Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP^r) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP^r. This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP^r, enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.Full sunlight is dangerous for plants, algae, and cyanobacteria. It can cause oxidative damages leading to the destruction of the photosynthetic apparatus and to cell death. A short-term photoprotective mechanism developed by oxygenic photosynthetic organisms is the reduction of excitation energy being funneled into the photochemical reaction centers by dissipating excess energy as heat at the level of the antennae (Niyogi and Truong, 2013). In plants and green algae, this mechanism involves the membrane chlorophyll antennae, the light-harvesting complex (for review, see Horton et al., 1996; Horton and Ruban, 2005; Jahns and Holzwarth, 2012), and in cyanobacteria, the extramembrane phycobiliprotein-containing antennae, the phycobilisomes (PBSs; for review, see Kirilovsky and Kerfeld, 2012; Kirilovsky, 2014). Despite these differences in composition and structure of their antennae, carotenoids have an essential role in both plants and cyanobacteria. In plants, high irradiance leads to acidification of the lumen that triggers conformational changes in the light-harvesting complexes and in their organization in the membrane, switching the light-harvesting complex into an effective energy-dissipating form. In cyanobacteria, high irradiance photoactivates a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), that acts as the stress sensor and the energy quencher. In both cases, changes in pigment-pigment interactions (carotenoid-chlorophyll, carotenoid-bilin, chlorophyll-chlorophyll) enable thermal dissipation of excitation energy via three different possible mechanisms: excitation energy transfer (Ruban et al., 2007; Berera et al., 2013), charge transfer (Holt et al., 2005; Tian et al., 2011), or excitonic interactions between the pigments (Bode et al., 2009).The study of the photoactivation of the OCP and its interaction with the phycobilisome is essential to elucidate the mechanism of energy quenching in cyanobacterial photoprotection. The OCP is a soluble 35-kD protein constituted by an α-helical N-terminal domain (residues1–165) and an α-helix/β-sheet C-terminal domain (residues 190–317; Kerfeld et al., 2003; Wilson et al., 2010; Fig. 1A). A flexible linker of 25 amino acids connects both domains. The ketocarotenoid 3′-hydroxyechinenone (3′-hECN), having a carbonyl (keto) group in one of the rings and a hydroxyl group in the other one, spans both domains of the protein, with the carbonyl group residing in a hydrophobic pocket of the C-terminal domain. Tyr-201 and Trp-288 interact via hydrogen bonds to the carotenoid keto group. In the dark, the OCP is orange (OCP^o). Absorption of blue-green light by the carotenoid induces conformational changes in the carotenoid and in the protein converting the orange form into the active red form (OCP^r; Wilson et al., 2008; Fig. 1C). The photoconversion reaction has a very low quantum yield, and the rate of OCP^r accumulation largely depends on light intensity (Wilson et al., 2008). Thus, accumulation of the red form occurs only under high irradiance (Wilson et al., 2008). Both OCP^o and OCP^r are energetically suitable to quench PBS fluorescence and excitation energy (Polívka et al., 2013; Niedzwiedzki et al., 2014), but only OCP^r is able to bind to the PBS and dissipate most of the excess energy as heat (Gwizdala et al., 2011). In OCP^o, strong interactions exist between the N- and C-terminal globular domains, including salt bridges