How the west was won: genetic reconstruction of rapid wolf recolonization into Germany’s anthropogenic landscapes

Following massive persecution and eradication, strict legal protection facilitated a successful reestablishment of wolf packs in Germany, which has been ongoing since 2000. Here, we describe this recolonization process by mitochondrial DNA control-region sequencing, microsatellite genotyping and sex identification based on 1341 mostly non-invasively collected samples. We reconstructed the genealogy of German wolf packs between 2005 and 2015 to provide information on trends in genetic diversity, dispersal patterns and pack dynamics during the early expansion process. Our results indicate signs of a founder effect at the start of the recolonization. Genetic diversity in German wolves is moderate compared to other European wolf populations. Although dispersal among packs is male-biased in the sense that females are more philopatric, dispersal distances are similar between males and females once only dispersers are accounted for. Breeding with close relatives is regular and none of the six male wolves originating from the Italian/Alpine population reproduced. However, moderate genetic diversity and inbreeding levels of the recolonizing population are preserved by high sociality, dispersal among packs and several immigration events. Our results demonstrate an ongoing, rapid and natural wolf population expansion in an intensively used cultural landscape in Central Europe.Subject terms: Conservation biology, Population genetics     

A 3D-DNA Molecule Made of PlayMais

More than 60 years have passed since the work of Rosalind Franklin, James Watson, and Francis Crick led to the discovery of the 3D-DNA double-helix structure. Nowadays, due to the simple and elegant architecture of its double helix, the structure of DNA is widely known. The biological role of the DNA molecule (e.g., genetic information), however, along with the cellular mechanisms involving the DNA double helix (e.g., DNA replication) are topics that have not yet reached a broader public. In this educational article, we aim to provide a way for schoolchildren to live a three-dimensional experience that focuses on the DNA double helix structure. Moreover, taking advantage of an engaging and visual protocol, students will experience an overview of its biological implications. To do so, starting from a gene sequence, students will have the opportunity to build their own 3D-DNA double helix structure using PlayMais flakes.     

Putting the Squeeze on Plasmodesmata: A Role for Reticulons in Primary Plasmodesmata Formation

