Self-organization is a key tool for the construction of functional nanomaterials. We have recently established a novel method for the self-organization of biomolecular building blocks and nanoparticles. Towards this goal, protein containers, engineered with opposite surface charge, are used as an atomically precise ligand shell for the assembly of inorganic nanoparticles.[1] The assembly of these protein-nanoparticle composites yields highly ordered nanoparticle superlattices with unprecedented precision. The structure of the protein scaffold can be tuned with external stimuli such as metal ion concentration.[2] Importantly, these composite materials show catalytic activity inside the porous material.[3] Along these lines, the protein containers used as a scaffold offer a viable route towards renewable materials.[4]

For the formation of biohybrid materials, the inorganic cargo has to be encapsulated into the protein containers. Here, we demonstrate that the highly specific cargo-loading mechanism of the bacterial nanocompartment encapsulin can be employed for encapsulation of artificial cargo such as inorganic nanoparticles.[5] For this purpose, gold nanoparticles were decorated with cargo-loading peptides. By lock-and-key interaction between the peptides and the peptide-binding pockets on the inner container surface, the nanoparticles are encapsulated with extremely high efficiency. Most notably, the supramolecular peptide binding is independent from external factors such as ionic strength.[5] Cargo-loading peptides may serve as generally applicable tool for efficient and specific encapsulation of cargo molecules into a protein compartment. Moreover, these nanoparticle protein-container composites are suitable for applications as building blocks in materials, exploiting the plasmonic properties of gold nanoparticles for light manipulation or sensing.

[1] M. Künzle, T. Eckert, T. Beck, J. Am. Chem. Soc. 2016, 138, 12731-12734.
[2] M. Künzle, T. Eckert, T. Beck, Inorg. Chem. 2018, 57, 13431-13436.
[3] M. Lach, M. Künzle, T. Beck, Chem. Eur. J. 2017, 23, 17482-17486.
[4] a) M. Künzle, M. Lach, T. Beck, Dalton Transactions 2018, 47, 10382-10387; b) M. Lach, M. Künzle, T. Beck, Biochemistry 2019, 58, 140-141.
[5] M. Künzle, J. Mangler, M. Lach, T. Beck, Nanoscale 2018, 10, 22917-22926.

In recent years, the genome of microorganisms from various habitats, such as industrial
waste areas, areas rich in vegetable oils or in soils contaminated with oil, has been
analyzed. This has allowed us to identify enzymes with functions that offer enormous
potential for various applications in the industrial sector, from catalysis to remediation.
Much of this knowledge has been leveled by bacteria of the genus Pseudomonas, since their
metabolic versatility has been involved in a large number of biotechnological applications.
Lipases catalyze the hydrolysis of triacylglycerides whose products are fatty acids and
glycerol. These enzymes have a catalytic triad consisting of a serine, an acid residue
(glutamic acid or aspartic acid) and a histidine. In addition, lipases have a preserved
structure known as a lid. This lid is a mobile element that discovers the active site.
It has been observed that lipase 2 (lip2) of Pseudomonas alcaligenes has a sequence
identity of 48% with the lipase of P. aeruginosa, while the region of the lid has high
identity with lids described in other halophilic or psychrophilic bacteria such as
Marinobacter mobilis, Oleiphilus messinensis, Oleispira antartica or Hahellaceae
bacterium, which makes it different from other lipases described until now.
The lip 2 gene of P. alcaligenes was cloned into the Pet 28a vector and was expressed in
Escherichia coli BL21 cells in order to obtain a crystallographic structure that allows us to
describe and characterize the possible structural changes of the enzyme, as well as the
possible implications of these changes in the stability and catalysis of lip 2 of P.
alcaligenes.