between residues Trp-277-Asn-104 and Arg-155-Glu-244 (Kerfeld et al., 2003; Wilson et al., 2010). Upon photoactivation, these bonds are broken, leading to the solvent exposure of Arg-155, which plays an essential role in OCP binding to PBS (Wilson et al., 2012; Fig. 1C). The PBSs from Synechocystis sp. PCC 6803 (used in this work and hereafter simply referred to as Synechocystis) are formed by a core of allophycocyanin (APC) trimers. These trimers are organized in three cylinders from which rods containing phycocyanin hexamers radiate (for review, see Grossman et al., 1993; MacColl, 1998; Adir, 2005). OCP^r binds to one APC trimer, and its open structure allows the interaction between the OCP carotenoid and one APC bilin (Wilson et al., 2012). The first site of energy and fluorescence quenching is an APC trimer emitting at 660 nm (Tian et al., 2011, 2012, 2013; Takahashi et al., 2013). Once OCP^r is attached to PBS, thermal dissipation increases and less energy arrives at both photosystems (Wilson et al., 2006; Rakhimberdieva et al., 2010; Gorbunov et al., 2011). When the light becomes less intense, full antenna capacity is required. The Fluorescence Recovery Protein (FRP) is essential for this process. FRP accelerates the OCP^r to OCP^o dark conversion and facilitates OCP detachment from PBS (Boulay et al., 2010; Gwizdala et al., 2011; Sutter et al., 2013). The active FRP is a nonchromophorylated dimer that interacts with the C-terminal domain of the OCP^r (Sutter et al., 2013).Open in a separate windowFigure 1.A and B, Structure of the OCP from Synechocystis sp. PCC 6803 (Protein Data Bank identifier: 3MG1). The OCP monomer is represented in the orange state. The N-terminal arm (residues 1–22; red) interacts with the C-terminal domain (residues 196–315; sky blue). The Pro-22 and the Asp-6 are marked in blue. The N-terminal domain (residues 22–165) is green in the figure, and the linker between N-terminal and C-terminal domains is colored in violet. C, Model of photoactivation. Upon light absorption, the orange OCP^o is converted into the active red OCP^r. Changes in the carotenoid conformation induce conformational changes in the C-terminal domain, leading to the breakage of the interactions between the N-terminal and C-terminal domains and the opening of the protein.Previously, it has been demonstrated that the N-terminal globular domain of the OCP (green in Fig. 1, A and B) is a constitutively active energy quencher (Leverenz et al., 2014). Thus, its interaction with the C-terminal globular domain is essential for inhibiting OCP binding to PBS and energy quenching under low irradiance. This process must be tightly regulated. Little is known about this regulation. One possibility is that the N-terminal arm of the protein (red in Fig. 1, A and B), which in OCP^o interacts with the C-terminal globular domain, could have a role in this regulation.According to the OCP structure Asp-6, could form a hydrogen bond with Arg-229, which could stabilize the closed form of OCP^o. Pro-22 is located at the bent junction between the N-terminal arm and the N-terminal globular domain. It has been proposed that a cis-trans Pro isomerization could be involved in OCP photoactivation (Gorbunov et al., 2011), suggesting that Pro-22 isomerization could help the movement of the N-terminal arm and its detachment from the C-terminal domain during OCP photoactivation. In this work, we studied the effect of deleting the N-terminal arm and the mutations Asp-6-Leu and Pro-22-Val on photoactivity and OCP interaction with PBS and FRP (for the position of the N-terminal arm in the structure of OCP^o, see in Fig. 1, A and B).     

Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.     

Specificity of the Cyanobacterial Orange Carotenoid Protein: Influences of Orange Carotenoid Protein and Phycobilisome Structures

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.The cyanobacterial Orange Carotenoid Protein (OCP) is a photoactive soluble protein of 35 kD that binds a ketocarotenoid, 3′-hydroxyechinenone (hECN). It is present in the majority of phycobilisome (PB)-containing cyanobacterial strains (Kirilovsky and Kerfeld, 2012, 2013). The PBs are light-harvesting extramembrane complexes formed by a core from which rods radiate. The core and rods are constituted of water-soluble blue and red phycobiliproteins, which covalently attach bilins (for review, see Glazer, 1984; Grossman et al., 1993; MacColl, 1998; Tandeau de Marsac, 2003; Adir, 2005). The OCP was first described by Holt and Krogmann (1981), and its structure was determined in 2003 (Kerfeld et al., 2003). However, its function was discovered only in 2006 (Wilson et al., 2006) and its photoactivity in 2008 (Wilson et al., 2008). The OCP is essential in a photoprotective mechanism that decreases the energy arriving at the reaction centers under high irradiance. Strong light induces thermal dissipation of the energy absorbed by the PBs, resulting in a decrease of PB fluorescence emission and of energy transfer from the PBs to the reaction centers (Wilson et al., 2006). This process, which is light intensity dependent, is induced by blue or green light but not by orange or red light (Rakhimberdieva et al., 2004; Wilson et al., 2006). The absorption of strong blue-green light by the OCP induces changes in the conformation of the carotenoid, converting the inactive orange dark form (OCP^o) into an active red form (OCP^r; Wilson et al., 2008). In OCP^o, the hECN is in an all-trans-configuration (Kerfeld et al., 2003; Polívka et al., 2005). In OCP^r, the apparent conjugation length of the carotenoid increases, resulting in a less distorted, more planar structure (Wilson et al., 2008). Fourier transform infrared spectra showed that conformational changes in the protein are also induced (Wilson et al., 2008) that are essential for the induction of the photoprotective mechanism. Only OCP^r is able to bind to the core of PBs and to induce thermal energy dissipation (Wilson et al., 2008; Punginelli et al., 2009; Gorbunov et al., 2011; Gwizdala et al., 2011). Since the photoactivation of the OCP has a very low quantum yield (0.03; Wilson et al., 2008), the concentration of activated protein is zero in darkness and very low under low-light conditions (Wilson et al., 2008; Gorbunov et al., 2011). Thus, the photoprotective mechanism functions only under high-light conditions.The crystal structures of the Arthrospira maxima OCP and of the Synechocystis PCC 6803 (hereafter Synechocystis) OCP were solved in 2003 and 2010, respectively (Kerfeld et al., 2003; Wilson et al., 2010). These structures, assumed to correspond to the dark OCP^o form, are essentially identical. The OCP consists of an all-α-helical N-terminal domain (residues 1–165), unique to cyanobacteria, and an α-helical/β-sheet C-terminal domain that is a member of the Nuclear Transport Factor2 superfamily (residues 191–320; Synechocystis numbering). Both domains are joined by a linker (residues 166–190; Synechocystis numbering) that appears to be flexible. The hECN molecule spans the N- and C-terminal domains of the protein, with its carbonyl end embedded in and hydrogen bonded to two absolutely conserved residues (Tyr-201 and Trp-288) in the C-terminal domain. The carotenoid is almost entirely buried; only 3.4% of the 3′ hECN is solvent exposed (Kerfeld et al., 2003). Synechocystis OCP can also bind with high-affinity echinenone (ECN) and zeaxanthin. While the ECN OCP is photoactive, the zeaxanthin OCP is photoinactive (Punginelli et al., 2009), indicating the importance of the carotenoid carbonyl group for photoactivity. The largest interface through which the two domains interact and through which the carotenoid passes is stabilized by a small number of hydrogen bonds, including one formed between Arg-155 and Glu-244 (Wilson et al., 2010). This salt bridge stabilizes the closed structure of OCP^o. Upon illumination, protein conformational changes cause the breakage of this bond and the opening of the protein (Wilson et al., 2012). Arg-155, which becomes more exposed upon the separation of the two domains, is essential for the OCP^r binding to the PBs (Wilson et al., 2012).After exposure to high irradiance, when the light intensity decreases, recovery of full antenna capacity and fluorescence requires another protein, the Fluorescence Recovery Protein (FRP; Boulay et al., 2010). The active form of this soluble 13-kD protein is a dimer (Sutter et al., 2013). It interacts with the OCP^r C-terminal domain (Boulay et al., 2010; Sutter et al., 2013). This accelerates the red-to-orange OCP conversion and helps the OCP to detach from the PB (Boulay et al., 2010; Gwizdala et al., 2011).Genes encoding the full-length OCP are found in the vast majority of cyanobacteria but not in all; 90 of 127 genomes recently surveyed contain at least one gene for a full-length OCP (Kirilovsky and Kerfeld, 2013). The genomes of Synechococcus elongatus