33 research outputs found
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
Postmodification via Thiol-click Chemistry Yields Hydrophilic Trityl-nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization
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)âphenanthroline 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 FriedelâCrafts alkylation of indoles and the DielsâAlder 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 WholeâCell 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)âphenanthroline (Cu(II)âphen) complex. The ArMs are spontaneously
assembled by addition of Cu(II)âphen 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 FriedelâCrafts
alkylation of indoles. The ArM assembly in E. coli is further supported by a combination of cellâ
fractionation and inhibitor experiments and confirmed by inâcell solidâstate 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 FriedelâCrafts alkylation of a variety of indoles. The unique aspect of this wholeâcell 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
Challenging the Neoliberal Truth of Student as Consumer: Rethinking Student as Public Intellectual
The space-time variations in water vapor in the upper troposphere and lower stratosphere (UTLS) over the Asian summer monsoon (ASM) regions are investigated using six years of observations from the Aura-MLS to understand the ASM role in transporting water vapor to the tropical stratosphere. While some previous studies indicated that the ASM plays a role in dehydrating the lower stratosphere, the present investigations revealed that the ASM region plays an active role in hydrating rather than dehydrating the tropical lower stratosphere. The analysis also shows that only during the month of August (ASM) is moist air pumped into the lower stratosphere over the two key geographical locations (India and Western Pacific). The air parcel from the tropical tropopause takes approximately 10 - 12 months to reach the mid-stratosphere with an ascent rate of 2.8 Ă 10-4 m s-1. Thus it is envisaged that the present results will have important implications in understanding the exchange processes across the tropopause and its role in stratosphere chemistry