Primary plasmodesmata (PD) arise at cytokinesis when the new cell plate forms. During this process, fine strands of endoplasmic reticulum (ER) are laid down between enlarging Golgi-derived vesicles to form nascent PD, each pore containing a desmotubule, a membranous rod derived from the cortical ER. Little is known about the forces that model the ER during cell plate formation. Here, we show that members of the reticulon (RTNLB) family of ER-tubulating proteins in Arabidopsis (Arabidopsis thaliana) may play a role in the formation of the desmotubule. RTNLB3 and RTNLB6, two RTNLBs present in the PD proteome, are recruited to the cell plate at late telophase, when primary PD are formed, and remain associated with primary PD in the mature cell wall. Both RTNLBs showed significant colocalization at PD with the viral movement protein of Tobacco mosaic virus, while superresolution imaging (three-dimensional structured illumination microscopy) of primary PD revealed the central desmotubule to be labeled by RTNLB6. Fluorescence recovery after photobleaching studies showed that these RTNLBs are mobile at the edge of the developing cell plate, where new wall materials are being delivered, but significantly less mobile at its center, where PD are forming. A truncated RTNLB3, unable to constrict the ER, was not recruited to the cell plate at cytokinesis. We discuss the potential roles of RTNLBs in desmotubule formation.Plasmodesmata (PD), the small pores that connect higher plant cells, are complex structures of about 50 nm in diameter. Each PD pore is lined by the plasma membrane and contains an axial endoplasmic reticulum (ER)-derived structure known as the desmotubule (Overall and Blackman, 1996; Maule, 2008; Tilsner et al., 2011). The desmotubule is an enigmatic structure whose function has not been fully elucidated. The small spiraling space between the desmotubule and the plasma membrane, known as the cytoplasmic sleeve, is almost certainly a conduit for the movement of small molecules (Oparka et al., 1999). Some reports, however, suggest that the desmotubule may also function in cell-to-cell trafficking, providing an ER-derived pathway between cells along which macromolecules may diffuse (Cantrill et al., 1999). The desmotubule is one of the most tightly constricted membrane structures found in nature (Tilsner et al., 2011), but the forces that generate its intense curvature are not understood. In most PD, the desmotubule is a tightly furled tube of about 15 nm in diameter in which the membranes of the ER are in close contact along its length. The desmotubule may balloon out in the region of the middle lamella into a central cavity, but at the neck regions of the PD pore it is tightly constricted (Robinson-Beers and Evert, 1991; Ding et al., 1992; Glockmann and Kollmann, 1996; Overall and Blackman, 1996; Ehlers and Kollmann, 2001). Studies of PD using GFP targeted to the ER lumen (e.g. GFP-HDEL) have shown that GFP is excluded from the desmotubule due to the constriction of ER membranes in this structure (Oparka et al., 1999; Crawford and Zambryski, 2000; Martens et al., 2006; Guenoune-Gelbart et al., 2008). Therefore, lumenal GFP is unable to move between plant cells unless the membranes of the desmotubule become relaxed in some way. On the other hand, dyes and some proteins inserted into the ER membrane can apparently move through the desmotubule, either along the membrane or through the lumen, at least under some conditions (Grabski et al., 1993; Cantrill et al., 1999; Martens et al., 2006; Guenoune-Gelbart et al., 2008).Recently, a number of proteins have been described in mammalian, yeast, and plant systems that induce extreme membrane curvature. Among these are the RETICULONS (RTNs), integral membrane proteins that induce curvature of the ER to form tubules (Voeltz et al., 2006; Hu et al., 2008; Tolley et al., 2008, 2010; Sparkes et al., 2010). In animals, RTNs have been shown to be involved in a wide array of endomembrane-related processes, including intracellular transport and vesicle formation, and as RTNs can also influence axonal growth, they may have roles in neurodegenerative disorders such as Alzheimer’s disease (Yang and Strittmatter, 2007). Arabidopsis (Arabidopsis thaliana) has 21 RTN homologs, known as RTNLBs (Nziengui et al., 2007; Sparkes et al., 2010), considerably more than in yeast or mammals, but most have not been examined. RTNLBs contain two unusually long hydrophobic helices that form reentrant loops (Voeltz et al., 2006; Hu et al., 2008; Sparkes et al., 2010; Tolley et al., 2010). These are thought to induce membrane curvature by the molecular wedge principle (Hu et al., 2008; Shibata et al., 2009). When RTNLBs are overexpressed transiently in cells expressing GFP-HDEL, the ER becomes tightly constricted and GFP-HDEL is excluded from the lumen of the constricted ER tubules (Tolley et al., 2008, 2010), a situation similar to that which occurs in desmotubules (Oparka et al., 1999; Crawford and Zambryski, 2000; Martens et al., 2006). In vitro studies with isolated membranes have shown that the degree of tubulation is proportional to the number and spacing of RTNLB proteins in the membrane (Hu et al., 2008). For example, to constrict the ER membrane into a structure of 15 nm, the diameter of a desmotubule, would require RTNLBs to be inserted every 2 nm or less along the desmotubule axis (Hu et al., 2008), potentially making the desmotubule an extremely protein-rich structure (Tilney et al., 1991). Interestingly, a number of RTNLB proteins appear in the recently described PD proteome (Fernandez-Calvino et al., 2011), suggesting that RTNLBs are good candidates for proteins that model the cortical ER into desmotubules.Primary PD form at cytokinesis during the assembly of the cell plate (Hawes et al., 1981; Hepler, 1982). Of the numerous studies devoted to the structure of the cell plate, very few have examined the behavior of the ER during cytokinesis. During mitosis, elements of the ER are located in the spindle apparatus, separated from the cytoplasm (Hepler, 1980). Just prior to cytokinesis, there is a relative paucity of ER in the region destined to become the cell plate (Hepler, 1980; Hawes et al., 1981). The studies of Hawes et al. (1981) and Hepler (1982), exploiting heavy-metal impregnation of the ER, showed that during the formation of the new cell plate, strands of cortical ER are inserted across the developing wall, between the Golgi-derived vesicles that deposit wall materials. These ER strands become increasingly thinner during formation of the desmotubule, eventually excluding heavy metal stains from the ER lumen (Hepler, 1982). The center of the desmotubule often appears electron opaque in transmission electron microscopy images and has been referred to as the central rod (Overall and Blackman, 1996). This structure may consist of proteins that extend from the inner ER leaflets or may correspond to head groups of the membrane lipids themselves. In the fully formed primary PD, the desmotubule remains continuous with the cortical ER that runs close to the new cell wall (Hawes et al., 1981; Hepler, 1982; Oparka et al., 1994).Here, we show that two of the RTNLBs present in the PD proteome, RTNLB3 and RTNLB6, become localized to the cell plate during the formation of primary PD. These RTNLBs remain associated with the desmotubule in fully formed PD and are immobile, as evidenced by fluorescence recovery after photobleaching (FRAP) studies. A truncated version of RTNLB3, in which the second hydrophobic region was deleted (Sparkes et al., 2010), was not recruited to the cell plate at cytokinesis. We suggest that RTNLBs play an important role in the formation of primary PD and discuss mechanisms by which these proteins may model the ER into desmotubules.     

