Terminal Subunits Research Articles

Phalloidin and DNase I-bound F-actin pointed end structures reveal principles of filament stabilization and disassembly

Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament turnover, but the underlying mechanisms remain incompletely understood. Here, we present three cryo-EM structures of the F-actin pointed end in the presence and absence of phalloidin or DNase I. The two terminal subunits at the undecorated pointed end adopt a twisted conformation. Phalloidin can still bind and bridge these subunits, inducing a conformational shift to a flattened, F-actin-like state. This explains how phalloidin prevents depolymerization at the pointed end. Interestingly, two DNase I molecules simultaneously bind to the phalloidin-stabilized pointed end. In the absence of phalloidin, DNase I binding would disrupt the terminal actin subunit packing, resulting in filament disassembly. Our findings uncover molecular principles of pointed end regulation and provide structural insights into the kinetic asymmetry between the actin filament ends.

Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle’s terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC’s TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

A single septin from a polyextremotolerant yeast recapitulates many canonical functions of septin hetero-oligomers.

Morphological complexity and plasticity are hallmarks of polyextremotolerant fungi. Septins are conserved cytoskeletal proteins and key contributors to cell polarity and morphogenesis. They sense membrane curvature, coordinate cell division, and influence diffusion at the plasma membrane. Four septin homologues are conserved from yeasts to humans, the systems in which septins have been most studied. But there is also a fifth family of opisthokont septins that remain biochemically mysterious. Members of this family, Group 5 septins, appear in the genomes of filamentous fungi, but are understudied due to their absence from ascomycete yeasts. Knufia petricola is an emerging model polyextremotolerant black fungus that can also serve as a model system for Group 5 septins. We have recombinantly expressed and biochemically characterized KpAspE, a Group 5 septin from K. petricola. This septin--by itself in vitro--recapitulates many functions of canonical septin hetero-octamers. KpAspE is an active GTPase that forms diverse homo-oligomers, binds shallow membrane curvatures, and interacts with the terminal subunit of canonical septin hetero-octamers. These findings raise the possibility that Group 5 septins govern the higher-order structures formed by canonical septins, which in K. petricola cells form extended filaments, and provide insight into how septin hetero-oligomers evolved from ancient homomers.

ApcE plays an important role in light-induced excitation energy dissipation in the Synechocystis PCC6803 phycobilisomes

Phycobilisomes (PBs) play an important role in cyanobacterial photosynthesis. They capture light and transfer excitation energy to the photosynthetic reaction centres. PBs are also central to some photoprotective and photoregulatory mechanisms that help sustain photosynthesis under non-optimal conditions. Amongst the mechanisms involved in excitation energy dissipation that are activated in response to excessive illumination is a recently discovered light-induced mechanism that is intrinsic to PBs and has been the least studied. Here, we used single-molecule spectroscopy and developed robust data analysis methods to explore the role of a terminal emitter subunit, ApcE, in this intrinsic, light-induced mechanism. We isolated the PBs from WT Synechocystis PCC 6803 as well as from the ApcE-C190S mutant of this strain and compared the dynamics of their fluorescence emission. PBs isolated from the mutant (i.e., ApcE-C190S-PBs), despite not binding some of the red-shifted pigments in the complex, showed similar global emission dynamics to WT-PBs. However, a detailed analysis of dynamics in the core revealed that the ApcE-C190S-PBs are less likely than WT-PBs to enter quenched states under illumination but still fully capable of doing so. This result points to an important but not exclusive role of the ApcE pigments in the light-induced intrinsic excitation energy dissipation mechanism in PBs.

Obtaining Spatial Variations in Cabernet Sauvignon (Vitis vinifera L.) Wine Flavonoid Composition and Aromatic Profiles by Studying Long-Term Plant Water Status in Hyper-Arid Seasons

