Biology subjects

Uchanski, T.

Publications and source records attributed to Uchanski, T..

3 recordsLinked to original sources

bioRxiv2024

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs were identified in mammals, disfunction of which was associated with a series of life-threatening diseases. Advances in structural studies of RyRs are limited by the need for large amounts of native tissue or eukaryotic cell cultures. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification starting from isolated membranes to cryo-EM grade protein is completed within four hours on the bench and produces protein usable for cryo-EM as we show by solving the structures of rabbit RyR1 and bovine and mouse RyR2 solubilized in detergent, reconstituted into lipid nanodiscs or liposomes. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in the cases where the amount of starting material is limited.

biochemistry↗

bioRxiv2022

ChILL & DisCO to discover competitive, connective and allosteric Nanobodies that modulate the SOS1-RAS protein-protein interactions and tune the nucleotide exchange rate

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce novel and broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse Nanobodies that either stabilize or disrupt PPIs in a single experiment. We applied ChILL and DisCO to identify competitive, connective or fully allosteric Nanobodies that inhibit or facilitate the formation of the SOS1*RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro and RAS signalling in cellulo. One of these connective Nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as an innovative pharmacophore for extending the repertoire of potential leads.

bioengineering↗

bioRxiv2019

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 [A] resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAA {beta}3 homopentameric receptor bound to its small-molecule agonist histamine.

biochemistry↗