Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein have featured in the construction of nanoreactors synthetic organelles, relatively little is known about underlying molecular parameters that govern stability flux through their pores. In this work, we systematically designed 24 variants Thermotoga maritima encapsulin cage, featuring pores different sizes charges. Twelve pore were...

Subcellular compartments often serve to store nutrients or sequester labile toxic compounds. As bacteria mostly do not possess membrane-bound organelles, they have rely on protein-based compartments. Encapsulins are one of the most prevalent compartmentalization strategies found in prokaryotes. Here, we show that desulfurase encapsulins can and large amounts crystalline elemental sulfur. We determine 1.78-angstrom cryo-EM structure a 24-nanometer desulfurase-loaded encapsulin. Elemental...

Abstract Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated specific motifs known as targeting peptides (TPs), though the structural basis for encapsulation largest encapsulin class,...

Encapsulins are a class of microbial protein compartments defined by the viral HK97-fold their capsid protein, self-assembly into icosahedral shells, and dedicated cargo loading mechanism for sequestering specific enzymes. often misannotated traditional sequence-based searches yield many false positive hits in form phage capsids. Here, we develop an integrated search strategy to carry out large-scale computational analysis prokaryotic genomes with goal discovering exhaustive curated set all...

Encapsulins are microbial protein nanocages capable of efficient self-assembly and cargo enzyme encapsulation. Due to their favorable properties, including high thermostability, protease resistance, robust heterologous expression, encapsulins have become popular bioengineering tools for applications in medicine, catalysis, nanotechnology. Resistance against physicochemical extremes like temperature low pH is a highly desirable feature many biotechnological applications. However, no...

Enzyme nanoreactors are nanoscale compartments consisting of encapsulated enzymes and a selectively permeable barrier. Sequestration colocalization can increase catalytic activity, stability, longevity, highly desirable features for many biotechnological biomedical applications enzyme catalysts. One promising strategy to construct is repurpose protein nanocages found in nature. However, protein-based often exhibit decreased partially caused by mismatch shell selectivity the substrate...

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite strong preference for icosahedral symmetry, we show simple point mutations virus-like capsid drive assembly unique symmetry-reduced structures. Starting with encapsulin Myxococcus xanthus , 180-mer bacterial adopts well-studied viral HK97 fold, use mass photometry and...

Encapsulins are self-assembling protein compartments found in prokaryotes and specifically encapsulate dedicated cargo enzymes. The most abundant encapsulin class Dye-decolorizing Peroxidases (DyPs). It has been previously suggested that DyP encapsulins involved oxidative stress resistance bacterial pathogenicity due to DyPs' inherent ability reduce detoxify hydrogen peroxide while oxidizing a broad range of organic co-substrates. Here, we report the structural biochemical analysis widely...

In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes from GC-rich Gram-positive bacteria, particularly the phylum Actinobacteria, are often difficult to overexpress purify soluble form using standard Escherichia coli expression systems. Here, we present protocol heterologously nanocompartments encapsulin-containing Streptomyces coelicolor. Successful production begins with transfer...

Terpenoids are the largest class of natural products, found across all domains life. One most abundant bacterial terpenoids is volatile odorant 2-methylisoborneol (2-MIB), partially responsible for earthy smell soil and musty taste contaminated water. Many 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in model bacterium Streptomyces griseus. Here, we revise function EshA, now referred as Sg Enc, show that it Family 2B encapsulin shell...

Abstract Protein nanocages play crucial roles in sub‐cellular compartmentalization and spatial control all domains of life have been used as biomolecular tools for applications biocatalysis, drug delivery, bionanotechnology. The ability to their assembly state under physiological conditions would further expand practical utility. To gain such control, we introduced a peptide capable triggering conformational change at key structural position the largest known encapsulin nanocompartment. We...