The spatial variability in vineyard soil might negatively affect wine composition, leading to inhomogeneous flavonoid composition and aromatic profiles. In this study, we investigated the spatial variability in wine chemical composition in a Cabernet Sauvignon (Vitis vinifera L.) vineyard in 2019 and 2020. Because of the tight relationships with soil profiles, mid-day stem water potential integrals (Ψstem Int) were used to delineate the vineyard into two zones, including Zone 1 with relatively higher water stress and Zone 2 with relatively lower water stress. Wine from Zone 2 generally had more anthocyanins in 2019. In 2020, Zone 1 had more anthocyanins and flavonols. Zone 2 had more proanthocyanidin extension and terminal subunits as well as total proanthocyanidins in 2020. According to the Principal Component Analyses (PCA) for berry and wine chemical composition, the two zones were significantly different in the studied wine aromatic compounds. In conclusion, this study provides evidence of the possibility of managing the spatial variability of both wine flavonoid composition and aromatic profiles through connecting vineyard soil variability to grapevine season-long water status.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The ‘self-acting’ (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the ‘interface-acting’ (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and ‘poisoning’ plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

MpaR-driven expression of an orphan terminal oxidase subunit supports Pseudomonas aeruginosa biofilm respiration and development during cyanogenesis.

Cyanide is an inhibitor of heme-copper oxidases, which are required for aerobic respiration in all eukaryotes and many prokaryotes. This fast-acting poison can arise from diverse sources, but mechanisms by which bacteria sense it are poorly understood. We investigated the regulatory response to cyanide in the pathogenic bacterium Pseudomonas aeruginosa, which produces cyanide as a virulence factor. Although P. aeruginosa has the capacity to produce a cyanide-resistant oxidase, it relies primarily on heme-copper oxidases and even makes additional heme-copper oxidase proteins specifically under cyanide-producing conditions. We found that the protein MpaR controls expression of cyanide-inducible genes in P. aeruginosa and elucidated the molecular details of this regulation. MpaR contains a DNA-binding domain and a domain predicted to bind pyridoxal phosphate (vitamin B6), a compound that is known to react spontaneously with cyanide. These observations provide insight into the understudied phenomenon of cyanide-dependent regulation of gene expression in bacteria.

Coordination Chemistry of Open-Ring Hemiporphyrazines

Hemiporphyrazines, phthalocyanine analogues consisting of two isoindoline subunits and two pyridine or other aromatic subunits, have attracted considerable interest not only from the viewpoint of their fundamental properties but also in materials science. Since the first report on hemiporphyrazine about 70 years ago, various types of hemiporphyrazines have been synthesized to date. However, when comparing the rich chemistry of porphyrins and other porphyrinoids, the structural diversity of hemiporphyrazines and azaporphyrinoids is still limited. In particular, much less attention has been paid for acyclic hemiporphyrazines, which is in sharp contrast to many reports on acyclic porphyrinoids. Herein we report synthesis of open-ring hemiporphyrazines and their coordination chemistry. Helical metal complexes (1M ) and dinuclear metal complexes ((2M)2 ) were obtained depending on the terminal subunit of the open-hemiporphyrazine ligands. The effects of the central metal ion on molecular and electronic structures will be discussed in the presentation. Figure 1

Structures of the free and capped ends of the actin filament.

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Coupling Hydrophilic Interaction Chromatography and Reverse-Phase Chromatography for Improved Direct Analysis of Grape Seed Proanthocyanidins.

Acid-catalyzed depolymerization is recognized as the most practical method for analyzing subunit composition and the polymerization degree of proanthocyanidins, involving purification by removing free flavan-3-ols, as well as acid-catalyzed cleavage and the identification of cleavage products. However, after the removal of proanthocyanidins with low molecular weights during purification, the formation of anthocyanidins from the extension subunits accompanying acid-catalyzed cleavage occurred. Thus, grape seed extract other than purified proanthocyanidins was applied to acid-catalyzed depolymerization. Hydrophilic interaction chromatography was developed to quantify free flavan-3-ols in grape seed extract to distinguish them from flavan-3-ols from terminal subunits of proanthocyanidins. Reverse-phase chromatography was used to analyze anthocyanidins and cleavage products at 550 and 280 nm, respectively. It is found that the defects of the recognized method did not influence the results of the subunit composition, but both altered the mean degree of polymerization. The established method was able to directly analyze proanthocyanidins in grape seed extract for higher accuracy and speed than the recognized method.

Pseudouridimycin-A Potent Nucleoside Inhibitor of the RNA Polymerase Beta Prime Subunit of Streptococcus pyogenes.

