1. Nonequilibrium, Single-Molecule Studies of Protein Unfolding Ching-Hwa Kiang, Rice University, DMR-0505814 We used the atomic force microscope to manipulate and unfold individual molecules of the titin I27 domain and reconstructed its free energy surface using Jarzynski's equality. The free energy surface for both the stretching and unfolding was reconstructed using an exact formula that relates the nonequilibrium work fluctuations to the molecular free energy. The unfolding free energy barrier, i.e. the activation energy, was directly obtained from experimental data for the first time. This work demonstrates that it is now possible to obtain free energy surfaces for molecular systems, including systems where only nonequilibrium work can be measured. N. C. Harris, Y. Song, and C.-H. Kiang, (2006) submitted. Stretching single protein molecules

2. Nonequilibrium, Single-Molecule Studies of Protein Unfolding Ching-Hwa Kiang, Rice University, DMR-0505814 The advance in nano-scale instrumentation has made it possible to manipulate and observe reactions at the single-molecule level. “Seeing is believing” has provided basis for proof of principles and gaining important insight into the complex biological world. We want to explore biological molecular properties and interactions using novel nano-tools such as atomic force microscope to study single bio-molecules. Knowledge of accurate thermodynamic properties of protein systems is important for biophysical and biochemical understandings of molecular processes, expanding understanding of biology and medicine under physiological conditions. The challenge lies on how to relate data from single-molecule measurements to fundamental and physiologically relevant properties. The biggest problem is that while manipulating the molecules, we have perturbed the system and forced it to undergo transitions through a non-equilibrium process. For example, in studying the heart muscle protein titin, we stretch the molecule faster than what happens during heart muscle contraction and relaxation. The first step is to determine the quantities at zero force limit. Application of the recently derived nonequilibrium work theorem to such problem should allow us to extract useful information that are not accessible with conventional methods.

3. Education: Four graduate (Nolan Harris, Eric Botello, both minority, Yang Song, and Chad Richard), two undergraduate (Jacob Sargent and Casey Wang, female) and three postdoc (Dr. Leiming Li, Dr. Wei Liao, and Dr. Fang-Chi Hsu, female) worked on research supported by this NSF Award. Students and postdoc background ranges from physics, chemistry, to bioengineering, making the research environment truly multidisciplinary. Social Impact: Single-molecule manipulation and measurements allow us to probe many complex cellular assemblies and biological processes on a nanometer scale. The mechanical properties of biomolecules are functions underlying the structures, and are crucial to many biological processes. For example, mechanical properties of muscle proteins, such as titin and dystrophin, play an important role in muscle contraction, and malfunction of the proteins have been directly linked to many heart and muscular diseases. We are developing a method that can be used as a reliable technique to be used to determine a wide range of fundamental biomolecular properties that are important in understanding diseases. Nonequilibrium, Single-Molecule Studies of Protein Unfolding Ching-Hwa Kiang, Rice University, DMR-0505814 Image: http://www.oxford- personal-trainer.co.uk

4. The research project supported by NSF is interdisciplinary because it uses physical techniques to solve biological problems. Graduate and undergraduate students, as well as postdoctoral fellows were trained to use the state-of-the-art atomic force microscopy and spectroscopy techniques. The research group members came from different disciplines, ranging from physics, chemistry, to engineering. Interactions with the Texas Medical Center on a regular basis have made the multidisciplinary approach to science beneficial to my team members as they advance the field of biophysics at Rice and beyond. Nonequilibrium, Single-Molecule Studies of Protein Unfolding Ching-Hwa Kiang, Rice University, DMR-0505814