Explanatory power by vagueness. Challenges to the strong prior hypothesis on hallucinations exemplified by the Charles-Bonnet-Syndrome.

Predictive processing models are often ascribed a certain generality in conceptually unifying the relationships between perception, action, and cognition or the potential to posit a 'grand unified theory' of the mind. The limitations of this unification can be seen when these models are applied to specific cognitive phenomena or phenomenal consciousness. Our article discusses these shortcomings for predictive processing models of hallucinations by the example of the Charles-Bonnet-Syndrome. This case study shows that the current predictive processing account omits essential characteristics of stimulus-independent perception in general, which has critical phenomenological implications. We argue that the most popular predictive processing model of hallucinatory conditions - the strong prior hypothesis - fails to fully account for the characteristics of nonveridical perceptual experiences associated with Charles-Bonnet-Syndrome. To fill this explanatory gap, we propose that the strong prior hypothesis needs to include reality monitoring to apply to more than just veridical percepts.

Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Incorporation of an Unnatural Amino Acid as a Domain-Specific Fluorescence Probe in a Two-Domain Protein.

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid p-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein.

Prolyl isomerization as a molecular memory in the allosteric regulation of the signal adapter protein c-CrkII.

c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.

Dimeric Structure of the Bacterial Extracellular Foldase PrsA.

Secretion of proteins into the membrane-cell wall space is essential for cell wall biosynthesis and pathogenicity in Gram-positive bacteria. Folding and maturation of many secreted proteins depend on a single extracellular foldase, the PrsA protein. PrsA is a 30-kDa protein, lipid anchored to the outer leaflet of the cell membrane. The crystal structure of Bacillus subtilis PrsA reveals a central catalytic parvulin-type prolyl isomerase domain, which is inserted into a larger composite NC domain formed by the N- and C-terminal regions. This domain architecture resembles, despite a lack of sequence conservation, both trigger factor, a ribosome-binding bacterial chaperone, and SurA, a periplasmic chaperone in Gram-negative bacteria. Two main structural differences are observed in that the N-terminal arm of PrsA is substantially shortened relative to the trigger factor and SurA and in that PrsA is found to dimerize in a unique fashion via its NC domain. Dimerization leads to a large, bowl-shaped crevice, which might be involved in vivo in protecting substrate proteins from aggregation. NMR experiments reveal a direct, dynamic interaction of both the parvulin and the NC domain with secretion propeptides, which have been implicated in substrate targeting to PrsA.

Control of protein function by prolyl isomerization.

BACKGROUND: Prolyl cis/trans isomerizations have long been known as critical and rate-limiting steps in protein folding. RESULTS: Now it is clear that they are also used as slow conformational switches and molecular timers in the regulation of protein activity. Here we describe several such proline switches and how they are regulated. CONCLUSIONS AND GENERAL SIGNIFICANCE: Prolyl isomerizations can function as attenuators and provide allosteric systems with a molecular memory. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Cardiovascular magnetic resonance assessment of the aortic valve stenosis: an in vivo and ex vivo study.

BACKGROUND: Aortic valve area (AVA) estimation in patients with aortic stenosis may be obtained using several methods. This study was undertaken to verify the cardiovascular magnetic resonance (CMR) planimetry of aortic stenosis by comparing the findings with invasive catheterization, transthoracic (TTE) as well as tranesophageal echocardiography (TEE) and anatomic CMR examination of autopsy specimens. METHODS: Our study was performed in eight patients with aortic valve stenosis. Aortic stenosis was determined by TTE and TEE as well as catheterization and CMR. Especially, after aortic valve replacement, the explanted aortic valves were examined again with CMR ex vivo model. RESULTS: The mean AVA determined in vivo by CMR was 0.75 ± 0.09 cm(2) and ex vivo by CMR was 0.65 ± 0.09 cm(2) and was closely correlated (r = 0.91, p < 0.001). The mean absolute difference between AVA derived by CMR ex vivo and in vivo was -0.10 ± 0.04 cm(2). The mean AVA using TTE was 0.69 ± 0.07 with a significant correlation between CMR ex vivo (r = 0.85, p < 0.007) and CMR in vivo (r = 0.86, p < 0.008). CMR ex vivo and in vivo had no significant correlation with AVA using Gorlin formula by invasive catheterization or using planimetry by TEE. CONCLUSION: In this small study using an ex vivo aortic valve stenosis model, the aortic valve area can be reliably planimetered by CMR in vivo and ex vivo with a well correlation between geometric AVA by CMR and the effective AVA calculated by TTE.

