Intrinsic tryptophan fluorescence quenching by iodine in non-canonical amino acid reveals alteration of the hydrogen bond network in the photoactive orange carotenoid protein.

The Orange Carotenoid Protein (OCP) regulates cyanobacterial photosynthetic activity through photoactivation in intense light. A hydrogen bonding network involving the keto-carotenoid oxygen and Y201 and W288 residues prevents the spontaneous activation of dark-adapted OCP. To investigate the role of the hydrogen bonds in OCP photocycling, we introduced non-canonical amino acids near the keto-carotenoid, particularly iodine at the meta-position of Y201. This modification significantly increased the yield of red OCP photoproducts, albeit with a shorter lifetime. Changes in tryptophan fluorescence during photocycling influenced by the presence of iodine near W288 revealed interactions between Y201 and W288 in the absence of the carotenoid in the C-domain. We propose that upon the relaxation of red states, a ternary complex with the carotenoid is formed. Analysis of spectral signatures and interaction energies indicates that the specific iodo-tyrosine configuration enhances interactions between the carotenoid and W288.

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs' regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

The role of the local environment on the structural heterogeneity of carotenoid ß-ionone rings.

Our analysis of the X-ray crystal structure of canthaxanthin (CAN) showed that its ketolated ß-ionone rings can adopt two energetically equal, but structurally distinct puckers. Quantum chemistry calculations revealed that the potential energy surface of the ß-ionone ring rotation over the plane of the conjugated π-system in carotenoids depends on the pucker state of the ß-ring. Considering different pucker states and ß-ionone ring rotation, we found six separate local minima on the potential energy surface defining the geometry of the keto-ß-ionone ring-two cis and one trans orientation for each of two pucker states. We observed a small difference in energy and no difference in relative orientation for the cis-minima, but a pronounced difference for the position of trans-minimum in alternative pucker configurations. An energetic advantage of ß-ionone ring rotation from a specific pucker type can reach up to 8 kJ/mol ([Formula: see text]). In addition, we performed the simulation of linear absorption of CAN in hexane and in a unit cell of the CAN crystal. The electronic energies of [Formula: see text] transition were estimated both for the CAN monomer and in the CAN crystal. The difference between them reached [Formula: see text], which roughly corresponds to the energy gap between A and B pucker states predicted by theoretical estimations. Finally, we have discussed the importance of such effects for biological systems whose local environment determines conformational mobility, and optical/functional characteristics of carotenoid.

Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin ß-Subunit Production in E. coli.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin ß-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

α-Crystallin Domains of Five Human Small Heat Shock Proteins (sHsps) Differ in Dimer Stabilities and Ability to Incorporate Themselves into Oligomers of Full-Length sHsps.

The α-crystallin domain (ACD) is the hallmark of a diverse family of small heat shock proteins (sHsps). We investigated some of the ACD properties of five human sHsps as well as their interactions with different full-length sHsps. According to size-exclusion chromatography, at high concentrations, the ACDs of HspB1 (B1ACD), HspB5 (B5ACD) and HspB6 (B6ACD) formed dimers of different stabilities, which, upon dilution, dissociated to monomers to different degrees. Upon dilution, the B1ACD dimers possessed the highest stabilities, and those of B6ACD had the lowest. In striking contrast, the ACDs of HspB7 (B7ACD) and HspB8 (B8ACD) formed monomers in the same concentration range, which indicated the compromised stabilities of their dimer interfaces. B1ACD, B5ACD and B6ACD transiently interacted with full-length HspB1 and HspB5, which are known to form large oligomers, and modulated their oligomerization behavior. The small oligomers formed by the 3D mutant of HspB1 (mimicking phosphorylation at Ser15, Ser78 and Ser82) effectively interacted with B1ACD, B5ACD and B6ACD, incorporating these α-crystallin domains into their structures. The inherently dimeric full-length HspB6 readily formed heterooligomeric complexes with B1ACD and B5ACD. In sharp contrast to the abovementioned ACDs, B7ACD and B8ACD were unable to interact with full-length HspB1, the 3D mutant of HspB1, HspB5 or HspB6. Thus, their high sequence homology notwithstanding, B7ACD and B8ACD differ from the other three ACDs in their inability to form dimers and interact with the full-length small heat shock proteins. Having conservative primary structures and being apparently similar, the ACDs of the different sHsps differ in terms of their dimer stabilities, which can influence the heterooligomerization preferences of sHsps.

