In this proposal the PIs will study how proteins fold in an active 3-dimensional structure. A fundamental prediction following from the landscape theory of protein folding is that protein folding can occur rapidly downhill on a multidimensional folding funnel. In this project the PIs will use a combination of single molecule fluorescence (SMF) and microfluidic methods to more directly explore downhill folding. The project will focus on the following specific goals. 1) Measure folding/unfolding rates of multiple putative downhill folding proteins using an ultrafast microfluidic mixer and ensemble fluorescence. 2) Directly probe the downhill character of folding for single protein molecules. Microfluidic mixing will trigger the folding/unfolding, and SMF will be used to follow the distribution of populations and dynamics during the transition. Non-equilibrium SMF/microfluidics methods, specifically with respect to time resolution and triggering will be improved for these experiments. 3) In order to further improve the time-resolution of the triggering, the PIs will also explore methods to photo-initiate folding of single protein molecules trapped in a new microfluidic device. The main intellectual merit of the proposed activity is to provide the first direct observations of this so-far elusive process, and provide a substantial test of the landscape theory of protein folding. These experiments will also be used to better examine many so-far hidden details of folding energy landscapes. The combined microfluidics/SMF methods developed during the project will be useful for a wide range of single-molecule investigations where the fast non-equilibrium kinetics needs to be resolved, and thus will be of general utility to the scientific community. The tools and general insights from this project will be disseminated widely. Undergraduate and post-doctoral students will receive training in evolving state-of-the-art technologies during the course of this interdisciplinary project involving two institutions and components from physics, biology and chemistry. Results from the project will also be incorporated into classes.
Proteins perform and facilitate an incredible breadth of essential functions in living organisms, ranging from chemistry to structure, transport and regulation. Protein molecules encode a large degree of complexity in terms of their structures (shapes) and dynamics (movements). It is increasingly being recognized that delineating this complex physics of proteins is critical to understanding many aspects of their function, and that experimental tests of underlying physical theories are critical in forming such an understanding. Along these lines, in this project, we have been developing microfluidic and single-molecule methods aimed to provide enhanced tests of fast protein processes such as downhill folding. Single-molecule experiments have increasingly been revolutionizing multiple areas of science in recent years. They provide new and powerful ways to probe proteins, by interrogating one molecule at a time to extract the most detailed information. Microfluidic techniques enable tight control of physical and chemical environments on the micrometer scale, providing several important benefits for single-molecule experiments. These include enhanced mixing, generation of concentration gradients, and rapid changes of the medium, all done using very small quantities of sample. Our work synergistically used single-molecule and microfluidic methods that resulted in several exciting developments. In one key example, we developed improved rapid micro-mixing methods to abruptly initiate folding reactions, while using fluorescence to detect the conformations of the individual proteins. In this experiment, it is possible to monitor how quickly and in what way the protein structure changes, helping to understand the physics of the reaction. In other work, we devised a method to improve the data collection and speed of single-molecule experiments used to probe structural changes in proteins. In yet another set of experiments, we collaboratively tested a theoretical prediction about structural complexity of a protein dimer. Our results directly confirmed the prediction and also provided new and interesting insight into the system. Overall, the research results from the project included developments in microfluidic mixing methods for studying fast processes at single-molecule and collective levels, methods to improve single-molecule detection in rapid processes, initial work on initiation of biochemical reactions by rapid laser-aided cooling and applications to fast-folding and other proteins. Broader impacts of the project have emerged in multiple directions. These include contributions to improved scientific methods and infrastructure, multidisciplinary teaching and training of students and young scientists, and dissemination of scientific knowledge related to the project. The methods and insights from the project have implications in several areas of modern biological physics and biology. In particular, the methods from the project can be used to study the physics of a variety of molecules that are key components of the cellular machinery, hence contributing to the infrastructure of important areas of science. More generally in the long term, such research is anticipated to continue to provide an improved fundamental understanding of the molecular physics of living systems, and hence positively contribute to society. Teaching, training and outreach activities related to the project have had significant positive impacts on multiple students and post-doctoral trainees, and thus will contribute to their developing successful research careers. It should be noted that the research was intrinsically cross-disciplinary and cutting-edge, providing an excellent training ground for the next generation of scientists. Results of the work have been widely disseminated through published original papers and reviews in high-quality and well-read journals, and presentations in seminars and at local, national and international meetings.