Initiation of phage infection by partial unfolding and prolyl isomerization.

Infection of Escherichia coli by the filamentous phage fd starts with the binding of the N2 domain of the phage gene-3-protein to an F pilus. This interaction triggers partial unfolding of the gene-3-protein, cis → trans isomerization at Pro-213, and domain disassembly, thereby exposing its binding site for the ultimate receptor TolA. The trans-proline sets a molecular timer to maintain the binding-active state long enough for the phage to interact with TolA. We elucidated the changes in structure and local stability that lead to partial unfolding and thus to the activation of the gene-3-protein for phage infection. Protein folding and TolA binding experiments were combined with real-time NMR spectroscopy, amide hydrogen exchange measurements, and phage infectivity assays. In combination, the results provide a molecular picture of how a local unfolding reaction couples with prolyl isomerization not only to generate the activated state of a protein but also to maintain it for an extended time.

Unique proline-rich domain regulates the chaperone function of AIPL1.

Human aryl hydrocarbon receptor (AHR) interacting protein (AIP) and AIP like 1 (AIPL1) are cochaperones of Hsp90 which share 49% sequence identity. Both proteins contain an N-terminal FKBP-like prolyl peptidyl isomerase (PPIase) domain followed by a tetratricopeptide repeat (TPR) domain. In addition, AIPL1 harbors a unique C-terminal proline-rich domain (PRD). Little is known about the functional relevance of the individual domains and how these contribute to the association with Hsp90. In this study, we show that these cochaperones differ from other Hsp90-associated PPIase as their FKBP domains are enzymatically inactive. Furthermore, in contrast to other large PPIases, AIP is inactive as a chaperone. AIPL1, however, exhibits chaperone activity and prevents the aggregation of non-native proteins. The unique proline-rich domain of AIPL1 is important for its chaperone function as its truncation severely affects the ability of AIPL1 to bind non-native proteins. Furthermore, the proline-rich domain decreased the affinity of AIPL1 for Hsp90, implying that this domain acts as a negative regulator of the Hsp90 interaction besides being necessary for efficient binding of AIPL1 to non-native proteins.

The prolyl isomerase SlyD is a highly efficient enzyme but decelerates the conformational folding of a client protein.

Folding enzymes often use distinct domains for the interaction with a folding protein chain and for the catalysis of intrinsically slow reactions such as prolyl cis/trans isomerization. Here, we investigated the refolding reaction of ribonuclease T1 in the presence of the prolyl isomerase SlyD from Escherichia coli to examine how this enzyme catalyzes the folding of molecules with an incorrect trans proline isomer and how it modulates the conformational folding of the molecules with the correct cis proline. The kinetic analysis suggests that prolyl cis → trans isomerization in the SlyD-bound state shows a rate near 100 s(-1) and is thus more than 10(4)-fold accelerated, relative to the uncatalyzed reaction. As a consequence of its fast binding and efficient catalysis, SlyD retards the conformational folding of the protein molecules with the correct cis isomer, because it promotes the formation of the species with the incorrect trans isomer. In the presence of ≥1 µM SlyD, protein molecules with cis and trans prolyl isomers refold with identical rates, because SlyD-catalyzed cis/trans equilibration is faster than conformational folding. The cis or trans state of a particular proline is thus no longer a determinant for the rate of folding.

Structural and energetic basis of infection by the filamentous bacteriophage IKe.