Structural basis for the recognition by 14-3-3 proteins of a conditional binding site within the oligomerization domain of human nucleophosmin.

Nucleophosmin 1 (NPM1) is a multifunctional protein regulating ribosome biogenesis, centrosome duplication and chromatin remodeling. Being a major nucleolar protein, NPM1 can migrate to the nucleus and the cytoplasm, which is controlled by changes of NPM1 oligomerization and interaction with other cell factors. NPM1 forms a stable pentamer with its N-terminal structured domain, where two nuclear export signals and several phosphorylation sites reside. This domain undergoes dissociation and disordering upon Ser48 phosphorylation in the subunit interface. Recent studies indicated that Ser48 is important for NPM1 interaction with other proteins including 14-3-3, the well-known phosphoserine/phosphothreonine binders, but the structural basis for 14-3-3/NPM1 interaction remained unaddressed. By fusing human 14-3-3ζ with an NPM1 segment surrounding Ser48, which was phosphorylated inside Escherichia coli cells by co-expressed protein kinase A, here we obtained the desired protein/phosphopeptide complex and determined its crystal structure. While biochemical data indicated that the interaction is driven by Ser48 phosphorylation, the crystallographic 14-3-3/phosphopeptide interface reveals an NPM1 conformation distinctly different from that in the NPM1 pentamer. Given the canonical phosphopeptide-binding mode observed in our crystal structure, Ser48 emerges as a conditional binding site whose recognition by 14-3-3 proteins is enabled by NPM1 phosphorylation, disassembly and disordering under physiological circumstances.

Modulation of Aptamer-Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA's complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA's complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn't affecting the G-qaudruplex's integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD.

Several signaling pathways control phosphorylation of the proapoptotic protein BAD and its phosphorylation-dependent association with 14-3-3 proteins in the cytoplasm. The stability of the 14-3-3/BAD complex determines the cell fate: unphosphorylated BAD escapes from 14-3-3, migrates to the mitochondria and initiates apoptosis. While the 14-3-3/BAD interaction represents a promising drug target, it lacks structural characterization. Among several phosphosites identified in vivo, Ser75 and Ser99 of human BAD match the consensus sequence RXXpSXP recognized by 14-3-3 and, therefore, represent canonical 14-3-3-binding sites. Yet, BAD contains other serines phosphorylatable in vivo, whose role is less understood. Here, we report a 2.36 Å crystal structure of 14-3-3ζ complexed with a BAD fragment which includes residues Ser74 and Ser75, both being substrates for protein kinases. While the BAD peptide is anchored to 14-3-3 by phosphoserine as expected, the BAD peptide was unexpectedly phosphorylated at Ser74 instead of Ser75, revealing noncanonical binding within the amphipathic groove and leading to a one-step positional shift and reorganization of the interface. This observation exemplifies plasticity of the amphipathic 14-3-3 groove in accommodating various peptides and suggests the redundancy of Ser74 and Ser75 phosphosites with respect to binding of BAD to 14-3-3.

Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins.

Many major protein-protein interaction networks are maintained by 'hub' proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that 'read' the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273-1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

Design, expression, purification and crystallization of human 14-3-3ζ protein chimera with phosphopeptide from proapoptotic protein BAD.

14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the "chimeric" approach for studies of other relevant 14-3-3 complexes.

Fluorescence recovery protein: a powerful yet underexplored regulator of photoprotection in cyanobacteria†.

