Biology subjects

Kerfeld, C. A.

Publications and source records attributed to Kerfeld, C. A..

10 recordsLinked to original sources

bioRxiv2024

Structure Characterization of Bacterial Microcompartment Shells via X-ray Scattering and Coordinate Modeling: Evidence for adventitious capture of cytoplasmic proteins

Bacterial microcompartments (BMCs) are self-assembling, protein shell structures that are widely investigated across a broad range of biological and abiotic chemistry applications. A central challenge in BMC research is the targeted capture of enzymes during shell assembly. While crystallography and cryo-EM techniques have been successful in determining BMC shell structures, there has been only limited success in visualizing the location of BMC-captured enzyme cargo. Here, we demonstrate the opportunity to use small angle X-ray scattering (SAXS) and pair density distribution function (PDDF) measurements combined with quantitative comparison to coordinate structure models as an approach to characterize BMC shell structures in solution conditions directly relevant to biochemical function. Using this approach, we analyzed BMC shells from Haliangium ochraceum that were isolated following expression in E. coli. The analysis allowed BMC shell structures and the extent of encapsulated enzyme cargo to be identified. Notably, the results demonstrate that HO-BMC shells adventitiously capture significant amounts of cytoplasmic cargo during assembly in E. coli. Our findings highlight the utility of SAXS/PDDF analysis for evaluating BMC architectures and enzyme encapsulation, offering valuable insights for designing BMC shells as platforms for biological and abiotic catalyst capture within confined environments.

biophysics↗

bioRxiv2024

The new phycobilisome linker protein ApcI regulates high light adaptation in Synechocystis sp. PCC 6803

Phycobilisomes are versatile cyanobacterial antenna complexes that harvest light energy to drive photosynthesis. These complexes can also adapt to various light conditions, dismantling under high light to prevent photo-oxidation and arranging in rows under low light to increase light harvesting efficiency. Light quality also influences phycobilisome structure and function, as observed under far-red light exposure. Here we describe a new, phycobilisome linker protein, ApcI (previously hypothetical protein sll1911), expressed specifically under red light. We characterized ApcI in Synechocystis sp. PCC 6803 using mutant strain analyses, phycobilisome binding experiments, and protein interaction studies. Mutation of apcI conferred high light tolerance to Synechocystis sp. PCC 6803 compared to wild type with reduced energy transfer from phycobilisomes to the photosystems. Binding experiments revealed that ApcI replaces the linker protein ApcG at the membrane-facing side of the phycobilisome core using a paralogous C-terminal domain. Additionally, the N-terminal extension of ApcI was found to interact with photosystem II. Our findings highlight the importance of phycobilisome remodeling for adaptation under different light conditions. The characterization of ApcI provides new insights into the mechanisms by which cyanobacteria optimize light-harvesting in response to varying light environments.

plant biology↗

bioRxiv2024

Electrochemical cofactor recycling of bacterial microcompartments

Bacterial microcompartments (BMCs) are prokaryotic organelles that consist of a protein shell which sequesters metabolic reactions in its interior. While most of the substrates and products are relatively small and can permeate the shell, many of the encapsulated enzymes require cofactors that must be regenerated inside. We have analyzed the occurrence of an enzyme previously assigned as a cobalamin (vitamin B12) reductase and, curiously, found it in many unrelated BMC types that do not employ B12 cofactors. We propose NAD+ regeneration as a new function of this enzyme and name it MNdh, for Metabolosome NADH dehydrogenase. Its partner shell protein BMC-TSE assists in passing the generated electrons to the outside. We support this hypothesis with bioinformatic analysis, functional assays, EPR spectroscopy, protein voltammetry and structural modeling verified with X-ray footprinting. This discovery represents a new paradigm for the BMC field, identifying a new, widely occurring route for cofactor recycling and a new function for the shell as separating redox environments.