Filamentous phage use the two N-terminal domains of their gene-3-proteins to initiate infection of Escherichia coli. One domain interacts with a pilus, and then the other domain binds to TolA at the cell surface. In phage fd, these two domains are tightly associated with each other, which renders the phage robust but non-infectious, because the TolA binding site is inaccessible. Activation for infection requires partial unfolding, domain disassembly and prolyl isomerization. Phage IKe infects E. coli less efficiently than phage fd. Unlike in phage fd, the pilus- and TolA-binding domains of phage IKe are independent of each other in stability and folding. The site for TolA binding is thus always accessible, but the affinity is very low. The structures of the two domains, analysed by X-ray crystallography and by NMR spectroscopy, revealed a unique fold for the N-pilus-binding domain and a conserved fold for the TolA-binding domain. The absence of an activation mechanism as in phage fd and the low affinity for TolA probably explain the low infectivity of phage IKe. They also explain why, in a previous co-evolution experiment with a mixture of phage fd and phage IKe, all hybrid phage adopted the superior infection mechanism of phage fd.

Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD.

SlyD is a bacterial two-domain protein that functions as a molecular chaperone, a prolyl cis/trans isomerase, and a nickel-binding protein. This review summarizes recent findings about the molecular enzyme mechanism of SlyD. The chaperone function located in one domain of SlyD is involved in twin-arginine translocation and increases the catalytic efficiency of the prolyl cis/trans isomerase domain in protein folding by two orders of magnitude. The C-terminal tail of SlyD binds Ni2+ ions and supplies them for the maturation of [NiFe] hydrogenases. A combined biochemical and biophysical analysis revealed the molecular basis of the delicate interplay of the different domains of SlyD for optimal function.

Yersinia enterocolitica and Photorhabdus asymbiotica ß-lactamases BlaA are exported by the twin-arginine translocation pathway.

In general, ß-lactamases of medically important Gram-negative bacteria are Sec-dependently translocated into the periplasm. In contrast, ß-lactamases of Mycobacteria spp. (BlaC, BlaS) and the Gram-negative environmental bacteria Stenotrophomonas maltophilia (L2) and Xanthomonas campestris (Bla(XCC-1)) have been reported to be secreted by the twin-arginine translocation (Tat) system. Yersinia enterocolitica carries 2 distinct ß-lactamase genes (blaA and blaB) encoding BlaA(Ye) and the AmpC-like ß-lactamase BlaB, respectively. By using the software PRED-TAT for prediction and discrimination of Sec from Tat signal peptides, we identified a functional Tat signal sequence for Yersinia BlaA(Ye). The Tat-dependent translocation of BlaA(Ye) could be clearly demonstrated by using a Y. enterocolitica tatC-mutant and cell fractionation. Moreover, we could demonstrate a unique unusual temperature-dependent activity profile of BlaA(Ye) ranging from 15 to 60 °C and a high 'melting temperature' (T(M)=44.3°) in comparison to the related Sec-dependent ß-lactamase TEM-1 (20-50°C, T(M)=34.9 °C). Strikingly, the blaA gene of Y. enterocolitica is present in diverse environmental Yersinia spp. and a blaA homolog gene could be identified in the closely related Photorhabdus asymbiotica (BlaA(Pa); 69% identity to BlaA(Ye)). For BlaA(Pa) of P. asymbiotica, we could also demonstrate Tat-dependent secretion. These results suggest that Yersinia BlaA-related ß-lactamases may be the prototype of a large Tat-dependent ß-lactamase family, which originated from environmental bacteria.

A kinetic approach to determining the conformational stability of a protein that dimerizes after folding.

A strongly stabilized form of the ß1 domain of the streptococcal protein G, termed Gß1-M2, was previously obtained by an in vitro selection method for stabilized protein variants. It contains four substitutions, but how they contribute to the Gibbs free energy of denaturation (ΔG(D)) could not be determined, because, unlike the wild-type protein, Gß1-M2 dimerizes in a spectroscopically silent reaction. Here we determined the ΔG(D) of the folded Gß1-M2 monomer by using a kinetic approach that uncouples the folding of the monomer from dimerization. The conformational equilibration of the monomer is faster than dimer formation, and therefore, its stability constant could be determined from the ratio of the rate constants for monomer unfolding and refolding. In this approach, double-mixing experiments were essential for uncovering the unfolding kinetics of the transient Gß1-M2 monomer and the association of the monomers after their folding. The analysis revealed that the selected substitutions stabilize the Gß1-M2 monomer by 15 kJ mol(-1) in an additive fashion. The combination of single- and double-mixing kinetic experiments thus allowed us to determine the thermodynamic stability of a transient species that is inaccessible in equilibrium experiments. It can be applied for proteins in which monomer folding and oligomerization are kinetically uncoupled.