Cyanobacteria utilize an elegant photoprotection mechanism mediated by the photoactive Orange Carotenoid Protein (OCP), which upon binding dissipates excess energy from light-harvesting complexes, phycobilisomes. The OCP activity is efficiently regulated by its partner, the Fluorescence Recovery Protein (FRP). FRP accelerates OCP conversion to the resting state, thus counteracting the OCP-mediated photoprotection. Behind the deceptive simplicity of such regulation is hidden a multistep process involving dramatic conformational rearrangements in OCP and FRP, the details of which became clearer only a decade after the FRP discovery. Yet many questions regarding the functioning of FRP have remained controversial. In this review, we summarize the current knowledge and understanding of the FRP role in cyanobacterial photoprotection as well as its evolutionary history that presumably lies far beyond cyanobacteria.

Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic insights into the differences in substrate specificity within the PPEP family.

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei, which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases.

Oligomeric state of αB-crystallin under crowded conditions.

Small heat shock proteins (sHsps) are molecular chaperones preventing protein aggregation. Dynamics of quaternary structure plays an important role in the chaperone-like activity of sHsps. However, an interrelation between the oligomeric state and chaperone-like activity of sHsps remains insufficiently characterized. Most of the accumulated data were obtained in dilute protein solutions, leaving the question of the oligomeric state of sHsps in crowded intracellular media largely unanswered. Here, we analyzed the effect of crowding on the oligomeric state of αB-crystallin (αB-Cr) using analytical ultracentrifugation. Marked increase in the sedimentation coefficient of αB-Cr was observed in the presence of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP) and trimethylamine N-oxide (TMAO) at 48 °C. An especially pronounced effect was detected for the PEG and TMAO mixture, where the sedimentation coefficient (s20,w) of αB-Cr increased from 10.7 S in dilute solution up to 40.7 S in the presence of crowding agents. In the PEG + TMAO mixture, addition of model protein substrate (muscle glycogen phosphorylase b) induced dissociation of large αB-Cr oligomers and formation of complexes with smaller sedimentation coefficients, supporting the idea that, under crowding conditions, protein substrates can promote dissociation of large αB-Cr oligomers.

Hybrid coupling of R-phycoerythrin and the orange carotenoid protein supports the FRET-based mechanism of cyanobacterial photoprotection.

To regulate the effectiveness of photosynthesis and photoprotection cyanobacteria utilize a system consisting of only few components. Photoactivation of the orange carotenoid protein (OCP) enables its interaction with a specific, yet controversial site in the core of the light-harvesting antenna, the phycobilisome (PBS). The resulting delivery of a quenching carotenoid molecule to the antenna pigments leads to thermal dissipation of the excitation energy absorbed by the latter, and, consequently, to depression of the photosynthetic activity. The nature of the OCP-induced PBS fluorescence quenching mechanism remains debatable, however, specific protein-protein interactions between PBS and photoactivated OCP should provide a unique environment for interactions between the excitation energy donor and acceptor. Here we questioned whether the Förster theory of resonance energy transfer can explain PBS quenching by OCP even at their very small spectral overlap and whether in model systems, the absence of specific protein-protein interactions of OCP with a donor of energy can be compensated by a better spectral overlap. Hybridization of algal R-phycoerythrin with cyanobacterial OCP by chemical crosslinking results in a significant decrease of R-phycoerythrin fluorescence lifetime, irrespective of the OCP photoactivation status. Supported by structural considerations, this indicates that FRET may be the essence of cyanobacterial photoprotection mechanism.

Structural peculiarities of keto-carotenoids in water-soluble proteins revealed by simulation of linear absorption.

