2D IR as a tool for protein kinetics, it can be applied to other systems as well. Undergraduate and graduate students and postdocs involved in this study are being trained to work in a collaborative interdisciplinary and inter-institutional environment. Participation of underrepresented groups derives from exchange programs among Oberlin, Howard University, Stony Brook, and Wisconsin. Cyber-infrastructure components include web-conferences to discuss research progress and dissemination of user-friendly software for the theoretical calculation of spectra. An undergraduate physical chemistry lab experiment is being developed to show how FTIR spectroscopy can be used as a tool for protein secondary structure determination; the experiment will be tested in the physical chemistry lab courses at Wisconsin, Stony Brook, Oberlin, and Howard, and then made available to the broader academic community. Institutions that lack the needed instrumentation will be able to incorporate this experiment into their curriculum by taking advantage of the web-based remote access features being developed.
Nearly everything on our planet, including ourselves, are dictated by molecules. Some are simple, like the air that we breath. Others are complex, like the proteins that flex our muscles. All are so small that we cannot see them with even the best light microscopes. But it is important to "see" them, because we need to know what they look like. Molecules function because of their shapes. A drug works because it fits like a key into a keyhole, locking the door to the disease. A plastic is hard, because each molecule is entwined with the others around it. Thus, as a society, we need to know the shapes of molecules. And since they cannot be seen, we need to devise ways of deducing their shapes. Over the past 50 years, scientists have developed many novel ways to deduce the shapes of molecules, which scientists call molecular structure. Some methods work well on molecules that quickly tumble, like air. Another method feels their shape with a finely tipped needle. But there is no single method that works for all molecules. Two particularly challenging classes of molecules are ones that are on surfaces, like the proteins in the walls of our cells, and molecules that move, like the proteins that flex our muscles. In these cases, and others, the best structural methods cannot be applied. As a result, how do we design a drug to fit to it or a different molecule to wrap around it? We would have to rely on luck. And sometimes we are lucky, but molecules are so complicated and subtle, that most often we are not. This grant from the National Science Foundation funded a team of four researchers to develop a promising new tool for studying the structures of molecules. The technique is called two-dimensional infrared spectroscopy, because it produces an abstract picture of a molecule by the way it reflects two pulses of heat from a laser, kind of like how an ultrasound can imagine a fetus in a woman’s uterus. What is most important, is that 2D IR spectroscopy works well where the other methods fail, such as for surface bound molecules and dynamic molecules. Our grant was focused on learning how to better interpret the abstract picture into a molecular shape. The interpretation is not trivial, just like the signal measured by an ultrasound probe is not actually a picture, but has to be digitally processed into an image. We made tremendous progress. We have now developed, arguably, the most accurate method for interpreting 2D IR spectra. And, we have demonstrated its application to several proteins on a surface membrane, with unprecedented results. Now that it is proven, it can be applied by anyone in the world to study their particular problem at hand. And there are lots of important applications, from fighting the influenza virus to developing new solar cells, because these types of problematic molecules exist nearly everywhere.
