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Lagarias, J. Clark

Publications and source records attributed to Lagarias, J. Clark.

14 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Cyanobacteriochromes: A Rainbow of Photoreceptors

Widespread phytochrome photoreceptors use photoisomerization of linear tetrapyrrole (bilin) chromophores to measure the ratio of red to far-red light. Cyanobacteria also contain distantly related cyanobacteriochrome (CBCR) proteins that share the bilin-binding GAF domain of phytochromes but sense other colors of light. CBCR photocycles are extremely diverse, ranging from the near-UV to the near-IR. Photoisomerization of the bilin triggers photoconversion of the CBCR input, thereby modulating the biochemical signaling state of output domains such as histidine kinase bidomains that can interface with cellular signal transduction pathways. CBCRs thus can regulate several aspects of cyanobacterial photobiology, including phototaxis, metabolism of cyclic nucleotide second messengers, and optimization of the cyanobacterial light-harvesting apparatus. This review examines spectral tuning, photoconversion, and photobiology of CBCRs and recent developments in understanding their evolution and in applying them in synthetic biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Elucidating the origins of phycocyanobilin biosynthesis and phycobiliproteins

Terrestrial ecosystems and human societies depend on oxygenic photosynthesis, which began to reshape our atmosphere approximately 2.5 billion years ago. The earliest known organisms carrying out oxygenic photosynthesis are the cyanobacteria, which use large complexes of phycobiliproteins as light-harvesting antennae. Phycobiliproteins rely on phycocyanobilin (PCB), a linear tetrapyrrole (bilin) chromophore, as the light-harvesting pigment that transfers absorbed light energy from phycobilisomes to the chlorophyll-based photosynthetic apparatus. Cyanobacteria synthesize PCB from heme in two steps: A heme oxygenase converts heme into biliverdin IXα (BV), and the ferredoxin-dependent bilin reductase (FDBR) PcyA then converts BV into PCB. In the current work, we examine the origins of this pathway. We demonstrate that PcyA evolved from pre-PcyA proteins found in nonphotosynthetic bacteria and that pre-PcyA enzymes are active FDBRs that do not yield PCB. Pre-PcyA genes are associated with two gene clusters. Both clusters encode bilin-binding globin proteins, phycobiliprotein paralogs that we designate as BBAGs (bilin biosynthesis-associated globins). Some cyanobacteria also contain one such gene cluster, including a BBAG, two V4R proteins, and an iron–sulfur protein. Phylogenetic analysis shows that this cluster is descended from those associated with pre-PcyA proteins and that light-harvesting phycobiliproteins are also descended from BBAGs found in other bacteria. We propose that PcyA and phycobiliproteins originated in heterotrophic, nonphotosynthetic bacteria and were subsequently acquired by cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

GUN4 appeared early in cyanobacterial evolution

Photosynthesis relies on chlorophylls, which are synthesized via a common tetrapyrrole trunk pathway also leading to heme, vitamin B12, and other pigmented cofactors. The first committed step for chlorophyll biosynthesis is insertion of magnesium into protoporphyrin IX by magnesium chelatase. Magnesium chelatase is composed of H-, I-, and D-subunits, with the tetrapyrrole substrate binding to the H-subunit. This subunit is rapidly inactivated in the presence of substrate, light, and oxygen, so oxygenic photosynthetic organisms require mechanisms to protect magnesium chelatase from similar loss of function. An additional protein, GUN4, binds to the H-subunit and to tetrapyrroles. GUN4 has been proposed to serve this protective role via its ability to bind linear tetrapyrroles (bilins). In the current work, we probe the origins of bilin binding by GUN4 via comparative phylogenetic analysis and biochemical validation of a conserved bilin-binding motif. Based on our results, we propose that bilin-binding GUN4 proteins arose early in cyanobacterial evolution and that this early acquisition represents an ancient adaptation for maintaining chlorophyll biosynthesis in the presence of light and oxygen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Tetrapyrrole biosynthesis and signaling (chlorophyll, heme, and bilins)

Chlamydomonas utilizes the cyclic tetrapyrroles chlorophyll, heme, siroheme, vitamin B12 and linear tetrapyrrole (bilin) metabolites as cofactors for photosynthetic light harvesting, electron transport, redox reactions, mitigating oxidative stress and signaling. Furthermore, with the exception of vitamin B12, Chlamydomonas can synthesize and degrade all of these tetrapyrroles. In this chapter, we review what is currently known about how Chlamydomonas biosynthesizes and metabolizes tetrapyrroles, how these processes are regulated and the various functions of tetrapyrroles in Chlamydomonas.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

