Biotherapeutic approaches against cardio-metabolic dysfunctions based on extracellular vesicles.

Among the different pathways involved in the cell-to-cell communication, extracellular vesicles (EVs) are defined as key players in the transport of different signalling molecules, such as lipids, proteins, and RNA, from the originating cells to specific target cells. The biogenesis and composition of EVs are complex and confer them a unique ability to more effectively reach tissues and cells as compared to other types of synthetic carriers. Owing to these properties, EVs have been suggested as new therapeutic tools for personalized medicine. Since cardiometabolic diseases have reached pandemic proportions, new therapies are needed to be developed. In this context, EVs appear as promising therapeutic tools against cardiometabolic disorders associated with obesity and diabetes. This review focuses on the latest research on preclinical applications of EVs for cardiometabolic diseases, and draw primarily on our experience in this area.

Phenotypic switch of smooth muscle cells in paediatric chronic intestinal pseudo-obstruction syndrome.

Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo-obstruction (CIPO) syndrome, a life-threatening intestinal motility disorder. We showed that CIPO-SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet-Derived Growth Factor Receptor-alpha (PDGFRA) expression. We showed that this modulation occurs without origin-related differences in CIPO circular and longitudinal-derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO-SMC towards an undifferentiated stage. The development of CIPO-SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

Long-term Outcome of Renal Transplantation in Patients with Congenital Lower Urinary Tract Malformations: A Multicenter Study.

BACKGROUND: Renal insufficiency can occur in patients with congenital lower urinary tract malformations (LUTM) even when managed during infancy. Data in the current literature concerning this subject remain sparse. The aim of this study was to report the feasibility and long-term results of renal transplantation during adulthood in patients with a congenital LUTM. METHODS: A retrospective multicenter study from 3 French renal transplant centers was conducted, including 123 transplantations on 112 patients with LUTM (1996-2016). Graft survival, patient survival, and complications were analyzed. Results were stratified according to the underlying uropathy and the type of initial management during childhood or before transplantation. RESULTS: In this study, patients suffering from posterior urethral valves (n = 49), spina bifida (n = 21), central neurogenic bladder (n = 13), bladder exstrophy (n = 14), prune belly syndrome (n = 12), Hinman syndrome (n = 6), urogenital sinus (n = 4), and other pathologies (n = 4) were included. The mean age at transplantation was 32.1 years old (±11.2). The mean follow-up period was 7.2 years. Patient survival at 1, 5, 10, and 15 years was 97.4%, 93.0%, 89.4%, and 80.0%, respectively. Graft survival at 1, 5, 10, 15, and 20 years was 96.6%, 87.6%, 77.3%, 60.6%, and 36.4%, respectively. Enterocystoplasty and continent urinary diversions exposed grafts to more frequent acute pyelonephritis (P = 0.02). There was no difference in graft survival when transplantation was performed on an enterocystoplasty or urinary diversions compared with a native bladder, provided a well-conducted bladder management. CONCLUSIONS: Even though enterocystoplasty and continent urinary diversions exposed grafts to more frequent acute graft pyelonephritis, patient and graft survival rates in LUTM at 10 years were similar to other kidney transplantations on native bladders.

Epithelial Splicing Regulatory Protein 1 (ESRP1) is a new regulator of stomach smooth muscle development and plasticity.

In vertebrates, stomach smooth muscle development is a complex process that involves the tight transcriptional or post-transcriptional regulation of different signalling pathways. Here, we identified the RNA-binding protein Epithelial Splicing Regulatory Protein 1 (ESRP1) as an early marker of developing and undifferentiated stomach mesenchyme. Using a gain-of-function approach, we found that in chicken embryos, sustained expression of ESRP1 impairs stomach smooth muscle cell (SMC) differentiation and FGFR2 splicing profile. ESRP1 overexpression in primary differentiated stomach SMCs induced their dedifferentiation, promoted specific-FGFR2b splicing and decreased FGFR2c-dependent activity. Moreover, co-expression of ESRP1 and RBPMS2, another RNA-binding protein that regulates SMC plasticity and Bone Morphogenetic Protein (BMP) pathway inhibition, synergistically promoted SMC dedifferentiation. Finally, we also demonstrated that ESRP1 interacts with RBPMS2 and that RBPMS2-mediated SMC dedifferentiation requires ESRP1. Altogether, these results show that ESRP1 is expressed also in undifferentiated stomach mesenchyme and demonstrate its role in SMC development and plasticity.

Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration.

Pressure effects reveal that changes in the redox states of the heme iron complexes in the sensor domains of two heme-based oxygen sensor proteins, EcDOS and YddV, have profound effects on their flexibility.