To prevent irreversible damage caused by an excess of incident light, the photosynthetic machinery of many cyanobacteria uniquely utilizes the water-soluble orange carotenoid protein (OCP) containing a single keto-carotenoid molecule. This molecule is non-covalently embedded into the two OCP domains which are interconnected by a flexible linker. The phenomenon of OCP photoactivation, causing significant changes in carotenoid absorption in the orange and red form of OCP, is currently being thoroughly studied. Numerous additional spectral forms of natural and synthetic OCP-like proteins have been unearthed. The optical properties of carotenoids are strongly determined by the interaction of their electronic states with vibrational modes, the surrounding protein matrix, and the solvent. In this work, the effects of the pigment-protein interaction and vibrational relaxation in OCP were studied by computational simulation of linear absorption. Taking into account Raman spectroscopy data and applying the multimode Brownian oscillator model as well as the cumulant expansion technique, we have calculated a set of characteristic microparameters sufficient to demarcate different carotenoid states in OCP forms, using the model carotenoids spheroidene and spheroidenone in methanol/acetone solution as benchmarks. The most crucial microparameters, which determine the effect of solvent and protein environment, are the Huang-Rhys factors and the frequencies of C[double bond, length as m-dash]C and C-C stretching modes, the low-frequency mode and the FWHM due to inhomogeneous line broadening. Considering the difference of linear absorption between spheroidene and spheroidenone, which remarkably resembles the photoinduced changes of OCP absorption, and applying quantum chemical calculations, we discuss structural and functional determinants of carotenoid binding proteins.

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP.

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP towards various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

Effect of the NBD-group position on interaction of fluorescently-labeled cholesterol analogues with human steroidogenic acute regulatory protein STARD1.

Steroidogenic acute regulatory protein (StAR, STARD1) is a key factor of intracellular cholesterol transfer to mitochondria, necessary for adrenal and gonadal steroidogenesis, and is an archetypal member of the START protein family. Despite the common overall structural fold, START members differ in their binding selectivity toward various lipid ligands, but the lack of direct structural information hinders complete understanding of the binding process and cholesterol orientation in the STARD1 complex in particular. Cholesterol binding has been widely studied by commercially available fluorescent steroids, but the effect of the fluorescent group position on binding remained underexplored. Here, we dissect STARD1 interaction with cholesterol-like steroids bearing 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group in different positions, namely, with 22-NBD-cholesterol (22NC), 25-NBD-cholesterol (25NC), 20-((NBDamino)-pregn-5-en-3-ol (20NP) and 3-(NBDamino)-cholestane (3NC). While being able to stoichiometrically bind 22NC and 20NP with high fluorescence yield and quantitative exhaustion of fluorescence of some protein tryptophans, STARD1 binds 25NC and 3NC with much lower affinity and poor fluorescence response. In contrast to 3NC, binding of 20NP leads to STARD1 stabilization and substantially increases the NBD fluorescence lifetime. Remarkably, in terms of fluorescence response, 20NP slightly outperforms commonly used 22NC and can thus be used for screening of various potential ligands by a competition mechanism in the future.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Erratum to: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.

Dissection of the deep-blue autofluorescence changes accompanying amyloid fibrillation.

Pathogenesis of numerous diseases is associated with the formation of amyloid fibrils. Extrinsic fluorescent dyes, including Thioflavin T (ThT), are used to follow the fibrillation kinetics. It has recently been reported that the so-called deep-blue autofluorescence (dbAF) is changing during the aggregation process. However, the origin of dbAF and the reasons for its change remain debatable. Here, the kinetics of fibril formation in model proteins were comprehensively analyzed using fluorescence lifetime and intensity of ThT, intrinsic fluorescence of proteinaceous fluorophores, and dbAF. For all systems, intensity enhancement of the dbAF band with similar spectral parameters (∼350 nm excitation; ∼450 nm emission) was observed. Although the time course of ThT lifetime (indicative of protofibrils formation) coincided with that of tyrosine residues in insulin, and the kinetic changes in the ThT fluorescence intensity (reflecting formation of mature fibrils) coincided with changes in ThT absorption spectrum, the dbAF band started to increase from the beginning of the incubation process without a lag-phase. Our mass-spectrometry data and model experiments suggested that dbAF could be at least partially related to oxidation of amino acids. This study scrutinizes the dbAF features in the context of the existing hypotheses about the origin of this spectral band.