Abstract Photoreceptors in the phytochrome superfamily use 15,16-photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties. Canonical phytochromes include master regulators of plant growth and development in which light signals trigger interconversion between a red-absorbing 15Z dark-adapted state and a metastable, far-red-absorbing 15E photoproduct state. Distantly related cyanobacteriochromes (CBCRs) carry out a diverse range of photoregulatory functions in cyanobacteria and exhibit considerable spectral diversity. One widespread CBCR subfamily typically exhibits a red-absorbing 15Z dark-adapted state similar to that of phytochrome that gives rise to a distinct green-absorbing 15E photoproduct. This red/green CBCR subfamily also includes red-inactive examples that fail to undergo photoconversion, providing an opportunity to study protein–chromophore interactions that either promote photoisomerization or block it. In this work, we identified a conserved lineage of red-inactive CBCRs. This enabled us to identify three substitutions sufficient to block photoisomerization in photoactive red/green CBCRs. The resulting red-inactive variants faithfully replicated the fluorescence and circular dichroism properties of naturally occurring examples. Converse substitutions restored photoconversion in naturally red-inactive CBCRs. This work thus identifies protein–chromophore interactions that control the fate of the excited-state population in red/green cyanobacteriochromes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Natural diversity provides a broad spectrum of cyanobacteriochrome-based diguanylate cyclases

Cyanobacteriochromes (CBCRs) are spectrally diverse photosensors from cyanobacteria distantly related to phytochromes that exploit photoisomerization of linear tetrapyrrole (bilin) chromophores to regulate associated signaling output domains. Unlike phytochromes, a single CBCR domain is sufficient for photoperception. CBCR domains that regulate the production or degradation of cyclic nucleotide second messengers are becoming increasingly well characterized. Furthermore, cyclic di-guanosine monophosphate (c-di-GMP) is a widespread small-molecule regulator of bacterial motility, developmental transitions, and biofilm formation whose biosynthesis is regulated by CBCRs coupled to GGDEF (diguanylate cyclase) output domains. In this study, we compare the properties of diverse CBCR-GGDEF proteins with those of synthetic CBCR-GGDEF chimeras. Our investigation shows that natural diversity generates promising candidates for robust, broad spectrum optogenetic applications in live cells. Since light quality is constantly changing during plant development as upper leaves begin to shade lower leaves—affecting elongation growth, initiation of flowering, and responses to pathogens, these studies presage application of CBCR-GGDEF sensors to regulate orthogonal, c-di-GMP-regulated circuits in agronomically important plants for robust mitigation of such deleterious responses under natural growing conditions in the field.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Bilin-dependent regulation of chlorophyll biosynthesis by GUN4

Significance Enzymes of the chlorophyll biosynthetic pathway, which bind protoporphyrin and Mg-porphyrins, are susceptible to damage by singlet oxygen production in the presence of light and oxygen. These studies show that heme-derived linear tetrapyrroles (bilins) both stimulate and protect the protoporphyrin-binding CHLH subunit of Mg chelatase, the first committed enzyme of the chlorophyll synthesis, from self-sensitized photodamage and turnover via formation of nonphotosensitizing GENOMES UNCOUPLED 4 (GUN4):bilin:porphyrin adducts, which deliver protoporphyrin to CHLH. GUN4:bilin adducts likely evolved to sustain chlorophyll biosynthesis in an oxic world, accounting for retention of bilin synthesis in nearly all oxygenic photosynthetic species on Earth.

photosynthesis↗
DOE OSTI2021

Crystal structure of a far-red–sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism

Significance Phytochromes are well-known far-red-light sensors found in plants that trigger adaptive responses to facilitate competition for light capture with neighboring plants. Red- and far-red sensing are also critical for cyanobacteria living in the far-red–enriched shade of plants. This work reports the crystal structure of a far-red–sensing cyanobacteriochrome, a distant cyanobacterial relative of phytochrome. These studies shed insights into the molecular basis of far-red-sensing by phycobilin-based photoreceptors. Owing to the deep tissue penetration of far-red light, far-red–sensing cyanobacteriochromes are promising protein scaffolds for developing genetically encoded photoswitches, optoacoustic contrast agents, and fluorescent probes for in situ imaging and optogenetic applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (~68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward ( 15Z P r → 15E P g ) and reverse ( 15E P g → 15Z P r ) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15Z P r photoproduct in AnPixJg2. Altogether, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φ p within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold

Significance Cyanobacteriochromes (CBCRs) are small cyanobacterial photoreceptors which are highly diversified and categorized into many lineages based on their primary sequences. In this study, we identified an atypical CBCR exhibiting an orange/green reversible photocycle. Step-by-step site-directed mutagenesis was performed on this native CBCR, and seven new photoconvertible variants were created. During this process, we identified residues crucial for each color tuning event. These seven molecules covering the shorter-wavelength blue-to-orange region would contribute to the future development of multicolored optogenetic tools and are complementary to recently developed molecules sensing longer wavelengths of light.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, in this study we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

59 BASIC BIOLOGICAL SCIENCES↗