The catalytic activity of a heme-based oxygen sensor phosphodiesterase from Escherichia coli (EcDOS) towards cyclic diGMP is regulated by the redox state of the heme iron complex in the enzyme's sensing domain and the association of external ligands with the iron center. Specifically, the Fe(II) complex is more active towards cyclic diGMP than the Fe(III) complex, and its activity is further enhanced by O2 or CO binding. In order to determine how the redox state and coordination of the heme iron atom regulate the catalytic activity of EcDOS, we investigated the flexibility of its isolated N-terminal heme-binding domain (EcDOS-heme) by monitoring its spectral properties at various hydrostatic pressures. The most active form of the heme-containing domain, i.e. the Fe(II)-CO complex, was found to be the least flexible. Conversely, the oxidized Fe(III) forms of EcDOS-heme and its mutants had relatively high flexibilities, which appeared to be linked to the low catalytic activity of the corresponding intact enzymes. These findings corroborate the suggestion, made on the basis of crystallographic data, that there is an inverse relationship between the flexibility of the heme-containing domain of EcDOS and its catalytic activity. The Fe(II)-CO form of the heme domain of a second heme-based oxygen sensor, diguanylate cyclase (YddV), was also found to be quite rigid. Interestingly, the incorporation of a water molecule into the heme complex of YddV caused by mutation of the Leu65 residue reduced the flexibility of this heme domain. Conversely, mutation of the Tyr43 residue increased its flexibility.

Temperature and pressure effects on C112S azurin: volume, expansivity, and flexibility changes.

The pressure-induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol(-1) deg(-1) fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure-jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure-induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures.

Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-ß peptide injection in rats.

Alzheimer's disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-ß protein (Aß). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aß fragment (25-35) oligomers (oAß(25-35)) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAß(25-35) and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAß(25-35) was still present in the brain after 6 weeks. oAß(25-35) injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAß(25-35) injection resulted in increased APP expression, Aß(1-42) generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aß, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.

Under pressure that splits a family in two. The case of lipocalin family.

The lipocalin family is typically composed of small proteins characterized by a range of different molecular recognition properties. Odorant binding proteins (OBPs) are a class of proteins of this family devoted to the transport of small hydrophobic molecules in the nasal mucosa of vertebrates. Among OBPs, bovine OBP (bOBP) is of great interest for its peculiar structural organization, characterized by a domain swapping of its two monomeric subunits. The effect of pressure on unfolding and refolding of native dimeric bOBP and of an engineered monomeric form has been investigated by theoretical and experimental studies under pressure. A coherent model explains the pressure-induced protein structural changes: i) the substrate-bound protein stays in its native configuration up to 330 MPa, where it loses its substrate; ii) the substrate-free protein dissociates into monomers at 200 MPa; and iii) the monomeric substrate-free form unfolds at 120 MPa. Molecular dynamics simulations showed that the pressure-induced tertiary structural changes that accompany the quaternary structural changes are mainly localized at the interface between the monomers. Interestingly, pressure-induced unfolding is reversible, but dimerization and substrate binding can no longer occur. The volume of the unfolding kinetic transition state of the monomer has been found to be similar to that of the folded state. This suggests that its refolding requires relatively large structural and/or hydrational changes, explaining thus the relatively low stability of the monomeric form of this class of proteins.

Effects of pressure and osmolytes on the allosteric equilibria of Salmonella typhimurium tryptophan synthase.

Osmolytes are common constituents of bacteria that may be produced or accumulate at high concentrations, up to 1 M, when cells are subjected to stresses like ionic strength and temperature. However, the effects of osmolytes on the allosteric properties of bacterial enzymes have rarely been examined. We have studied the effects of osmolytes and hydrostatic pressure on the allosteric equilibria of Salmonella typhimurium tryptophan (Trp) synthase. Trp synthase is a well-studied multienzyme complex with activity tightly regulated by allosteric interactions between the α- and ß-subunits. Trp synthase activity is affected by a wide range of physical parameters, including monovalent cations, pH, ligands, solvents, and hydrostatic pressure. Osmolytes, including betaine, taurine, sucrose, and polyethylene glycol, activate Trp synthase 2-3-fold in the absence of monovalent cations, indicating that osmolytes can stabilize the active closed conformation. However, in the presence of monovalent cations, osmolytes have only minor effects on activity and allosteric equilibria, but 1 M betaine stabilizes the Trp synthase-Ser-indoline complex against apparent pressure-induced subunit dissociation. Na(+) and K(+) are more effective at shifting the α-aminoacrylate-indoline quinonoid equilibrium toward the quinonoid side, with a K(Q) of 8-10, than NH(4)(+)(K(Q) ~ 2). Furthermore, pressure-jump experiments show that the mechanism of indoline reaction to form a quinonoid complex may be different for the NH(4)(+) enzyme than the Na(+) and K(+) forms. These results show that osmolytes have subtle but significant effects on the allosteric properties of Trp synthase, and these effects may be important in vivo.