and Thermosynechococcus elongatus, two cyanobacterial strains used as model organisms in photosynthesis and stress studies, do not contain a full-length ocp gene. These strains also lack FRP and β-carotene ketolase (involved in ketocarotenoid synthesis). As a consequence, these strains lack the OCP-related photoprotective mechanism and are more sensitive to fluctuating light intensities (Boulay et al., 2008).The core of the hemidiscoidal PBs of Synechocystis, the model organism routinely used for the study of the OCP-related photoprotective mechanism, consists of three cylinders, each one formed by four trimers of allophycocyanin (APC; Fig. 1; for review, see Glazer, 1984; Bryant, 1991; Grossman et al., 1993; MacColl, 1998; Adir, 2005). The APC trimers are predominantly assembled from a two-subunit heterodimer, αAPC-βAPC, which binds two phycocyanobilins, one in each subunit. Of the 12 total APC trimers in the PB core, eight are trimers of αAPC-βAPC. These trimers have a maximal emission at 660 nm (APC₆₆₀). The upper cylinder contains only APC₆₆₀ trimers. In contrast, each basal cylinder contains only two APC₆₆₀ trimers. Each basal cylinder also contains the following: (1) a trimer in which one αAPC subunit is replaced by a special αAPC-like subunit called ApcD, and (2) a trimer in which one β-subunit is replaced by ApcF, a βAPC-like subunit, and one α-subunit is replaced by the N-terminal domain of ApcE, an αAPC-like domain (Fig. 1). The trimers containing one or two of these special subunits have a maximal emission at 680 nm (APC₆₈₀). In each cylinder, the two external trimers are stabilized by an 8.7-kD linker protein.Open in a separate windowFigure 1.Schematic orthogonal projections of the various PB cores. In the PBs containing three or five cylinders, the top complete cylinder is formed by four αAPC-βAPC trimers emitting at 660 nm. Each of the basal cylinders of three types of PBs contains two αAPC-βAPC trimers emitting at 660 nm and two trimers emitting at 683 nm. In one of them, one αAPC is replaced by ApcD, and in the other one, αAPC-βAPC is replaced by the dimer ApcF-ApcE. In the five cylinder PBs, two additional semicylinders formed by two αAPC-βAPC trimers are present. In all the cylinders, the two external trimers include an 8.7-kD linker protein (ApcC).The C-terminal part of Synechocystis ApcE contains three interconnected repeated domains of about 120 residues (called Rep domains) that are similar to the conserved domains of rod linkers. Each Rep domain interacts with an APC trimer situated in different cylinders, which stabilizes the core of PB (Zhao et al., 1992; Shen et al., 1993; Ajlani et al., 1995; Ajlani and Vernotte, 1998). The ApcE protein also determines the number of APC cylinders that form the PB core (Capuano et al., 1991, 1993). Indeed, there are PBs containing only the two basal cylinders, as in S. elongatus (ex S. elongatus PCC 7942) and Synechococcus PCC 6301. In these strains, the approximately 72-kD ApcE possesses only two Rep domains. There also exist pentacylindrical cores in which, in addition to the three cylinders existing in Synechocystis PBs, there are two other cylinders, each formed by two APC₆₆₀ trimers, for example in Anabaena variabilis, Anabaena PCC 7120, and Mastigocladus laminosus (Glauser et al., 1992; Ducret et al., 1998). In the pentacylindrical PBs, ApcE (approximately 125 kD) contains four Rep domains (Capuano et al., 1993). Finally, ApcE is also involved in the interaction between the PB and the thylakoids.The bicylindric and tricylindric cores are surrounded by six rods formed generally by three hexamers of the blue phycocyanin (PC) or two PC hexamers and one hexamer containing phycoerythrin or phycoerythrincyanonin. The rods and the hexamers are stabilized by nonchromophorylated linker proteins. A linker protein, L^RC also stabilizes the binding of the rods to the core. The pentacylindric PBs can contain up to eight rods. The quantity and length of rods and the presence of phycoerythrin or phycoerythrocyanin at the periphery of the rods depends on environmental conditions like light intensity or quality (Kipe-Nolt et al., 1982; Glauser et al., 1992).The OCP probably binds to one of the APC₆₆₀ trimers (Tian et al., 2011, 2012; Jallet et al., 2012), and the presence of the rods stabilizes this binding to Synechocystis PBs (Gwizdala et al., 2011). The different structures of PBs in other strains could affect the binding of the OCP. Thus, we undertook a study about the relationship between the structure of PBs and OCP binding in preparation for introducing the OCP-related photoprotective mechanism into S. elongatus using Synechocystis genes. In this study, we used the in vitro reconstitution system developed by Gwizdala et al. (2011) with three different types of isolated PBs: Arthrospira platensis PCC 7345 (hereafter Arthrospira) PBs, having a tricylindrical core like Synechocystis PBs; Anabaena variabilis (hereafter Anabaena) PBs, having a pentacylindrical core; and S. elongatus PCC 7942 (hereafter Synechococcus) PBs, having a bicylindrical core. We also used two different OCPs, the Synechocystis OCP and the Arthrospira OCP. Each OCP was isolated from mutant Synechocystis cells overexpressing one or the other ocp gene with a C-terminal His tag.     