Streptococcus pyogenes (group A streptococcus, GAS), a Gram-positive bacterium, is a major cause of mild to severe life-threatening infections. Antibacterial resistance to penicillin and macrolides poses a major threat in the treatment of GAS and necessitates alternate drugs and newer antibiotics. In this direction, nucleotide-analog inhibitors (NIAs) have emerged as important antiviral, antibacterial, and antifungal agents. Pseudouridimycin (PUM), a nucleoside analogue inhibitor discovered from the soil bacterium Streptomyces sp., has proven to be effective against multidrug-resistant S. pyogenes. However, the mechanism of its activity remains elusive. In this study, subunits of the RNA polymerase of GAS have been identified as targets for PUM inhibition and the binding regions have been mapped to the N-terminal domain of the β' subunit, using computational methods. The antibacterial activity of PUM against macrolide-resistant GAS was evaluated. PUM showed effective inhibition at 0.1-1 μg/mL concentration, which was higher when compared to earlier reports. The molecular interaction between PUM and the RNA polymerase β'-N terminal subunit was investigated using isothermal titration calorimetry (ITC), circular dichorism (CD), and intrinsic fluorescence spectroscopy. The thermodynamic characterization by ITC showed an affinity constant of 6.175 × 105 M-1 denoting a moderate affinity. Fluorescence studies revealed that the interaction of protein-PUM was spontaneous in nature and follows a static quenching of tyrosine signals from the protein. The near- and far-UV CD spectral analysis concluded that PUM induced local tertiary structural changes in the protein, predominantly contributed by aromatic amino acids rather than notable changes in the secondary structure. Hence PUM could be a promising lead drug target for macrolide-resistant strains of S. pyogenes and enable eradication of pathogen in the host system.

Flammulina filiformis Pkac Gene Complementing in Neurospora crassa Mutant Reveals Its Function in Mycelial Growth and Abiotic Stress Response

Flammulina filiformis is a popular edible mushroom that easily suffers from heat and oxidative stresses. The cyclic adenylate-dependent protein kinase A (cAMP/PKA) pathway is the main signaling pathway in response to environmental stress, and the PKAC is the terminal catalytic subunit of this pathway. In this study, the Pkac gene was identified in F. filiformis, which was highly conserved in basidiomycetes and ascomycetes. The transcription analysis showed that the Pkac gene was involved in the mycelial growth and the fruiting body development of fungi. In Neurospora crassa, the Pkac gene deletion (ΔPkac) resulted in the slower growth of the mycelia. We complemented the F. filiformis FfPkac to N. crassa ΔPkac mutant to obtain the CPkac strain. The mycelial growth in the CPkac strain was restored to the same level as the WT strain. In addition, the FfPkac gene showed significantly up-regulated expression under heat and oxidative stresses. By analyzing the differentially expressed genes of ΔPkac and Cpkac with WT, respectively, seven downstream genes regulated by Pkac were identified and may be related to mycelial growth. They were mainly focused on microbial metabolism in diverse environments, mitochondrial biogenesis, protein translation and nucleocytoplasmic transport. RT-qPCR results confirmed that the expression patterns of these seven genes were consistent with FfPkac under heat and oxidative stresses. The results revealed the conserved functions of PKAC in filamentous fungi and its regulatory mechanism in response to heat and oxidative stresses.

The effects of regions and the wine aging periods on the condensed tannin profiles and the astringency perceptions of Cabernet Sauvignon wines.

This study sought to determine the effects of wine-producing regions and aging periods on the astringency and chemistry of condensed tannins of Cabernet Sauvignon dry red wines. A wine quality study was performed with 5 vintages of 32 Cabernet Sauvignon wines produced in four Chinese wine-producing regions, Hebei (H), Xinjiang (X), Inner Mongolia (NM), and Ningxia (NX). Condensed tannin profiles were assessed by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The (-)-epicatechin as the terminal subunit (tEC) is the major differential component between regions. Correlation analysis revealed that condensed tannin concentration and composition significantly affected the sensory evaluation of astringency. Condensed tannin concentrations were significantly and negatively correlated with wine aging periods. However, no significant correlation was found between aging periods and condensed tannin subunits (as mole%) composition. The current findings enhance the understanding of condensed tannins' chemical and astringency characteristics in Cabernet Sauvignon wines.

