Periodic Reporting for period 3 - TryptoBoost (Boosting tryptophan fluorescence with optical nanoantennas to watch label-free protein dynamics with single molecule resolution at high concentration) Reporting period: 2020-10-01 to 2022-03-31 Summary of the context and overall objectives of the project Proteins execute a broad range of functions that are central to life. Understanding these functions ultimately requires experiments at the single protein level to reveal dynamics and heterogeneities hidden in ensemble-averaged measurements. Currently, the preferred method to study single protein machineries is based on fluorescence techniques. However, fluorescence experiments suffer from major challenges: the need for external fluorescent labeling, weak signals and low non-physiological concentrations in the nanomolar range. These challenges severely limit the applicability and biological relevance of single molecule fluorescence on proteins. The TryptoBoost project aims to overcome all the previous challenges, and efficiently monitor single label-free proteins using their intrinsic tryptophan fluorescence enhanced by optical nanoantennas in the ultraviolet. Using the natural amino acid fluorescence rules out all drawbacks due to external labeling, while the optical nanostructures enable single protein analysis at the physiologically relevant micromolar concentrations thanks to the localization and enhancement of light-matter interactions at the nanoscale. More than 90% of all the 40,000 human proteins contain some aromatic amino acids which are fluorescent in the ultraviolet. The project breakthroughs exploring the UV autofluorescence of proteins without any external lable will benefit a broad range of biophysical, chemical, and medical applications. For instance, it will improve the development of therapeutic drugs, increase the detection sensitivity and read-out speed in analytical biosensing on chip, and provide new nanostructures to enhance ultraviolet photocatalysis. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far At the interface between physics, chemistry and nanosciences, we use aluminum nanometer-sized structures to manipulate the light at the nanoscale and enhance the detection of individual proteins without adding any external fluorescent label to them. We exploit the natural ultraviolet autofluorescence when proteins are illuminated in the deep ultraviolet. We show that aluminum nanoapertures can enhance the autofluorescence of label-free protein and enable their detection at the single molecule level. During our research, we discover the phenomenon of UV-induced corrosion of aluminum and show how surface passivation can prevent it and enable future applications of aluminum nanophotonics. While the limited ultraviolet photostability of proteins has been a major issue limiting this scientific field for many years, we show this problem can be solved using enzymatic oxygen scavengers and chemical reducing agents to promote the photostability of proteins and limit photobleaching. Using fluorescence spectroscopy, we illustrate some artefacts that the dyes can create and find some solution to avoid these issues. We also introduce new methods to quantify the local temperature around metal nanostructures in plasmonics. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) So far the minimum level of detection for protein autofluorescence requires that the protein contains about 24 tryptophan residues. This is already an improvement by more than six-fold as compared to the best state-of-the-art when the project started 2 years and a half ago. We will continue our scientific and technological development to develop better optical microscopes, better UV-resonant plasmonic nanostructures, better photochemical stabilization processes, with the major aim of achieving detection at the single tryptophan level. This ambitious goal requires careful optimization of several aspects, but the detection of several (less than a dozen) tryptophan residues should be fully reachable. We will also apply this new technology to protein investigations, notably proteins of the septin family to investigate their interactions with actin. Lastly, nano-optical trapping experiments will be developed further and combined with fluorescence spectroscopy to quantitatively characterize the trap performance and apply the nano-optical force to study single proteins.