In Vitro Reconstitution of the Cyanobacterial Photoprotective Mechanism Mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.     

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Tomosyn negatively regulates SNARE-dependent exocytic pathways including insulin secretion, GLUT4 exocytosis, and neurotransmitter release. The molecular mechanism of tomosyn, however, has not been fully elucidated. Here, we reconstituted SNARE-dependent fusion reactions in vitro to recapitulate the tomosyn-regulated exocytic pathways. We then expressed and purified active full-length tomosyn and examined how it regulates the reconstituted SNARE-dependent fusion reactions. Using these defined fusion assays, we demonstrated that tomosyn negatively regulates SNARE-mediated membrane fusion by inhibiting the assembly of the ternary SNARE complex. Tomosyn recognizes the t-SNARE complex and prevents its pairing with the v-SNARE, therefore arresting the fusion reaction at a pre-docking stage. The inhibitory function of tomosyn is mediated by its C-terminal domain (CTD) that contains an R-SNARE-like motif, confirming previous studies carried out using truncated tomosyn fragments. Interestingly, the N-terminal domain (NTD) of tomosyn is critical (but not sufficient) to the binding of tomosyn to the syntaxin monomer, indicating that full-length tomosyn possesses unique features not found in the widely studied CTD fragment. Finally, we showed that the inhibitory function of tomosyn is dominant over the stimulatory activity of the Sec1/Munc18 protein in fusion. We suggest that tomosyn uses its CTD to arrest SNARE-dependent fusion reactions, whereas its NTD is required for the recruitment of tomosyn to vesicle fusion sites through syntaxin interaction.     

Protein Fragments: Functional and Structural Roles of Their Coevolution Networks

Small protein fragments, and not just residues, can be used as basic building blocks to reconstruct networks of coevolved amino acids in proteins. Fragments often enter in physical contact one with the other and play a major biological role in the protein. The nature of these interactions might be multiple and spans beyond binding specificity, allosteric regulation and folding constraints. Indeed, coevolving fragments are indicators of important information explaining folding intermediates, peptide assembly, key mutations with known roles in genetic diseases, distinguished subfamily-dependent motifs and differentiated evolutionary pressures on protein regions. Coevolution analysis detects networks of fragments interaction and highlights a high order organization of fragments demonstrating the importance of studying at a deeper level this structure. We demonstrate that it can be applied to protein families that are highly conserved or represented by few sequences, enlarging in this manner, the class of proteins where coevolution analysis can be performed and making large-scale coevolution studies a feasible goal.     

Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

Spider silk is protein fibers with extraordinary mechanical properties. Up to now, it is still poorly understood how silk proteins are kept in a soluble form before spinning into fibers and how the protein molecules are aligned orderly to form fibers. Minor ampullate spidroin is one of the seven types of silk proteins, which consists of four types of domains: N-terminal domain, C-terminal domain (CTD), repetitive domain (RP) and linker domain (LK). Here we report the tertiary structure of CTD and secondary structures of RP and LK in aqueous solution, and their roles in protein stability, solubility and fiber formation. The stability and solubility of individual domains are dramatically different and can be explained by their distinct structures. For the tri-domain miniature fibroin, RP-LK-CTD_Mi, the three domains have no or weak interactions with one another at low protein concentrations (<1 mg/ml). The CTD in RP-LK-CTD_Mi is very stable and soluble, but it cannot stabilize the entire protein against chemical and thermal denaturation while it can keep the entire tri-domain in a highly water-soluble state. In the presence of shear force, protein aggregation is greatly accelerated and the aggregation rate is determined by the stability of folded domains and solubility of the disordered domains. Only the tri-domain RP-LK-CTD_Mi could form silk-like fibers, indicating that all three domains play distinct roles in fiber formation: LK as a nucleation site for assembly of protein molecules, RP for assistance of the assembly and CTD for regulating alignment of the assembled molecules.     

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Background

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.

Methodology/Results

FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.

Conclusion

Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.     

Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters

Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.The classical autotransporter (AT) family, also known as the type Va protein secretion system, represents the largest group of proteins secreted by Gram-negative bacteria and includes many virulence factors from important human pathogens (10, 17). Bacteria produce AT proteins as large polypeptide precursors, with their virulence activity (e.g., cytotoxins, adhesins, and proteases, etc.) present in a passenger domain flanked by an N-terminal signal peptide (sp) for Sec-dependent translocation across the bacterial inner membrane (IM) and a C-terminal domain of ∼30 to 40 kDa for insertion into the bacterial outer membrane (OM) (see Fig. 2A). A self-translocation model was originally proposed to explain the secretion mechanism of AT proteins across the OM, based mostly on data obtained with the IgA protease (IgAP) from Neisseria gonorrhoeae (43). In this model the C-terminal domain of ATs was supposed to fold in the OM as a β-barrel protein with an internal hydrophilic pore that could be used for the translocation of the passenger domain. The finding that the B subunit of cholera toxin (CtxB) should not have disulfide bonds for its secretion when fused as a heterologous passenger to the C-terminal domain of IgAP (30, 31) indirectly suggests passenger translocation in an unfolded conformation through a narrow channel expected for a β-barrel. Similar observations with the C-terminal domains of IcsA from Shigella flexneri (56) and AIDA-I from Escherichia coli (36) supported this model.Previous work done by our group challenged the original self-translocation model, since a 45-kDa C-terminal fragment of IgAP was shown to form oligomeric ring-shaped complexes with a central hydrophilic pore of ∼2 nm (63). In addition, this C-terminal fragment of IgAP was found to translocate folded immunoglobulin (Ig) domains with disulfide bonds to the bacterial surface, indicating that at least a ∼2-nm pore was being used for passenger secretion (61, 62). These data led us to propose a “multimeric” version of the self-translocation model in which the secretion of the passenger may occur through the central channel assembled by the oligomerization of the C-terminal domains in the OM. Studies with IcsA from S. flexneri (7, 46, 47, 64) and EspP from E. coli (53) also provided evidence indicating that native and heterologous passengers adopt folded or at least partially folded conformations in the periplasm before OM translocation. Conversely, a limited capacity for the translocation of folded native passengers with engineered disulfide bonds has been reported by studies with Hbp from E. coli (23) and pertactin from Bordetella pertussis (24). Crystallographic structures of the C-terminal domains of NalP from Neisseria meningitidis (41) and EspP from E. coli (2) revealed distinct β-barrel folding with 12 amphipathic β-strands and one N-terminal α-helix filling the central hydrophilic pore of the β-barrel. No indication of oligomerization was obtained with the crystallographic data. In addition, the putative protein-conducting channels of the EspP and NalP β-barrels (of ∼1 nm in diameter) were found to be closed due to the presence of the internal α-helix, which would impede the transport of passenger polypeptides (either folded or unfolded) through the reported structures. Thus, an alternative model was proposed for the assisted translocation of ATs (3, 41), in which the protein-conducting channel for secretion across the OM would be provided by the conserved Bam complex. The Bam complex is required for the insertion of β-barrel proteins (32), and the depletion of its essential component BamA (formerly YaeT in E. coli and Omp85 in Neisseria) prevents the insertion of several ATs in the OM (i.e., IcsA and SepA from S. flexneri, AIDA-I and Hbp from E. coli, and BrkA from B. pertussis) (21, 50). BamA was reported to form hydrophilic pores in lipid membranes in vitro (54) and to cross-link in vivo with the passenger domain of a slow-secretion mutant of EspP (19), which supports a role for BamA in translocation.Despite the above-described progress made in our understanding of ATs, their actual molecular mechanism of secretion remains uncertain. This is partially because the reported information is based on studies with different model AT proteins and nonhomogenous experimental approaches used by different laboratories, which sometimes produce data that are difficult to compare or may be conflicting. Here, we report a comparative study to determine conserved biochemical and functional properties found in AT C-terminal domains. Following a uniform experimental approach for six AT C-terminal domains selected from the gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria, we have investigated their capacities for the secretion of peptides and globular domains, their pore formation and oligomerization properties, and their requirement for an N-terminal α-helix for AT function and C-terminal domain stability. Our results shed light on the secretion mechanism of ATs from the conserved structural features found in their C-terminal domains.     

