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NANO2021-Nanoteknologi og nye materiale

Bridging biological and soft matter technologies to reveal the molecular mechanism of a bacterial colonization factor

Alternative title: Integrasjon av biologiske og "soft-matter" teknikker i studiet av den molekylære mekanisme bak bakteriell kolonisering

Awarded: NOK 5.2 mill.

Project Manager:

Project Number:

272201

Application Type:

Project Period:

2017 - 2021

Partner countries:

Structural biology has delivered some of the most important scientific breakthroughs in biology in recent time. The main engine in the field has been X-ray crystallography, fueled by highly brilliant X-rays produced by synchrotrons. However, proteins consist of up to 50% of hydrogen atoms that remain hidden to the eye of this technique; yet they play central roles for protein stability and function. Neutron radiation allows us to visualize these omnipresent and light, but immensely important atoms. In addition, neutrons are significantly less destructive to proteins than X-rays, thus allowing an experimental setting that is closer to the natural environment of the proteins. Spallation technology promises to provide neutron beams of higher intensity than ever before, with the most advanced facility, the European Spallation Source (ESS), under construction in Lund, Sweden. This project has employed neutron-radiation-based techniques to complement classical structural biological methods in order to study a common bacterial virulence factor of the water-borne bacterial family of vibrios. Specifically, we were interested in a colonization factor of cholera-bacteria. Similar proteins are also found in many other bacteria, and even in viruses. The techniques include neutron crystallography, a complementary technique to X-ray crystallography that visualizes hydrogens, giving insight into their role at the atomic level. Neutron reflectometry and small-angle neutron scattering (SANS) allow the characterization of the interaction of the protein (and its family members) with its natural crystallized substrate, chitin. We have performed experiments for all three of these techniques, however, the main focus has been on the application of neutron- and X-ray scattering techniques (SANS/SAXS) to characterize the structures in solution or suspension. To distinguish between the two interaction partners, we first developed an efficient protocol for protein deuteration, and determined the D2O match point for the protein's interaction partner chitin in collaboration with researchers at the Institute Laue-Langevin in Grenoble. Some of these experiments have also been performed in the USA, at the National Institute of Standards and Technology (NIST). We could show that the adhesin indeed lines up on chitin, however, not as regular as beads on a string. Our results suggest how protein binding may prepare the ground for microcolony formation of the bacteria. We also investigated the effect of ions on the protein structure and binding of chitin, which is the most abundant biomolecule in marine environments. Next in line is the neutron crystal structure of the deuterated protein. Promising crystals have already been obtained, which diffract X-rays to atomic resolution. Now the crystals need to grow bigger, to allow diffraction by neutrons. In addition to studying a chitin-binding adhesin of cholera-bacteria, we have investigated the structure and function of a closely related protein from Pseudomomas aeruginosa, a bacterium that is infamous for its prevalence in hospital settings and widespread antibiotic resistance. Using a combination of X-ray and neutron-based techniques, and functional studies, our aim was to draw a complete picture of the structure and function of these important medical targets - which will help to combat antibiotic resistance, solve problems of Norwegian food industry and benefit the production of renewable fuels. Moreover, this project has increased our competence in neutron-based structural biology technologies that will prepare us for experiments at the world's brightest neutron source, the ESS.

The two main effects of this project are: (1) It has increased our competence in neutron-based structural biology technologies that will prepare us and the users of our core facilities (NORCRYST) for exciting experiments at the world's brightest neutron source, the ESS. (2) The obtained insights into the structure and function of two bacterial virulence factors open new doors to combat antibiotic resistance.

Currently, neutron based technologies are applied mainly in soft matter and materials science, whereas their application to biological matter is limited. This proposal aims at establishing a platform of competence within neutron based methodologies relevant to the Life Sciences that will prepare us for applications at the European Spallation Source (ESS). Essential technologies of this neutron-based toolbox are neutron crystallography, neutron reflectometry and small-angle neutron scattering. Common to all these techniques is the requirement for protein deuteration. The development, establishment and consolidation of relevant know-how will be of significant value to the structure biological community in Oslo and the rest of Norway. The long term goal is to append neutron based scattering techniques to the synchrotron pipeline of structural biology today, complementing and enhancing future molecular models. As target, we selected the secreted colonization factor GbpA from Vibrio cholerae. This protein serves as adhesin to aquatic biofilms and human mucins, and enzymatically degrades chitin (and maybe other targets?) with its LPMO domain. As such, GbpA promises important insights into biofilm formation and clearance affecting fish and man, as well as exploitable results related to biofuels and hence bioeconomy. In this work, we will use cutting-edge technologies in collaboration with international partners, and will provide internationally competitive career development training to our young recruit.

Publications from Cristin

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NANO2021-Nanoteknologi og nye materiale