With co-funding from the Division of Molecular and Cellular Biosciences, the Office of International Science and Engineering, and the Office of Polar Programs, the Analytical and Surface Chemistry Program supports Prof. Ido Braslavsky of Ohio University to study interactions between specialized antifreeze proteins (AFPs) and ice using fluorescence microscopy techniques combined with a unique ice growth/flow microfluidic cell. With collaborators at Queens University (Canada), Yale University, and the University of California - San Diego, Dr. Braslavsky's group seeks to directly visualize AFPs on ice surfaces, and thus determine their location and concentrations under well defined conditions of growth, temperature, and solute flow. The overall aim is improved understanding of the mechanisms of AFPs, and more broadly of biomolecule influence on crystal growth.
Graduate and undergraduate students in the interdisciplinary Braslavsky group will participate in the research and will attend a Workshop on Ice Binding Proteins held in Canada, thus experiencing first-hand international scientific collaborations. The knowledge gained from the research will be incorporated into a new graduate Biophysics lab course being developed by the PI. Outreach activities to local schools through indoor and outdoor demonstrations at the Athens Public Library examine many aspects of ice growth in the natural environment. Results will also be incorporated into an exhibit "Ice as a Mineral" at the Yale Peabody Museum of Natural History (organized by collaborator John Wettlaufer). Broad societal impacts that may result include more reliable food supplies through prevention of frost damage, enhancement of food preservation technology, and better organ, tissue and blood preservation.
Antifreeze is not just for cars: Antifreeze proteins protect animals and plants from the cold by preventing ice crystals from getting too big. Animals and plants often need to cope with freezing temperatures. They do this using proteins that can bind to tiny crystals of ice and prevent them from getting bigger. These antifreeze proteins (AFPs) are found in fish, insects, fungi, bacteria, and plants. The influence of AFPs on ice includes the ability to stabilize ice crystals that form when water is cooled below its normal freezing point. They also inhibit the recrystallization of ice, where big crystals grow at the expense of small crystals. Recrystallization is often what causes damage in frozen tissue. In addition, AFPs change the shapes of ice crystals, leading to shapes like those in figure 1. The shaping of the ice can be used by the frozen dairy products industry in stabilizing ice cream texture. The project, "Experimental Study on the Interaction between Antifreeze Proteins and Ice" has helped us to understand the way AFPs inhibit ice growth and shape the ice crystals that do form. The project was perform by the groups of Prof. Ido Braslavsky at Ohio University, and at the Hebrew University of Jerusalem, in collaboration with Prof. Peter Davies from Queens University in Ontario, Canada, Prof John Wettlaufer from Yale University, and Prof. Alex Groismann from the University of California San Diego. We developed a unique combination of experimental tools to investigate the AFPs (see figure 2). A temperature-controlled microfluidic device made up of tiny channels allowed us to control minute amounts of liquid and tiny ice crystals. Fluorescent dyes attached to the AFP molecules allowed us to track AFP binding to ice crystals. We used a microscope to watch the growth of ice within solutions that contained AFP molecules so we could measure the shape of the crystals and where the AFP molecules attach. See www.ohio.edu/research/communications/coldcase.cfm for further details. Our investigations contributed to understanding how AFPs work. We found that there are two main ways of controlling the shape of ice crystals. AFPs that have a moderate ability to inhibit ice growth, control the shape by controlling the location of formation of additional ice. Strongly binding, hyperactive AFPs control ice crystal shape by controlling the melting of the crystals into their final shapes (see figure 2). This general finding will serve as new method to classify AFPs. The two main methods of controlling growth come from the ability of the hyperactive AFPs to block all directions of growth, while moderate-binding AFPs block only some directions of growth. In addition, we showed that AFPs bind to ice permanently and inhibit ice growth even when no additional proteins are available in the solution. AFPs can also prevent ice melting at temperatures above the regular melting point, thus allowing the state of superheated ice. These results were published in two papers in the prestigious scientific journal Proceedings of the National Academy of Sciences of the U.S.A. (PNAS) in 2010 (figure 3) and 2013. On both papers, the first author is a PhD student, Yeliz Celik, from Ohio University. These finding represent a significant contribution to the understanding of the AFP activity and give a direct proof to the long-debated irreversible binding of AFPs to ice surfaces. The PI also engaged in outreach and mentoring of graduate and undergraduate students. A PhD student graduated based on work directly related to the funded research. The project included the training of a postdoctoral fellow. An undergraduate student summer project in Israel allowed a student to experience international research. An international conference on Ice Binding Proteins was organized in Canada and was attended by collaborators and by students from the PI’s group. (See http://pldserver1.biochem.queensu.ca/IBP_meeting_2011/) The PI and the Co-PI developed and taught biophysics-inspired lab experiments involving laser tweezers and microfluidic devices that were used for demonstrations at Ohio University Department of Physics & Astronomy lab classes for undergraduates and graduate students as well as demonstrations at Open Houses in 2009 and 2011. The PI did outreach events for elementary school students in Athens, Ohio. In these hands-on classes, the students have been engaged in activities relevant to atmospheric physics phenomena such as the rainbow, snow and clouds. (Figure 4 and 5) To summarize, our research on AFPs resulted in a new understanding of AFP interaction with ice crystals, including their irreversible attachment, the way they shape crystals during melting, and their ability to allow superheated ice. Education of undergraduate students, as well as graduate and postdoctoral fellows was part of this award. Outreach activities engaged elementary school students in hands on science activities and with a direct interaction with a university level researchers. Better understanding of AFP interaction with ice has future applications in food science and cryopreservation of cell and tissue for medical use.