Folding mechanism of an extremely thermostable (ßα)(8)-barrel enzyme: a high kinetic barrier protects the protein from denaturation.

HisF, the cyclase subunit of imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima, is an extremely thermostable (ßα)(8)-barrel protein. We elucidated the unfolding and refolding mechanism of HisF. Its unfolding transition is reversible and adequately described by the two-state model, but 6 weeks is necessary to reach equilibrium (at 25 °C). During refolding, initially a burst-phase off-pathway intermediate is formed. The subsequent productive folding occurs in two kinetic phases with time constants of ~3 and ~20 s. They reflect a sequential process via an on-pathway intermediate, as revealed by stopped-flow double-mixing experiments. The final step leads to native HisF, which associates with the glutaminase subunit HisH to form the functional ImGPS complex. The conversion of the on-pathway intermediate to the native protein results in a 10(6)-fold increase of the time constant for unfolding from 89 ms to 35 h (at 4.0 M GdmCl) and thus establishes a high energy barrier to denaturation. We conclude that the extra stability of HisF is used for kinetic protection against unfolding. In its refolding mechanism, HisF resembles other (ßα)(8)-barrel proteins.

Conservation of the folding mechanism between designed primordial (ßα)8-barrel proteins and their modern descendant.

The (ßα)(8)-barrel is among the most ancient, frequent, and versatile enzyme structures. It was proposed that modern (ßα)(8)-barrel proteins have evolved from an ancestral (ßα)(4)-half-barrel by gene duplication and fusion. We explored whether the mechanism of protein folding has remained conserved during this long-lasting evolutionary process. For this purpose, potential primordial (ßα)(8)-barrel proteins were constructed by the duplication of a (ßα)(4) element of a modern (ßα)(8)-barrel protein, imidazole glycerol phosphate synthase (HisF), followed by the optimization of the initial construct. The symmetric variant Sym1 was less stable than HisF and its crystal structure showed disorder in the contact regions between the half-barrels. The next generation variant Sym2 was more stable than HisF, and the contact regions were well resolved. Remarkably, both artificial (ßα)(8)-barrels show the same refolding mechanism as HisF and other modern (ßα)(8)-barrel proteins. Early in folding, they all equilibrate rapidly with an off-pathway species. On the productive folding path, they form closely related intermediates and reach the folded state with almost identical rates. The high energy barrier that synchronizes folding is thus conserved. The strong differences in stability between these proteins develop only after this barrier and lead to major changes in the unfolding rates. We conclude that the refolding mechanism of (ßα)(8)-barrel proteins is robust. It evolved early and, apparently, has remained conserved upon the diversification of sequences and functions that have taken place within this large protein family.

Reprogramming the infection mechanism of a filamentous phage.

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.

Chaperone domains convert prolyl isomerases into generic catalysts of protein folding.

The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.

A remote prolyl isomerization controls domain assembly via a hydrogen bonding network.

Prolyl cis/trans isomerizations determine the rates of protein folding reactions and can serve as molecular switches and timers. In the gene-3-protein of filamentous phage, Pro-213 trans --> cis isomerization in a hinge region controls the assembly of the 2 domains N1 and N2 and, in reverse, the activation of the phage for infection. We elucidated the structural and energetic basis of this proline-limited domain assembly at the level of individual residues by real-time 2D NMR. A local cluster of inter-domain hydrogen bonds, remote from Pro-213, is stabilized up to 3,000-fold by trans --> cis isomerization. This network of hydrogen bonds mediates domain assembly and is connected with Pro-213 by rigid backbone segments. Thus, proline cis/trans switching is propagated in a specific and directional fashion to change the protein structure and stability at a distant position.