Abstract Protein capsids are a widespread form of compartmentalisation in nature. Icosahedral symmetry is ubiquitous derived from spherical viruses, as this geometry maximises the internal volume that can be enclosed within. Despite strong preference for icosahedral symmetry, we show simple point mutations virus-like capsid drive assembly novel symmetry-reduced structures. Starting with encapsulin Myxococcus xanthus , 180-mer bacterial adopts well-studied viral HK97 fold, use mass photometry...

Understanding the structure and structure–function relationships of membrane proteins is a fundamental problem in biomedical research. Given difficulties inherent to performing mechanistic biochemical biophysical studies vitro, we previously developed facile HeLa cell-based cell-free expression (CFE) system that enables efficient reconstitution full-length (FL) functional inner nuclear Sad1/UNC-84 (SUN) (i.e., SUN1 SUN2) supported lipid bilayers. Here, provide evidence suggests...

Terpenoids are the largest class of natural products, found across all domains life. One most abundant bacterial terpenoids is volatile odorant 2-methylisoborneol (2-MIB), partially responsible for earthy smell soil and musty taste contaminated water. Many 2-MIB biosynthetic gene clusters were thought to encode a conserved transcription factor, named EshA in model bacterium

Summary Viruses exploit host cytoskeletal elements and motor proteins for trafficking through the dense cytoplasm. Yet molecular mechanism that describes how viruses connect to machinery is unknown. Here, we demonstrate first example of viral microtubule from purified components: HIV-1 hijacking transport machinery. We discover directly binds retrograde microtubule-associated motor, dynein, not via a cargo adaptor, as previously suggested. Moreover, show motility supported by multiple,...

Sarcomeric myosins have the remarkable ability to form regular bipolar thick filaments that, together with actin thin filaments, constitute fundamental contractile unit of skeletal and cardiac muscle. This has been established for over 50 years yet a molecular model filament not attained. In part this is due lack detailed coiled-coil that constitutes myosin rod. The self-assemble resides in C-terminal section known as light meromyosin (LMM) which exhibits strong salt-dependent aggregation...

Abstract Protein shells or capsids are a widespread form of compartmentalization in nature. Viruses use protein to protect and transport their genomes while many cellular organisms for varied metabolic purposes. These protein-based compartments often exhibit icosahedral symmetry consist small number structural components with defined roles. Encapsulins prevalent strategy prokaryotes. All encapsulins studied thus far single shell that adopts the viral HK97-fold. Here, we report...

Abstract Intracellular compartmentalization is essential for all cells and enables the regulation optimization of metabolism 1 . One main functions subcellular compartments storage nutrients 2–4 As bacteria do generally not possess membrane-bound organelles, they often have to rely on functionally analogous protein-based 2,5–7 Encapsulin nanocompartments are one most prevalent strategies found in prokaryotes 5,8 Here we show that desulfurase encapsulins represent a novel sulfur compartment...

Abstract Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization Streptomyces noursei enzyme AlbAB, cyclodipeptide oxidase (CDO) carrying out α,β-dehydrogenations during biosynthesis antibiotic albonoursin. We show that AlbAB is megadalton heterooligomeric filament containing covalently bound flavin mononucleotide cofactors....

Abstract Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein have recently been featured in the construction of nanoreactors synthetic organelles, relatively little is known about underlying molecular parameters that govern cage stability flux through their pores. In this work, we systematically designed 24-member library variants based on T. maritima encapsulin, each featuring pores different...

Abstract Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated specific motifs known as targeting peptides (TPs), though the structural basis for encapsulation largest encapsulin class,...

Encapsulin nanocompartments are a class of prokaryotic organelles that enable specialized biochemistry through the sequestration enzymatic cargo within selectively permeable protein shell. Although encapsulin systems utilize variety cargos, shell has only ever been shown to be composed single type protomer. Here, we report structural characterization and proposed assembly dynamics an nanocompartment with novel two-component from Streptomyces lydicus. Separate heterologous expression...