Functional Domains in the Retroviral Transmembrane Protein

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967–6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.     

Structural and Functional Analysis of Multi-Interface Domains

A multi-interface domain is a domain that can shape multiple and distinctive binding sites to contact with many other domains, forming a hub in domain-domain interaction networks. The functions played by the multiple interfaces are usually different, but there is no strict bijection between the functions and interfaces as some subsets of the interfaces play the same function. This work applies graph theory and algorithms to discover fingerprints for the multiple interfaces of a domain and to establish associations between the interfaces and functions, based on a huge set of multi-interface proteins from PDB. We found that about 40% of proteins have the multi-interface property, however the involved multi-interface domains account for only a tiny fraction (1.8%) of the total number of domains. The interfaces of these domains are distinguishable in terms of their fingerprints, indicating the functional specificity of the multiple interfaces in a domain. Furthermore, we observed that both cooperative and distinctive structural patterns, which will be useful for protein engineering, exist in the multiple interfaces of a domain.     

Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein

In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.     

Disentangling the Roles of Functional Domains in the Aggregation and Adsorption of the Multimodular Sea Star Adhesive Protein Sfp1

Marine Biotechnology - Sea stars can adhere to various underwater substrata using an adhesive secretion of which Sfp1 is a major component. Sfp1 is a multimodular protein composed of four subunits...     

MALDI Top-Down Sequencing: Calling N- and C-Terminal Protein Sequences with High Confidence and Speed

The ability to match Top-Down protein sequencing (TDS) results by MALDI-TOF to protein sequences by classical protein database searching was evaluated in this work. Resulting from these analyses were the protein identity, the simultaneous assignment of the N- and C-termini and protein sequences of up to 70 residues from either terminus. In combination with de novo sequencing using the MALDI-TDS data, even fusion proteins were assigned and the detailed sequence around the fusion site was elucidated. MALDI-TDS allowed to efficiently match protein sequences quickly and to validate recombinant protein structures—in particular, protein termini—on the level of undigested proteins.