Mapping the spatiotemporal dynamics of proteins is a fundamental and critical challenge for understanding and addressing biomedical problems. Unfortunately, current tools cannot observe key structural motions, because they (1) require frozen, static samples, the dynamics of which are non-physiological; and/or (2) lack the necessary spatial resolution. Techniques such as NMR, x-ray crystallography and cryo-EM provide structural data without the time-ordering of the corresponding dynamics. Thus, these techniques cannot be applied to multi-step processes, such as drug binding. In contrast, single-molecule techniques follow individual proteins through these processes and, as a result, single-molecule Frster Resonance Energy Transfer (smFRET) has emerged as the state-of-the-art for observing protein dynamics. smFRET measures the ef?ciency of energy transfer between two dyes attached to key points on the protein structure. However, smFRET is limited to 3 nm spatial resolution, leaving nanoscale protein dynamics inaccessible. Consequently, a new, improved experimental tool is required. I propose to develop a new spectroscopic ruler that resolves dynamics with spatial resolution spanning nearly an order of magnitude (1-7 nm). Whereas smFRET measures the ef?ciency, I will measure the rate of energy transfer at the single-molecule level. To perform this mea- surement, I am developing a new technique, single-molecule Ultrafast Spectroscopy (smUS), by combining tools from two distinct ?elds, single-molecule biophysics and condensed-phase ultrafast spectroscopy. The signi?cant experimental challenges associated with both sets of tools require my unique background in these ?elds for successful implementation. By directly measuring the rate of energy transfer and using state-of-the-art theoretical frameworks to interpret the results, I introduce a new ability to probe conformational dynamics down to <1 nm. This technique is applicable to any protein, peptide, DNA or RNA that can be functionally labelled, and will be made broadly available to the research community. In my lab, we focus on receptor proteins. In 60% of drug delivery, receptor proteins bind ligands to initiate microscopic motions. Despite the importance of these motions, their dynamics are inaccessible with existing tools due to limitations in resolution, sensitivity, or experimental conditions. My proposed tech- nique resolves these motions with 1 nm spatial resolution and 10 ms temporal resolution to reveal the interplay between microscopic mechanisms and macroscopic function. This unprecedented resolution uniquely enables the study of therapeutic targets. Thus, we envision this approach uncovering the microscopic underpinnings of a broad range of biomedical problems.
In protein function, including drug delivery, microscopic motions as small as one nanometer often drive macroscopic phenomena and uncovering these motions would dramatically improve the speed, ef?cacy and cost of drug discovery. The key obstacle is that current technologies lack the spatial resolution to monitor these motions. We overcome this obstacle by developing and applying a new nanometer distance assay that directly accesses protein motion with unprecedented spatiotemporal resolution under physiological conditions.
