Passera, A. | bioRxiv Science

bioRxiv2023

A Fast Interferometric Beam Shaper for Multi-Emitter 3D MINFLUX

Beams of light that feature an intensity zero are essential to a variety of optical microscopy methods. Super-resolution techniques like STED and RESOLFT, together with localization strategies like MINFLUX and MINSTED, rely on accurate and fast displacements of such beams and their zeros. Extending these methods to the third dimension requires axial deflection, which, in contrast to lateral deflection, remains technologically challenging on the microsecond scale. Here, we present a fast general-purpose beam-shaping polarization interferometer that, instead of displacing the entire beam, enables such axial deflections by deforming the beam shape to deflect its zero. Based on this approach, we showcase a four-channel dual-color excitation system for three-dimensional MINFLUX imaging and tracking. We include first demonstrations of improved MINFLUX localization schemes that utilize the combination of distinct beam shapes and three-dimensional multi-emitter tracking. We believe that the presented approach will facilitate the broader adoption of three dimensional MINFLUX and provides a versatile basis for future implementations of advanced single-molecule localization methods.

biophysics↗

bioRxiv2023

Simultaneous enhancement of multiple functional properties using evolution-informed protein design

Designing optimized proteins is important for a range of practical applications. Protein design is a rapidly developing field that would benefit from approaches that enable many changes in the amino acid primary sequence, rather than a small number of mutations, while maintaining structure and enhancing function. Homologous protein sequences contain extensive information about various protein properties and activities that have emerged over billions of years of evolution. Evolutionary models of sequence co-variation, derived from a set of homologous sequences, have proven effective in a range of applications including structure determination and mutation effect prediction. In this work we apply one of these models (EVcouplings) to computationally design highly divergent variants of the model protein TEM-1 {beta}-lactamase, and characterize these designs experimentally using multiple biochemical and biophysical assays. Nearly all designed variants were functional, including one with 84 mutations from the nearest natural homolog. Surprisingly, all functional designs had large increases in thermostability and most had a broadening of available substrates. These property enhancements occurred while maintaining a nearly identical structure to the wild type enzyme. Collectively, this work demonstrates that evolutionary models of sequence co-variation (1) are able to capture complex epistatic interactions that successfully guide large sequence departures from natural contexts, and (2) can be applied to generate functional diversity useful for many applications in protein design.

synthetic biology↗