Betel Nut (Areca Catechu) Extract Against Vancomycin- Resistant Enterococcus

Vancomycin-resistant Enterococcus (VRE) is a bacterium that is resistant to various antibiotics, especially vancomycin. The VRE resistance mechanism is caused by a change of amino acid residue in the terminal subunit of peptide NAM/NAG, D-alanyl- D-alanine, which is the vancomycin site. D-alanyl-D-alanine and D-alanyl-D-serine variations only provide one site for vancomycin which is used for four spot hydrogen interactions. These changes cause the affinity to decrease by 1000 times so antibiotics cannot perform their functions. Betel nut is known for its action as a natural remedy. Betel nuts have antimicrobial properties against gram-positive and gram-negative bacteria. It is thought that this is through their phenolic compounds. The aim of this research was to determine the antimicrobial activity of betel nuts against VRE. The research was conducted in a microbiology laboratory in June-July 2021. The antimicrobial action of the betel nut was assessed using the microdilution method and scanning electron microscopy. Betel nut extract was prepared using water, n-hexane, and ethyl acetate. The control used was tetracycline. It was found that the concentrations that were able to inhibit VRE were 256 μg/mL of ethyl acetate extract, > 1024 μg/mL of water extract, 1024 μg/mL of n- hexane extract, and 128 μg/mL of tetracycline. The results showed a clear spot in the thin layer chromatography that was in contact with agar with VRE, and the clear spot belonged to the phenolic substances of the betel nut. The microscopy results showed that the VRE cells were destroyed when they came in contact with the betel nut extract. Therefore, we can conclude that the betel nut extract was able to inhibit VRE growth. These findings can be used to support research on alternative new drug compounds.
 Keywords: antimicrobial, betel nut, microdilution, SEM, VRE

Prediction of tannin profile in grape (Vitis vinifera L.) skins during berry maturation using a rapid mechanical puncture approach

Tannin structure and composition are variable during grape maturation, and crucially determine perceived astringency, body structure and aging capacity of red wines. This study investigated the evolution of condensed tannins (CTs) in grape skins as maturation progressed and the feasibility of using a rapid mechanical puncture approach for assessing the CTs profile. The results showed that the mean degree of polymerization (mDP), molecular mass (MM), and proportions of (–)-epigallocatechin in extension subunits (EGC_ext) and (–)-epicatechin-3-O-gallate in terminal subunits (ECG_term) of skins increased during grape maturation, while CTs content and the proportion of (–)-epicatechin-3-O-gallate in extension subunits decreased. The predictive models built by random forest for CTs content based on skin weight, mDP, MM_subunit, EGC_ext, and ECG_term obtained good results with high squared correlation coefficients of prediction and calibration (R2_P > 0.85 and R2_C ≈ 0.95). In addition, the classifications of CTs characteristics obtained from ripe and unripe samples were observed in different principal component spaces. This study indicated that the mechanical properties were useful for predicting skin CTs profile, estimating tannin maturity stages, and providing information for optimal harvesting and winemaking protocols.

Azulene-Embedded [n]Helicenes (n=5, 6 and 7).

Azulene is a non-benzenoid aromatic building block with unique chemical structure and physicochemical properties. By using the "bottom-up" synthetic strategy, we synthesized three azulene-embedded [n]helicenes ([n]AzHs, n=5, 6 and 7), in which one terminal azulene subunit was fused with n-2 benzene rings. P- and M-enantiomers were observed in the packing diagrams of [5]-, and [6]AzHs. However, P- and M-[7]AzHs could be isolated by recrystallization of the racemic mixture. These [n]AzHs were endowed with new properties through the azulene moiety such as low-lying first electric state (S1 ), small optical energy gap and anti-Kasha emission. [6]-, and [7]AzHs exhibit strong chiroptical responses with high absorption dissymmetry factor (gabs ) maxima of about 0.02, which is among the highest |gabs | values of helicenes in the visible range. These azulene-embedded [n]helicenes contribute to the non-benzenoid helicene library and allow the structure-property relationships to be better understood.

