The encapsulation of proteins in porous sol-gels has captured the attention of both applied and basic research scientists because the encapsulated proteins retain functional activity and exhibit enhanced stability. Despite the widespread interest in using this technology to develop new classes of robust biosensors, no in-depth biophysical study of protein response to sol-gel encapsulation has been carried out. The project proposed herein will systematically examine the impact of three promising encapsulation protocols on the conformation and dynamics of the well-characterized heme proteins, hemoglobin and myoglobin. We have probed the solution-phase conformation - secondary through quaternary - of these heme proteins with a myriad of laser-based time-resolved and steady state spectroscopic techniques, and are therefore uniquely qualified to examine the dynamic and static heme protein/sol-gel interactions. The spectroscopic tools at our fingertips include: CW UV resonance Raman, CW and time-resolved visible resonance Raman, time-resolved near IR absorption, time resolved fluorescence anisotropy and fluorescence lifetime measurements. Subtle as well as large-scale conformational changes and the associated dynamics are probed when combining these techniques. In addition, nanosecond, time-resolved absorption spectroscopy will primarily be used to characterize the ligand rebinding kinetics of the heme proteins in the sol-gels. It is anticipated that this comprehensive study will result in an algorithm for matching an encapsulation protocol to a particular biomedical or biophysical application.
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