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions,and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b₆f (cyt b₆f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the Calvin-Benson-Bassham cycle (Bassham et al., 1950) to fix CO₂. Alternatively, cyclic electron flow (CEF) between PSI and the cyt b₆f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO₂ fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues, and readjusts the ATP poise, leading to increased lumen acidification important for photoprotection (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF relies on the NAD(P)H dehydrogenase-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathways (Munekage et al., 2002, 2004; Petroutsos et al., 2009; Tolleter et al., 2011; Johnson et al., 2014). For both pathways, supercomplexes consisting of PSI-LIGHT HARVESTING COMPLEX I (LHCI) and components of the respective electron transfer routes have been identified. In Arabidopsis (Arabidopsis thaliana), a unique NAD(P)H dehydrogenase-PSI supercomplex with a molecular mass of more than 1,000 kD was discovered (Peng et al., 2008). From Chlamydomonas reinhardtii, Iwai et al. (2010) isolated a protein supercomplex composed of PSI-LHCI, LHCII, the cyt b₆/f complex, ferredoxin-NADPH oxidoreductase (FNR), and PROTON GRADIENT REGULATION-LIKE1 (PGRL1).PGRL1 and PGR5 interact physically in Arabidopsis and associate with PSI to allow the operation of CEF (DalCorso et al., 2008). Functional data suggest that PGRL1 might operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). The PGRL1-containing CEF supercomplex isolated from C. reinhardtii is capable of CEF under in vitro conditions in the presence of exogenously added soluble plastocyanin and ferredoxin (Iwai et al., 2010). Terashima et al. (2012) isolated a CEF supercomplex of similar composition from anaerobic growth conditions that was active in vitro and contained proteins such as the chloroplast-localized Ca²⁺ sensor CAS and ANAEROBIC RESPONSE1 (ANR1), which were also shown to be functionally important for efficient CEF in the alga. Notably, it was suggested that the onset of CEF in C. reinhardtii is redox controlled (Takahashi et al., 2013).It has been demonstrated that efficient CEF is crucial for successful acclimation to excess light (Munekage et al., 2004; Dang et al., 2014; Johnson et al., 2014; Kukuczka et al., 2014). The most rapid response to excess light, however, relies on a mechanism called nonphotochemical quenching (NPQ). The fastest constituent of NPQ is energy-dependent (qE) quenching, which operates at a time scale of seconds to minutes and regulates the thermal dissipation of excess absorbed light energy, thereby providing effective photoprotection. In vascular plants, the PSII protein PSII SUBUNIT S is essential for qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). CEF and qE are complementary for acclimation to excess light, as double mutants deficient in both mechanisms possess additive phenotypes and are highly sensitive to light (Kukuczka et al., 2014). Another constituent of NPQ is the quenching by state transitions. State transitions are important to balance the excitation energy between PSI and PSII (Bonaventura and Myers, 1969; Murata, 1969). Under light conditions where PSII is preferentially excited, both PSII core and LHCII proteins become phosphorylated (Lemeille and Rochaix, 2010). As a consequence, phosphorylated LHCII proteins detach from PSII and partly connect to PSI (state 2). Under conditions where PSI excitation is predominant, this process is reversed. LHCII proteins are dephosphorylated and associate with PSII (state 1). The extent of state transition between vascular plants such as Arabidopsis and C. reinhardtii differs significantly. The proportion of mobile LHCII antenna is about 80% in the alga, whereas in Arabidopsis, only 15% to 20% of LHCII is transferred to PSI under state 2 conditions (Lemeille and Rochaix, 2010). However, the large increase in PSI antenna size in C. reinhardtii has recently been challenged (Nagy et al., 2014; Ünlü et al., 2014): while 70% to 80% of mobile LHCII detached from PSII in response to transition to state 2 conditions, only a fraction of about 20% functionally attached to PSI.Phosphorylation of LHC proteins requires the function of the STT7 kinase or its ortholog STN7 in C. reinhardtii or Arabidopsis, respectively. In the absence of the STT7/STN7 kinase, the initiation of state transitions is blocked (Depège et al., 2003; Bellafiore et al., 2005). The mobile LHCII fraction of C. reinhardtii includes the two monomeric minor LHCII antenna proteins, CP26 and CP29 (encoded by lhcb5 and lhcb4 genes), and the major chlorophyll a/b binding protein of LHCII, LHCBM5 (Takahashi et al., 2006), but also the LHCSR3 protein was suggested to migrate during state transitions (Allorent et al., 2013). Takahashi et al. (2014) suggested that only CP29 and LHCBM5 directly associate with PSI to form the PSI-LHCI-LHCII supercomplex, while the binding of CP26 could occur indirectly or via the other two proteins. However, it is not yet known whether STT7 directly phosphorylates the LHCII proteins or if this takes place as part of a kinase cascade (Rochaix, 2007). Nevertheless, the direct interaction between STT7 and the LHCII proteins is quite likely, since none of the other chloroplast kinases was found to be specifically required for LHCII phosphorylation (Rochaix, 2014). The activity of the STT7 kinase is