Structural and functional studies of key enzyme (LpxC (UDP-3-O-acyl-N-acetylglucosamine deacetylase) from salmonella typhi involved in Gram-negative bacterial lipid A biosynthesis

Sudhir Kumar Pal¹, Sanjit Kumar^1*

¹ Centre for Bio Separation Technology, VIT University, Katpadi, Vellore, Tamil Nadu-632014

Abstract

Over the last decade, in the case of gram-negative bacteria the frequency of antibacterial resistance especially in ill and hospitalized patients (including multidrug resistance (MDR) and its association with severe infectious diseases) has increased at alarming rates. Salmonella typhi (gram negative bacteria) is an ubiquitous pathogen responsible for a number of diseases such as pneumonia, meningitis, etc. The increasing number of infections caused by MDR Salmonella typhi must be re-explored for novel treatment strategies. LpxC, a metal dependent amidase (highly active in the presence of Zn2+ ions (Kd ~60 pM)) is one such vital and rate-limiting enzyme committing the step of Lipid A (a strong human immuno-modulator bacterial endotoxin) biosynthesis. Many structures of LpxC enzyme in complex with different inhibitors are solved by various structural biology techniques but most of these inhibitors have been reported to have poor anti-microbial activity. Compounds with indole-2-carboxamide scaffold in their skeleton display various biological activities including anti-bacterial activity. We have done indole-2-carboxamide scaffold-based virtual screening and observed inhibitors like 0435 (-9.0 kcal/mol), 0436 (-9 kcal/mol), 1812 (-8.6 kcal/mol), 2584 (-8.5 kcal/mol), and 2545 (-8.4 kcal/mol) bound to the functional active site of LpxC.

β-Lactoglobulin (BLG) is a protein from the lipocalin family [1]. BLG is a milk protein with a natural affinity to fatty acids and retinol [2], however, it can bind with relatively high affinity but rather low selectivity a variety of biomolecules [3]. A conserved structural element of lipocalins is the eight-stranded antiparallel β-barrel which is the primary binding site for ligands [4]. Like many proteins from the lipocalin family, BLG can be modified by rational site-directed mutagenesis. Substitutions in the region of the β-barrel can be used to re-design the shape of the binding pocket to create new BLG variants with specific ligand preferences [5]. New variants (mutants) of β-lactoglobulin with increased affinity for tri-cyclic drugs (e.g. antipsychotics and antidepressants) may be used in the future as molecular transporters that selectively recognize and remove toxic compounds from the body.

New BLG mutants with substitutions at positions 39, 56, 58, 71, 92, 105, and 107 were expressed in E.coli. All proteins were purified by anion exchange and size-exclusion chromatography. Crystals were obtained by the vapor diffusion method in the hanging drop setup. New BLG mutants, possessing the different shape of the binding pocket, were co-crystalized with tricyclic drugs (e.g. fluphenazine, clomipramine, and chlorpromazine) and selected fatty acids. X-ray diffraction data were collected at XtaLAB Synergy (Rigaku). Structures of BLG‑ligand complexes (2.5-1.5Å) were solved by molecular replacement. Determined crystal structures revealed that some substitutions reduced the length of the binding pocket preventing fatty acids from binding at this site. Crystallographic studies were supported by biophysical studies (circular dichroism) which allowed to determine the binding constant for selected protein-ligand complexes.

Advances in computational protein design have allowed for the development of new proteins with unique properties. Symmetric designer proteins have remarkable stability and can serve as versatile building blocks for the creation of macromolecular assemblies. Here we present the development and structural determination of SAKe: A new symmetric, stable protein building block with modifiable loops. Following the observation of pH induced 3D self-assembly, we engineered metal binding sites along the protein's internal rotational axis to fabricate 2D surface arrays. Using atomic force microscopy, we demonstrated Cu(II) dependent on-surface 2D self-assembly. Additionally, using dynamic light scattering and x-ray diffraction, we identified and characterized a SAKe mutant which shows in solution Zn(II) mediated nanocage formation. This work showcases a stable and highly modifiable SAKe protein scaffold, which holds promise as a building block for the creation of multi-functional macromolecular materials.