microbiology↗

bioRxiv2024

Orthogonality of shell proteins across BMC subclasses in cyanobacteria

Bacterial microcompartments (BMC) are protein-based organelles broadly distributed across all bacterial phyla and subclassified into [≥]60 functional variants. Despite their evolutionary and metabolic diversity, shell proteins that structurally compose the BMC surface are closely related across BMC classes. Herein, we sought to identify molecular and physiological features that could promote independent operation of more than one BMC type within the same cell by reducing inter-organelle cross-talk of shell proteins. We heterologously expressed shell proteins from the structurally well-defined BMC of Haliangium ochraceum (HO) within Synechococcus elongatus PCC 7942, a model cyanobacterium containing the {beta}-carboxysome. We find considerable cross-reactivity of the HO hexameric shell protein (HO BMC-H) with components of the {beta}-carboxysome; HO BMC-H can integrate into carboxysomes, disrupt its ultrastructural organization, and impair its associated CO2 fixation reactions. S. elongatus is unable to maintain the integrity of the {beta}-carboxysome over time when HO BMC-H is expressed in the absence of one or more of three broad strategies that act to increase the orthogonality between HO and carboxysome BMC shell proteins: i) reduced expression of promiscuous shell proteins; ii) sequestration of free HO BMC-H proteins via co-expression of other members of the same HO shell protein class, or; iii) heterologous expression of BMC positional system proteins McdAB (Maintenance of carboxysome distribution AB), revealing a putative moonlighting function of the McdAB protein family. Our results have implications for bacteria that encode more than one BMC within their genome and may have translational implications for the use of engineered BMCs for biotechnological applications.

synthetic biology↗

bioRxiv2024

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria which encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semi-permeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo, the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We use molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo. Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceum model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

biophysics↗

bioRxiv2023

Structural and quantum chemical basis for OCP-mediated quenching of phycobilisomes

Cyanobacteria employ large antenna complexes called phycobilisomes (PBS) for light harvesting. However, intense light triggers non-photochemical quenching, where the Orange Carotenoid Protein (OCP) binds to PBS, dissipating excess energy as heat. The mechanism of efficiently transferring energy from phycocyanobilins in PBS to canthaxanthin in OCP remains insufficiently understood. Using advanced cryogenic-electron microscopy, we unveiled the OCP-PBS complex structure at 1.6-2.1 [A] resolution, showcasing its inherent flexibility. Employing multiscale quantum chemistry, we disclosed the quenching mechanism. Identifying key protein residues, we clarified how canthaxanthins transition dipole moment in its lowest-energy dark state becomes large enough for efficient energy transfer from phycocyanobilins. Our energy transfer model offers a detailed understanding of the atomic determinants of light harvesting regulation and antenna architecture in cyanobacteria. One sentence summaryHigh-resolution cryo-EM structure of the OCP-PBS complex reveals intrinsic motions and enables the atomic simulation of the quenching mechanism

biophysics↗

bioRxiv2021

Structures of the Cyanobacterial Phycobilisome

The phycobilisome is an elaborate antenna that is responsible for light-harvesting in cyanobacteria and red-algae. This large macromolecular complex captures incident sunlight and transfers the energy via a network of pigment molecules called bilins to the photosynthetic reaction centers. The phycobilisome of the model organism Synechocystis PCC 6803 consists of a core to which six rods are attached but its detailed molecular architecture and regulation in response to environmental conditions is not well understood. Here we present cryo-electron microscopy structures of the 6.2 MDa phycobilisome from Synechocystis PCC 6803 resolved at 2.1 [A] (rods) to 2.7 [A] (core), revealing three distinct conformations, two previously unknown. We found that two of the rods are mobile and can switch conformation within the complex, revealing a layer of regulation not described previously. In addition, we found a novel linker protein in the structure, that may represent a long-sought subunit that tethers the phycobilisome to the thylakoid membrane. Finally, we show how excitation energy is transferred within the phycobilisome and correlate our structures with known spectroscopic properties. Together, our results provide detailed insights into the biophysical underpinnings of cyanobacterial light harvesting and lay the foundation for bioengineering of future phycobilisome variants and artificial light harvesting systems.