mainly determined by the redox status of the plastoquinone pool (Vener et al., 1997; Zito et al., 1999). The identification of a PROTEIN PHOSPHATASE 2C (PP2C)-type phosphatase responsible for the dephosphorylation of the LHCII proteins in Arabidopsis has been described by two studies in parallel pointing to the fact that this enzyme, called PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, acts directly on phosphorylated LHCII proteins, in particular when they are associated with the PSI-LHCI supercomplex (Pribil et al., 2010; Shapiguzov et al., 2010). Moreover, it is not known whether these phosphatases are constitutively active or if they are regulated by other means, for example through the redox state of the plastoquinone pool. Nonetheless, both enzymes are conserved in land plants and exhibit orthologous proteins in C. reinhardtii (Rochaix et al., 2012).Another kinase related to STN7/STT7 is encoded in the Arabidopsis and C. reinhardtii genomes and named STN8 and STATE TRANSITION-LIKE1 (STL1), respectively. STN8 is involved in PSII core subunit phosphorylation and influences the repair of PSII after photodamage (Bonardi et al., 2005; Vainonen et al., 2005). Remarkably, the disassembly of the PSII holocomplex is inhibited in STN7/STN8 double mutants (Tikkanen et al., 2008; Fristedt et al., 2009; Dietzel et al., 2011; Nath et al., 2013), suggesting that the phosphorylation of core subunits is required for PSII disassembly. It was further suggested that STN8 controls the transition between linear electron flow and CEF by the phosphorylation of PGRL1 in Arabidopsis (Reiland et al., 2011). As described for STN7, the activity of STN8 is probably regulated via the redox state of the plastoquinone pool (Bennett, 1991; Fristedt et al., 2009). Notably, the action of STN8 is counteracted by a chloroplast PP2C phosphatase (Samol et al., 2012), allowing for the fast reversibility of STN8-mediated acclimation responses. Thus, it appears that an intricate regulatory network of chloroplast protein kinases and phosphatases evolved in vascular plants and algae that drives the acclimation response to various environmental cues, including excess and changing light settings (Rochaix et al., 2012). As STN7/STT7 and STN8/STL1 kinase activities appear to be controlled by the redox poise of the plastoquinone pool, the plastoquinone pool would be a central player in these acclimation responses. On the other hand, the kinases themselves are subjected to phosphorylation (Reiland et al., 2009, 2011; Lemeille et al., 2010; Wang et al., 2013). However, the functional consequences of this phosphorylation are unknown.Recent comparative analyses revealed the presence of at least 15 distinct chloroplast protein kinases, suggesting an intricate kinase phosphorylation network in the chloroplast (Bayer et al., 2012). Generally, the phosphorylation of proteins is one of the most abundant posttranslational modifications. In complex eukaryotic systems, protein phosphorylation occurs most frequently on Ser followed by Thr residues, whereas protein phosphorylation of Tyr residues (1,800:200:1) is comparatively rare (Hunter, 1998; Mann et al., 2002). Protein phosphorylation is a general phenomenon in vivo; it is assumed that about one-third of all proteins are phosphorylated at a given time (Cohen, 2000; Ahn and Resing, 2001; Venter et al., 2001; Manning et al., 2002; Knight et al., 2003). A recent large-scale quantitative evaluation of human proteomic data strengthened the importance of protein phosphorylation for cellular function and human biology (Wilhelm et al., 2014). The C. reinhardtii and Arabidopsis genomes encode large kinase families (Arabidopsis Genome Initiative, 2000; Kerk et al., 2002; Merchant et al., 2007), supporting the view that protein phosphorylation also plays an important role in a plant’s life cycle. It is thus evident that the understanding of protein phosphorylation, including the specificity of residues phosphorylated or dephosphorylated in response to cellular as well as environmental factors, is one key to understanding the complex functional biological networks at the whole-system level. Likewise, it is crucial to design experimental setups allowing the linkage between phosphorylation events and particular physiological consequences to be elucidated.In this regard, we designed experiments to investigate STT7 kinase-dependent phosphorylation dynamics in C. reinhardtii in response to high light and anoxia, employing quantitative proteomics in conjunction with in-depth physiological characterization. These conditions are particularly interesting, as high light conditions are known to fully induce LHCSR3 protein expression and qE, while anoxia promotes CEF activity. Recently, it was demonstrated that qE and CEF are complementary and crucial in acclimation to these environmental cues (Kukuczka et al., 2014). Notably, LHCSR3 phosphorylation was suggested to depend on STT7 function (Bonente et al., 2011), while CEF supercomplex formation was found to be independent of STT7 kinase function (Takahashi et al., 2013), indicating that STT7 function might impact the acclimation to high light and anoxia in different ways. However, our quantitative proteomics and physiological data reveal that STT7-dependent variations in protein phosphorylation profiles have similar dramatic phenotypic consequences in both conditions, strongly suggesting that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments.     

