Featuring expert speakers from Instruct Centres across Europe, Instruct-ERIC Webinar Series: Structure Meets Function highlights some of the latest developments in structural biology, demonstrating how integrative methods are enabling scientists to decipher the mechanisms that underpin health and disease.
Watch the previous webinars in the series here.
The sixth webinar in the series will be hosted by Instruct Centre BE on 12 January 2021, 11:00 - 12:30 CET.
Webinar moderator: Jan Steyaert
VIB-VUB Center for Structural Biology, Brussel
Talk 1: Curli – structure, function and application of a bacterial amyloid pathway
Speaker: Han Remaut
Affiliation: VIB-VUB Center for Structural Biology, Brussel
Abstract: Curli are functional amyloid fibers assembled by many Gram-negative bacteria as part of an extracellular matrix that encapsulates the bacteria within a biofilm. A multicomponent secretion-assembly system ensures the safe transport of the aggregation-prone curli subunits across the periplasm and outer membrane, and coordinates subunit self-assembly into surface-associated amyloid fibers. To avoid the build-up of potentially toxic intracellular protein aggregates, the timing and location of the interactions of the different curli proteins are of paramount importance. In this webinar, I will discuss our molecular and structural insights in the nucleation and polymerisation of the curli subunit CsgA, and how this functional amyloid evolved to circumvent intracellular toxicity. Additionally, I will discuss the structural insights in the curli secretion channel CsgG and its accessory protein CsgF, how the pathway is thought to translocate curli subunits across a biological bilayer that is devoid of known hydrolysable energy sources or electrochemical gradiënts, and how the structural features of these proteins enable nanopore sensing applications such as DNA/RNA sequencing.
Talk 2: Protein formulation through automated screening of pH and buffer conditions using the Robotein® high throughput facility
Speaker: André Matagne
Affiliation: Centre for Protein Engineering, InBioS RU, Liege
Abstract: Among various factors, the direct environment (e. g. pH, buffer components, salts, additives, etc…) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins.
In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated: i) the protein stability, using differential scanning fluorimetry and ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine β-lactamases, two metallo-β-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (VHH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins.
Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein.