Bilin

[Shara Mchale]

This command is used to construct a bilin material. The bilin material simulates the modified Ibarra-Krawinkler deterioration model with bilinear hysteretic response. Note that the hysteretic response of this material has been calibrated with respect to more than 350 experimental data of steel beam-to-column connections and multivariate regression formulas are provided to estimate the deterioration parameters of the model for different connection types. These relationships were developed by Lignos and Krawinkler (2009, 2011) and have been adopted by PEER/ATC (2010). The input parameters for this component model can be computed interactively from this [link: ]. Use the module Component Model.


This command is used to construct a bilin material. The bilin material simulates the modified Ibarra-Medina-Krawinkler deterioration model with bilinear hysteretic response. Note that the hysteretic response of this material has been calibrated with respect to more than 350 experimental data of steel beam-to-column connections and multivariate regression formulas are provided to estimate the deterioration parameters of the model for different connection types. These relationships were developed by Lignos and Krawinkler (2009, 2011) and have been adopted by PEER/ATC (2010). NOTE: before you use this material make sure that you have downloaded the latest OpenSees version.



bilin

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Cyanobacteriochromes (CBCRs) are a subfamily of phytochrome photoreceptors found exclusively in photosynthetic cyanobacteria. Four CBCRs containing a second Cys in the insert region (insert-Cys) have been identified from the nonheterocystous cyanobacterium Microcoleus B353 (Mbr3854g4 and Mbl3738g2) and the nitrogen fixing, heterocystous cyanobacterium Nostoc punctiforme (NpF2164g3 and NpR1597g2). These insert-Cys CBCRs can sense light in the near-UV to orange range, but key residues responsible for tuning their colour sensitivity have not been reported. In the present study, near-UV/Green (UG) photosensors Mbr3854g4 (UG1) and Mbl3738g2 (UG2) were chosen for further spectroscopic analysis of their spectral sensitivity and tuning. Consistent with most dual-Cys CBCRs, both UGs formed a second thioether linkage to the phycocyanobilin (PCB) chromophore via the insert-Cys. This bond is subject to breakage and relinkage during forward and reverse photoconversions. Variations in residues equivalent to Phe that are in close contact with the PCB chromophore D-ring in canonical red/green CBCRs are responsible for tuning the light absorption peaks of both dark and photoproducts. This is the first time these key residues that govern light absorption in insert-Cys family CBCRs have been identified and characterised.


Cyanobacteriochromes (CBCRs) with single or multiple bilin-binding cGMP-specific phosphodiesterase, Adenylyl cyclase and FhlA (GAF) domains are cyanobacterial photosensory proteins that are distantly related to phytochromes (Phys). These proteins reversibly interconvert between dark-stable and photoproduct states upon photoisomerisation of their linear tetrapyrrole (bilin) chromophores1. Some Phys and CBCRs are involved in regulating light acclimation processes such as phototaxis2,3,4,5,6,7,8, chromatic acclimation9,10,11,12,13 and light-dependent cell aggregation14,15. Cyanobacterial Phys and CBCRs mostly use phycocyanobilin (PCB) as a chromophore precursor, and photoisomerisation of the 15,16-double bond of the bilin chromophore is the primary photochemical reaction during photoconversion (Supplementary Fig. S1). Unlike most cyanobacterial Phys that undergo photoconversion between a red-absorbing dark state and a far-red-absorbing photoproduct, photostates of CBCRs cover near-UV5,16,17, violet16,17,18, blue15,16,17,19,20,21,22,23, teal20,22,23, green115,17,19,23,24,25,26, orange16,17,20,23,26, red10,21,23,25,26 and far-red27 wavelengths.


The diverse photocycles observed in most single-Cys CBCRs are due to spectral tuning mechanisms such as protochromism12, hydration28, PCB/phycoviolobilin (PVB) isomerisation29 and trapped-twist30, all of which influence the conformation and configuration of the bound bilin chromophore. In the trapped-twist model, Phe residues conserved in red/green CBCRs (Supplementary Fig. S1) constrain chromophore movement after the primary photoisomerisation event, which effectively traps the D-ring in an unconjugated state. Unlike these single-Cys GAFs, dual-Cys CBCR GAFs use a second Cys residue in the highly conserved Asp-Xaa-Cys-Phe (DXCF) motif, the poorly conserved CXXR/K motif in the insertion loop (insert-Cys) or, in AM1_1186g2, a distinctive Cys residue located at the helix α3 region of AnPixJg2 to form a second thioether linkage to the C10 atom of the bilin chromophore (Supplementary Fig. S1). Except in the insert-Cys CBCR UV/blue photocycle protein 1 (UB1) NpR1597g2, these second thioether covalent bonds are not stable, and these labile bonds undergo reversible formation and breakage during photoconversion, resulting in spectral tuning with wavelength optima from near-UV to red5,16,17,21,31,32,33 (Supplementary Fig. S1). In some dual-Cys-containing DXCF-type GAFs, the PCB chromophore isomerises into the PVB form, which results in blue to green (or teal, yellow or orange) spectral tuning5,17,20,32.


Multiple sequence alignment of these GAFs against previously reported insert-Cys GAFs VO1 (NpF2164g3) and UB1 (NpR1597g2)16,17 (Fig. 1) revealed the presence of the putative insert-Cys (position IV). Interestingly, residues corresponding to Phe in the β1 notch (position I) of the distinctive red(R)/green(G) CBCR NpR3784 and its homologs34 and the β2 (position III) and α4 helix (position X) (Supplementary Fig. S1) that are involved in the trapped-twist mechanism of red/green and Teal-DXCF photocycles30 are only loosely conserved in insert-Cys CBCRs. Intriguingly, the insertion loop and the DXCF and CH motifs of VO1 from N. punctiforme are subject to light-dependent structural changes that include conversion from a random-coil structure in the dark state to a stable α-helical structure in the photoproduct, whereas the α-helical and β-sheet structure surrounding the D-ring of the chromophore remains almost unchanged during the dark-to-light transition35 (Fig. 1). Thus, it is probable that both specific and general features observed in the photocycles of insert-Cys GAFs are related to variation in loosely conserved amino acids in the insertion loop and other parts of the bilin-binding pocket, including residues in position I, III and X.


In the present work, we investigated the spectral properties (wavelength optima) in the photocycles of insert-Cys CBCRs using a series of recombinant UG variants produced in E. coli engineered for coproduction of the PCB bilin chromophore36. Application of thiol modifying reagents such as dithiothreitol (DTT), β-mercaptoethanol (βME) and iodoacetoamide (IAM) resulted in red-shifted intermediates relative to the dark or light states present during the U/G photoconversion cycle. We also identified variations in Phe equivalents at the β2 (position III) and α4 helix (position X) surrounding the PCB D-ring that are responsible for spectral tuning in UGs. Our results strongly indicate that the geometry of PCB is constrained by residues equivalent to key Phe residues (positions III and X), and this constraint is responsible for spectral tuning in insert-Cys CBCRs. Our findings might help to identify similar CBCRs in other species, and could assist engineering of the spectral properties of these light-sensitive proteins.


In insert-Cys-type GAFs, the type and number of amino acid residues in the insertion loop vary between subfamily members (Fig. 1), suggesting that this region is not critical for conserved functions other than providing the second thioether linkage to the bilin chromophore. However, contrary to our expectations, this region was required for efficient photoconversion. As shown in Fig. 5a, when the insertion loop was deleted by mutation (ΔI), normal chromophorylation was observed (Supplementary Fig. S3), but the efficiency of photoconversion between red- and green-absorbing photoproducts was significantly reduced (Supplementary Table S1). Even replacement of the Tyr residue of the DXCF motif with Cys (ΔI Y741C) to mimic the canonical DXCF motif failed to recover the photoconversion efficiency. The triple variant C713A Y741C L742F UG1, in which DTYL was replaced by the canonical DXCF motif present in dual-Cys CBCRs (the number of amino acids in the insert loop was unchanged), also exhibited an inefficient photoconversion comparable to the ΔI Y741C variants (Supplementary Table S1). Consistently, the C569A Y585C L586F UG2 variant was also unable to complete U/G photocycles, although the absorption spectra were more similar to those of dark-adapted Pu forms (Fig. 5b). Thus, the insert-Cys residue in the insert loop is required for efficient photoconversion.






The trapped-twist mechanism in the colour tuning of canonical red/green CBCRs predicts that chromophore constraint is the result of chromophore-protein interactions that determine the tilted geometry of the trapped chromophore. This model implies that a chromophore-binding pocket with a larger volume would constrain PCB conjugation only weakly, resulting in a species that absorbs at a longer wavelength than species with a tightly twisted geometry. To correlate amino acid residues equivalent to the β1 notch (I), β2 element (III) and α4 helix (X) with chromophore volume, in silico mutagenesis of these residues mimicking wild-type insert-Cys CBCRs and their variants was performed using the AnPixJg2 crystal structure in its dark state (PDB ID: 3W2Z) as template. The volume of the chromophore-binding pocket was then estimated as a proxy measuring changes in volume. Although only two or three of these residues were substituted to mimic respective insert-Cys CBCRs, the estimated volumes of the binding pockets of UG1, UG2 and UB1 were comparable, but significantly smaller than that of VO1 (Supplementary Table S2 and Fig. S8). Substitution of the β2 Phe with a smaller residue resulted in a substantial increase in the volume of the chromophore-binding pocket, whilst substitution of the β1 notch or helix α4 Phe residues had a much less pronounced effect. Interestingly, substitution of the helix α4 Phe had a significant impact on volume when the β2 Phe was replaced by a smaller residue, suggesting that the β2 residue is a key determinant of the volume of the chromophore-binding pocket. Overall, a larger chromophore pocket resulting from a smaller residue in the vicinity of the bilin D-ring leads to red-shifting of both dark and photoproduct states. Thus, in insert-Cys CBCRs, steric constraints in the chromophore pocket are likely determined by residues equivalent to the β2 Phe and helix α4 Phe. This in silico analysis should be corroborated in the future by determining crystal structures for wild-type and variant proteins.

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