Distribution of lithostathine in the mouse lemur brain with aging and Alzheimer's-like pathology.

We analyzed the cellular distribution of the pancreatic inflammatory protein lithostathine and its receptor EXTL3 in the brain of the lemurian primate Microcebus murinus which develops amyloid deposits along with aging. In adult animals (2-4.5 years old), lithostathine and EXTL3 immunoreactivities were largely distributed in the whole brain, and more intensively in almost all cortical layers and hippocampal formation. Lithostathine was observed in the perikarya and neurites of cortical neurons but also in glial cells in the border of the ventricle and the corpus callosum. In healthy aged animals (8-13 years old), highest densities of lithostathine-containing cells were observed, mainly in occipital and parietal cortex. In aged animals with Aß deposits, the increase in lithostathine immunoreactivity was lower as compared with aged animals. Noteworthy, lithostathine-immunopositive cells did almost never colocalize with Aß plaques. In conclusion, lithostathine immunoreactivity in adult Microcebus murinus appeared ubiquitous and particularly in visual, sensorial, and cognitive brain areas. Immunoreactivity increased with aging and appeared markedly affected in neuropathological conditions. Its possible neuroprotection or neurodegeneration role in Alzheimer pathology deserves therefore to be investigated.

Regenerating islet-derived 1α (Reg-1α) protein is new neuronal secreted factor that stimulates neurite outgrowth via exostosin Tumor-like 3 (EXTL3) receptor.

Regenerating islet-derived 1α (Reg-1α)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1α is overexpressed during the very early stages of Alzheimer disease, and Reg-1α deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1α in neural cells remains unknown. Here, we show that Reg-1α is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1α is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1α or addition of recombinant Reg-1α significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1α by siRNA and following inhibition of secreted Reg-1α by antibodies. Moreover, Reg-1α colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1α on neurite outgrowth, and Reg-1α is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1α regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.

Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid ß fragment in rats.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid ß protein (Aß) formed by pathological processing of the Aß precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aß fragment 25-35 (Aß(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aß(25-35) peptide was used as negative control. The aggregated forms of Aß peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aß(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aß(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aß(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy.

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.

Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.

Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium.

A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase. P-jump kinetics in the presence of Na(+), NH(4) (+), and Na(+) together with benzimidazole were also examined. The plots of lnk versus P are nonlinear and require a compressibility (beta(double dagger) (o)) term to obtain a good fit. beta(double dagger) (o) is positive for the Na(+) enzyme but negative for NH(4) (+) and Na(+) with benzimidazole. These results suggest that there is a large contribution of solvation to the kinetics of the conformational change of Trp synthase. The relaxation kinetics are also different if the P-jumps are made by increasing or decreasing pressure, suggesting that the enzyme conformations are ensembles of microstates.

Staphylococcal enterotoxin A: Partial unfolding caused by high pressure or denaturing agents enhances superantigenicity.

The effect of transient exposure of Staphylococcus aureus enterotoxin A (SEA) to high pressure and/or denaturing agents was examined by assessing the toxin superantigenicity and immunoreactivity, and by monitoring pressure-induced changes in fluorescence emission spectra. Pressurization of SEA at 600 MPa and 45 degrees C in Tris-HCl buffer (20 mM, pH 7.4) resulted in a marked increase in both T-cell proliferation (superantigenicity) and immunoreactivity. In opposite, pressurization at 20 degrees C did not change significantly SEA superantigenicity and immunoreactivity, indicating some toxin baro-resistance. Exposure of SEA to 8 M urea at atmospheric pressure or at 600 MPa and 20 degrees C, also led to a marked increase of superantigenicity (but not of immunoreactivity). In contrast, exposure of SEA to sodium-dodecylsulfate (30 mM) led to an increase of immunoreactivity with some effect on superantigenicity after pressurization at 45 degrees C only. High pressure up to 600 MPa induced spectral changes which at 20 degrees C were fully reversible upon decompression. At 45 degrees C, however, a sharp break of the centre of spectral mass mainly due to tryptophan residues was observed at 300 MPa, and irreversible spectral changes mainly related to tyrosine residues subsisted after pressure release, indicating a marked protein conformational transition. Urea 8 M further increased SEA structural changes at 600 MPa and 20 degrees C. These results indicate that SEA, under a combination of high pressure and mild temperature, as well as in the presence of urea, partly unfolds to a structure of strongly increased T-cell proliferative ability.

