4CNet: A Confidence-Aware, Contrastive, Conditional, Consistency Model for Robot Map Prediction in Multi-Robot Environments

Mobile robots in unknown cluttered environments with irregularly shaped obstacles often face sensing, energy, and communication challenges which directly affect their ability to explore these environments. In this paper, we introduce a novel deep learning method, Confidence-Aware Contrastive Conditional Consistency Model (4CNet), for mobile robot map prediction during resource-limited exploration in multi-robot environments. 4CNet uniquely incorporates: 1) a conditional consistency model for map prediction in irregularly shaped unknown regions, 2) a contrastive map-trajectory pretraining framework for a trajectory encoder that extracts spatial information from the trajectories of nearby robots during map prediction, and 3) a confidence network to measure the uncertainty of map prediction for effective exploration under resource constraints. We incorporate 4CNet within our proposed robot exploration with map prediction architecture, 4CNet-E. We then conduct extensive comparison studies with 4CNet-E and state-of-the-art heuristic and learning methods to investigate both map prediction and exploration performance in environments consisting of uneven terrain and irregularly shaped obstacles. Results showed that 4CNet-E obtained statistically significant higher prediction accuracy and area coverage with varying environment sizes, number of robots, energy budgets, and communication limitations. Real-world mobile robot experiments were performed and validated the feasibility and generalizability of 4CNet-E for mobile robot map prediction and exploration.Comment: 14 pages, 10 figure

In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation and inhibitor experiments, supplemented with in-cell solid-state NMR, confirmed the in-cell assembly. The ArM containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system requires no engineering of the microbial host, the protein scaffold or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis in biosynthesis, achieving a hybrid metabolism

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

Sensitivity Enhanced Solid-State NMR of Soluble Proteins in Cells

In the recent years, there have been no unique protein structure topologies deposited in the PDB, which may reflect the finiteness of unique protein structures that exist in nature. Moreover, traditional structural studies have neglected the cellular context of the biomolecules, as there were no techniques that enabled atomistic studies in a highly heterogeneous cellular environment. Developments in biomolecular NMR and cryo-EM techniques have fueled the conception and the growth of cellular structural biology as a field. While cryo-EM focusses on very large molecular machines and protein assemblies, NMR has tackled small and heterogenous proteins. The two techniques thus complement each other. An analogous complementarity also exists within NMR, where cellular studies using solution-state NMR has been limited to small and soluble proteins while solid-state NMR has enabled studies on rather large insoluble proteins. The motivation behind pursuing the research presented in this thesis is to design and implement solid-state NMR approaches which would enable the study of soluble and promiscuous proteins within the cellular milieu. Despite their soluble nature, promiscuous proteins have remained out of the reach for solution-state NMR as they interact with their binding partners leading to reduced tumbling in crowded cellular environments. We have developed and tested an innovative DNP-ssNMR approach which is compatible with both mammalian and bacterial cells

Sensitivity Enhanced Solid-State NMR of Soluble Proteins in Cells

In the recent years, there have been no unique protein structure topologies deposited in the PDB, which may reflect the finiteness of unique protein structures that exist in nature. Moreover, traditional structural studies have neglected the cellular context of the biomolecules, as there were no techniques that enabled atomistic studies in a highly heterogeneous cellular environment. Developments in biomolecular NMR and cryo-EM techniques have fueled the conception and the growth of cellular structural biology as a field. While cryo-EM focusses on very large molecular machines and protein assemblies, NMR has tackled small and heterogenous proteins. The two techniques thus complement each other. An analogous complementarity also exists within NMR, where cellular studies using solution-state NMR has been limited to small and soluble proteins while solid-state NMR has enabled studies on rather large insoluble proteins. The motivation behind pursuing the research presented in this thesis is to design and implement solid-state NMR approaches which would enable the study of soluble and promiscuous proteins within the cellular milieu. Despite their soluble nature, promiscuous proteins have remained out of the reach for solution-state NMR as they interact with their binding partners leading to reduced tumbling in crowded cellular environments. We have developed and tested an innovative DNP-ssNMR approach which is compatible with both mammalian and bacterial cells

When Small becomes Too Big : Expanding the Use of In-Cell Solid-State NMR Spectroscopy

Solution-state NMR spectroscopy has become a powerful tool to study soluble proteins in cells, provided that they tumble sufficiently fast. In addition, cryo-electron tomography (cryo-ET) has recently displayed a tremendous potential to probe structures of large proteins and assemblies in their native cellular environments. However, challenges remain to obtain atomic-level information in native cell settings for proteins that are small, disordered, or are strongly engaged in intermolecular interactions. In this Minireview, we discuss recent progress in using sensitivity enhanced solid-state NMR spectroscopy methods in the context of cellular structural biology

In vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)–phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel–Crafts alkylation of indoles and the Diels–Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole‐Cell Biocatalysis

Artificial metalloenzymes (ArMs), which are hybrids of catalytically active transition metal complexes and proteins, have emerged as promising approach to the creation of biocatalysts for reactions that have no equivalent in nature. Here we report the assembly and application in catalysis of ArMs in the cytoplasm of E. coli cells based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneously added copper(II)‐phenanthroline (Cu(II)‐phen) complex. The ArMs are spontaneously assembled by addition of Cu(II)‐phen to E. coli cells that express LmrR and it is shown that the ArM containing whole cells are active in the catalysis of the enantioselective vinylogous Friedel‐Crafts alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cell‐ fractionation and inhibitor experiments and confirmed by in‐cell solid‐state NMR. A mutagenesis study showed that the same trends in catalytic activity and enantioselectivity in response to mutations of LmrR were observed for the ArM containing whole cells and the isolated ArMs. This made it possible to perform a directed evolution study using ArMs in whole cells, which gave rise to a mutant, LmrR_A92E_M8D that showed increased activity and enantioselectivity in the catalyzed vinylogous Friedel‐Crafts alkylation of a variety of indoles. The unique aspect of this whole‐cell ArM system is that no engineering of the microbial host, the protein scaffold or the cofactor is required to achieve ArM assembly and catalysis. This makes this system attractive for applications in whole cell biocatalysis and directed evolution, as demonstrated here. Moreover, our findings represent important step forward towards achieving the challenging goal of a hybrid metabolism by integrating artificial metalloenzymes in biosynthetic pathways