Chemo- and thermosensory responsiveness of Grueneberg ganglion neurons relies on cyclic guanosine monophosphate signaling elements

Neurons of the Grueneberg ganglion (GG) in the anterior nasal region of mouse pups respond to cool temperatures and to a small set of odorants. While the thermosensory reactivity appears to be mediated by elements of a cyclic guanosine monophosphate (cGMP) cascade, the molecular mechanisms underlying the odor-induced responses are unclear. Since odor-responsive GG cells are endowed with elements of a cGMP pathway, specifically the transmembrane guanylyl cyclase subtype GC-G and the cyclic nucleotide-gated ion channel CNGA3, the possibility was explored whether these cGMP signaling elements may also be involved in chemosensory GG responses. Experiments with transgenic mice deficient for GC-G or CNGA3 revealed that GG responsiveness to given odorants was significantly diminished in these knockout animals. These findings suggest that a cGMP cascade may be important for both olfactory and thermosensory signaling in the GG. However, in contrast to the thermosensory reactivity, which did not decline over time, the chemosensory response underwent adaptation upon extended stimulation, suggesting that the two transduction processes only partially overlap.     

Complement opsonization enhances friend virus infection of B cells and thereby amplifies the virus-specific CD8+ T cell response

B cells are one of the targets of Friend virus (FV) infection, a well-established mouse model often used to study retroviral infections in vivo. Although B cells may be effective in stimulating cytotoxic T lymphocyte responses, studies involving their role in FV infection have mainly focused on neutralizing antibody production. Here we show that polyclonal activation of B cells promotes their infection with FV both in vitro and in vivo. Furthermore, we demonstrate that complement opsonization of Friend murine leukemia virus (F-MuLV) enhances infection of B cells, which correlates with increased potency of B cells to activate FV-specific CD8(+) T cells.     

Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET

The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.