Azulene‐Embedded [n]Helicenes (n=5, 6 and 7)

AbstractAzulene is a non‐benzenoid aromatic building block with unique chemical structure and physicochemical properties. By using the “bottom‐up” synthetic strategy, we synthesized three azulene‐embedded [n]helicenes ([n]AzHs, n=5, 6 and 7), in which one terminal azulene subunit was fused with n‐2 benzene rings. P‐ and M‐enantiomers were observed in the packing diagrams of [5]‐, and [6]AzHs. However, P‐ and M‐[7]AzHs could be isolated by recrystallization of the racemic mixture. These [n]AzHs were endowed with new properties through the azulene moiety such as low‐lying first electric state (S1), small optical energy gap and anti‐Kasha emission. [6]‐, and [7]AzHs exhibit strong chiroptical responses with high absorption dissymmetry factor (gabs) maxima of about 0.02, which is among the highest |gabs| values of helicenes in the visible range. These azulene‐embedded [n]helicenes contribute to the non‐benzenoid helicene library and allow the structure–property relationships to be better understood.

An adequate regulated deficit irrigation strategy improves wine astringency perception by altering proanthocyanidin composition in Cabernet Sauvignon grapes

Regulated deficit irrigation (RDI) is an agricultural practice that can enhance grapevine berry and wine quality. However, the effects of water deficit on concentration and composition of proanthocyanidins (PAs) and wine astringency are often limited and contrasting. Therefore, the profiles of PAs were investigated in Cabernet Sauvignon grown at 60% (RDI-1), 70% (RDI-2), 80% (RDI-3), and 100% (conventional irrigation, CI) of estimated evapotranspiration in 2016 and 2017. The treatments, replicated three times, were laid out in a randomized block design where any border effects were avoided. Our results showed that compared with CI, grapes grown under RDI had greater total concentrations of phenolic compounds. Skin PAs content increased in response to the RDI, especially in RDI-1 and RDI-2, while the seed PAs content was relatively insensitive to RDI. Hierarchical cluster analysis indicated that higher percentage of galloylation, molecular mass, mean degree of polymerization and the special extension subunits, such as percentage of (–)-epicatechin-3-O-gallate (%ECG) and percentage of (–)-epicatechin (%EC), led to greater astringency intensity in the wine. However, RDI-1 decreased perceived astringency due to the decreased %ECG and %EC of PAs extension subunits, and increased %ECG of PAs terminal subunits. Our study suggests that the irrigation levels at RDI-2 and RDI-3 can significantly influence the PAs compositions and improve wine astringency perception.

Spectroscopic and Photophysical Investigation of Model Dipyrroles Common to Bilins: Exploring Natural Design for Steering Torsion to Divergent Functions.

Biliproteins are a unique class of photosynthetic proteins in their diverse, and at times, divergent biophysical function. The two contexts of photosynthetic light harvesting and photoreception demonstrate characteristically opposite criteria for success, with light harvesting demanding structurally-rigid chromophores which minimize excitation quenching, and photoreception requiring structural flexibility to enable conformational isomerization. The functional plasticity borne out in these two biological contexts is a consequence of the structural plasticity of the pigments utilized by biliproteins―linear tetrapyrroles, or bilins. In this work, the intrinsic flexibility of the bilin framework is investigated in a bottom-up fashion by reducing the active nuclear degrees of freedom through model dipyrrole subunits of the bilin core and terminus free of external protein interactions. Steady-state spectroscopy was carried out on the dipyrrole (DPY) and dipyrrinone (DPN) subunits free in solution to characterize their intrinsic spectroscopic properties including absorption strengths and nonradiative activity. Transient absorption (TA) spectroscopy was utilized to determine the mechanism and kinetics of nonradiative decay of the dipyrrole subunits, revealing dynamics dominated by rapid internal conversion with some Z→E isomerization observable in DPY. Computational analysis of the ground state conformational landscapes indicates enhanced complexity in the asymmetric terminal subunit, and the prediction was confirmed by heterogeneity of species and kinetics observed in TA. Taken together, the large oscillator strengths (f ∼ 0.6) of the dipyrrolic derivatives and chemically-efficient spectral tunability seen through the ∼100 nm difference in absorption spectra, validate Nature's "selection" of multi-pyrrole pigments for light capture applications. However, the rapid deactivation of the excited state via their natural torsional activity when free in solution would limit their effective biological function. Comparison with phytochrome and phycocyanin 645 crystal structures reveals binding motifs within the in vivo bilin environment that help to facilitate or inhibit specific inter-pyrrole twisting vital for protein operation.