biochemistry↗

bioRxiv2021

Structure of the Quenched Cyanobacterial OCP-Phycobilisome Complex

Photoprotection is an essential mechanism in photosynthetic organisms to balance the harvesting of light energy against the risks of photodamage. In cyanobacteria, photoprotective non-photochemical quenching relies on the interaction between a photoreceptor, the Orange Carotenoid Protein (OCP), and the antenna, the phycobilisome (PBS). Here we report the first structure of the OCP-PBS complex at 2.7 [A] overall resolution obtained by cryo-electron microscopy. The structure shows that the 6.2 MDa PBS is quenched by four 34 kDa OCP organized as two dimers. The complex also reveals that the structure of the active form of the OCP is drastically different than its resting, non-quenching form, with an [~]60 [A] displacement of its regulatory domain. These results provide a high-resolution blueprint of the structural basis of the protective quenching of excess excitation energy that enables cyanobacteria to harvest light energy and fix CO2 across environmentally diverse and dynamic surface of our planet.

biochemistry↗

bioRxiv2021

A Catalog of the Diversity and Ubiquity of Metabolic Organelles in Bacteria

Bacterial microcompartments (BMCs) are organelles that segregate segments of metabolic pathways, which are incompatible with surrounding metabolism. In contrast to their eukaryotic counterparts, the selectively permeable membrane of BMCs, the shell, is composed of protein. While the sequestered enzymes vary among functionally distinct BMCs, the proteins that form diverse BMC shells are structurally homologous; this enables the bioinformatic identification of the organelles by locating genes encoding shell proteins, which are typically proximal to those for the encapsulated enzymes. With recent advances in genome-resolved metagenomics and the emphasis on "microbial dark matter", many new genome sequences from diverse and obscure bacterial clades have become available. We find the number of identifiable BMC loci has increased twenty-fold since the last comprehensive census of 2014. Moreover, the addition of new types we uncovered doubles the number of distinct BMC types known. These expand the range of catalysis encapsulated in BMCs, underscoring that there is dark biochemistry that is compartmentalized in bacterial organelles yet to be discovered through genome sequencing. Our comprehensive catalog of BMCs provides a framework for their identification, correlation with bacterial niche adaptation, and experimental characterization, and broadens the foundation for the development of BMC-based nanoarchitectures for biomedical and bioengineering applications.

microbiology↗

bioRxiv2017

Synthetic OCP Heterodimers are Photoactive and Recapitulate the Fusion of Two Primitive Carotenoproteins in the Evolution of Cyanobacterial Photoprotection

The Orange Carotenoid Protein (OCP) governs photoprotection in the majority of cyanobacteria. It is structurally and functionally modular, comprised of a C-terminal regulatory domain (CTD), an N-terminal effector domain (NTD) and a ketocarotenoid; the chromophore spans the two domains in the ground state and translocates fully into the NTD upon illumination. Using both the canonical OCP1 and the presumably more primitive OCP2 from Fremyella diplosiphon, we show that an NTD-CTD heterodimer forms when the domains are expressed as separate polypeptides. The carotenoid is required for the heterodimeric association, assembling an orange complex which is stable in the dark. Both OCP1 and OCP2 heterodimers are photoactive, undergoing light-driven heterodimer dissociation, but differ in their ability to reassociate in darkness, setting the stage for bioengineering photoprotection in cyanobacteria as well as for developing new photoswitches for biotechnology. Additionally, we reveal that homodimeric CTD can bind carotenoid in the absence of NTD, and name this truncated variant the C-terminal domain-like Carotenoid Protein (CCP). This finding supports the hypothesis that the OCP evolved from an ancient fusion event between genes for two different carotenoid-binding proteins ancestral to the NTD and CTD. We suggest that the CCP and its homologs constitute a new family of carotenoproteins within the NTF2-like superfamily found across all kingdoms of life.

plant biology↗