Pressure effects on the structure and stability of the hyperthermophilic trehalose/maltose-binding protein from Thermococcus litoralis.

In this work, we investigated the effect of pressure on the structure and stability of the recombinant D-trehalose/D-maltose-binding protein isolated from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). The spectroscopic results obtained both in the absence and in the presence of maltose or trehalose revealed that the TMBP-Mal complex exhibits a larger structural stability under high pressure values than TMBP-Tre complex. In addition, the results also pointed out that pressure induces reversible denaturation transitions of the protein structure. By combining the fluorescence results obtained with 8-anilino-1-naphtalene sulfonate as extrinsic probe and the intrinsic indolic fluorescence of TMBP, it is evident that the protein structural changes above 400 MPa that involve the exposure to the solvent of a large portion of the hydrophobic protein domains are preceded by a partially unfolded protein structural state. The spectroscopic results have been interpreted and discussed by taking into account the X-ray structure of the protein and, in particular, the interactions of maltose and trehalose within the three-dimensional structure of TMBP.

Asymmetric kinetics of protein structural changes.

Thermodynamic and kinetic understanding of structural transformations in proteins is critical to new developments in medicine and biotechnology. These fields often require the design of mechanism-based modulators of protein function. Researchers increasingly consider these structural changes-such as folding/unfolding or shuttling between active and inactive states-within the energy landscape concept that supposes a high-dimensional, rugged conformational surface. The unevenness, or asperity, of this conformational surface results from energetic barriers and kinetic traps. However, for a large number of protein reactions, such as reversible folding/unfolding, the literature only reports simple two-state transitions, which calls into question the use of a more complex energy landscape model. The question is: are these reactions really that simple, or are we misled by a biased experimental approach? In this Account, we argue in favor of the latter possibility. Indeed, the frequently employed temperature-jump method only allows recording protein structure changes in the heating direction. Under those conditions, it might not be possible to detect other kinetic pathways that could have been taken in the cooling direction. Recently, however, we have developed bidirectional pressure- and temperature-jump methods, which can offer new insights. Here, we show the potential of these methods both for studying protein folding/unfolding reactions, taking ribonuclease A as model, and for studying functionally relevant protein conformational changes, using the open/closed allosteric transition of tryptophan synthase. For example, the heating and cooling temperature-jump induced kinetics involved in the folding/unfolding conformational surface of ribonuclease A is illustrated above. In both of our model systems, the kinetic transition states of several reaction steps were path-dependent, i.e. the rates and thermodynamic activation parameters depend on the direction of the applied pressure and temperature perturbation. This asymmetry suggests that proteins cope with external stress by adapting their structure to form different ensembles of conformational substates. These states are distinguished by their activation enthalpy and entropy barriers, which can be strongly negative in the folding direction. Based on our analysis of activation compressibility and heat capacity, hydration and packing defects of the kinetic transition states are also very important for determining the reaction path. We expect that a more generalized use of this experimental approach should allow researchers to obtain greater insight into the mechanisms of physiologically relevant protein structural changes.

Pressure and temperature jump relaxation kinetics of the conformational change in Salmonella typhimurium tryptophan synthase L-serine complex: large activation compressibility and heat capacity changes demonstrate the contribution of solvation.

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex. The intense fluorescence emission of the Tryptophan synthase L-Ser external aldimine complex at 495 nm, with 420 nm excitation, provides a probe of the conformational state of Trp synthase. P-jump measurements allowed the determination of rate constants for the reactions in the presence of Na(+), Na(+) with benzimidazole (BZI), and NH4(+). The data require a compressibility term, beta(o)(double dagger), to obtain good fits, especially for the NH4(+) and BZI/Na(+) data. The compressibility changes are consistent with changes in solvation in the transition state. The transition state for the relaxation is more similar in volume to the closed aminoacrylate complex in the presence of Na(+), while it is more similar to the open external aldimine in the presence of NH4(+). Differences between the relaxations for positive and negative P-jumps may arise from changing relative populations of microstates with pressure. T-jump experiments of the Na(+) form of the tryptophan synthase-L-Ser complex show large changes in rate and amplitude over the temperature range from 7 to 45 degrees C. The Arrhenius plots show strong curvature, and hence require a heat capacity term, DeltaC(p)(double dagger), to obtain good fits. The values of DeltaC(p)(double dagger) are very large and negative (-3.6 to -4.4 kJ mol(-1) K(-1)). These changes are also consistent with large changes in solvation in the transition state for interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.