Thermodynamics of the helix-coil transition: Binding of S15 and a hybrid sequence, disulfide stabilized peptide to the S-protein.

Pancreatic ribonuclease A may be cleaved to produce two fragments: the S-peptide (residues 1-20) and the S-protein (residues 21-124). The S-peptide, or a truncated version designated as the S15 peptide (residues 1-15), combines with the S-protein to produce catalytically active complexes. The conformation of these peptides and many of their analogues is predominantly random coil at room temperature; however, they populate a significant fraction of helical form at low temperature under certain solution conditions. Moreover, they adopt a helical conformation when bound to the S-protein. A hybrid sequence, disulfide-stabilized peptide (ApaS-25), designed to stabilize the helical structure of the S-peptide in solution, also combines with the S-protein to yield a catalytically active complex. We have performed high-precision titration microcalorimetric measurements to determine the free energy, enthalpy, entropy, and heat capacity changes for the binding of ApaS-25 to S-protein within the temperature range 5-25 degrees C. The thermodynamic parameters for both the complex formation reactions and the helix-to-coil transition also were calculated, using a structure-based approach, by calculating changes in accessible surface area and using published empirical parameters. A simple thermodynamic model is presented in an attempt to account for the differences between the binding of ApaS-25 and the S-peptide. From this model, the thermodynamic parameters of the helix-to-coil transition of S15 can be calculated.

TTF-1 and HNF-3beta in the developing tracheoesophageal fistula: further evidence for the respiratory origin of the distal esophagus'.

PURPOSE: Using an established rat model of esophageal atresia with tracheoesophageal fistula (EA-TEF), the authors have studied the organogenesis of this congenital anomaly. The authors previously have proposed that the "distal esophagus" actually is of respiratory lineage. In this report this hypothesis is tested by examining the expression of two foregut patterning transcription factors, thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3beta (HNF-3beta), within the developing TEF. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 to 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. Immunohistochemistry was performed using polyclonal antibodies to TTF-1 and HNF-3beta. RESULTS: TTF-1 is a homeodomain protein that previously has been shown to be expressed in the lung and trachea but not in the gastrointestinal tract, and which, when deleted in a developing lung, results in a mouse with no peripheral lung parenchyma. TTF-1 was expressed strongly in the lung, fistula, and distal esophagus, but not in the proximal esophagus. HNF-3beta is a forkhead transcription factor important in foregut patterning that binds and activates the TTF-1 promotor sequence. HNF-3beta was expressed globally in the fistula and lung as well as the esophagus. CONCLUSIONS: The expression of the lung-specific transcription factor TTF-1 within the TEF strongly implies that the "distal esophagus" is a respiratory-derived structure and thus supports our theory of TEF organogenesis. The conservation of HNF-3beta expression both in the TEF as well as the normal developing trachea and esophagus suggests that global foregut patterning is intact in the formation of this anomaly, and the defect lies at the level of the respiratory versus gastrointestinal commitment.

The ontogeny of TGF-beta1, -beta2, -beta3, and TGF-beta receptor-II expression in the pancreas: implications for regulation of growth and differentiation.

BACKGROUND/PURPOSE: The transforming growth factor-beta (TGF-beta) cytokines are important regulators of growth and differentiation in multiple mammalian organ systems. Recent studies suggest that they may play a significant role in the regulation of pancreatic organogenesis. The authors proposed to examine the ontogeny of expression of the TGF-beta cytokine isoforms (TGF-beta1, beta2, and beta3), as well as that of the type II TGF-beta receptor (TbetaRII), in the pancreas. We hypothesized that their patterns of expression might help to clarify the manner in which they influence the development of this organ. METHODS: Embryos from pregnant CD-1 mice were harvested on gestational days 12.5, 15.5, and 18.5. Microdissection was performed on the embryos to isolate their pancreases. The pancreases were fixed, frozen embedded, and sectioned with a cryostat. Immunohistochemistrywas performed using polyclonal antibodies to TGF-beta1, beta2, and beta3, and TbetaRII. RESULTS: The patterns of expression of TGF-beta1, beta2, and beta3 were similar throughout gestation. They were all present, though weakly, early in the development of the pancreas, in the E12.5 epithelial cells. Their expression persisted and became localized to the acinar cells later in gestation. TbetaRII staining was present in both the E12.5 epithelial cells and the surrounding mesenchyme. As the pancreas developed, TbetaRII became strongly expressed in the ductal epithelial cells with only minimal staining in the acinar and endocrine cells. CONCLUSIONS: TGF-betas may play a role in regulating pancreatic organogenesis. Our data suggest that they may be required for the normal development of acini. As in other cell systems, TGF-beta1 may act as a suppressor of pancreatic cellular growth and differentiation. The localization of TbetaRII to the mature ductal epithelium may indicate a need for ongoing regulation of growth and differentiation in the pancreatic ducts beyond the fetal period.

Epithelio-mesenchymal interactions in the developing mouse pancreas: morphogenesis of the adult architecture.

BACKGROUND/PURPOSE: The mammalian pancreas is thought to develop through a complex interaction between the budding epithelium and the surrounding mesenchyme. The exact nature of this interaction is unclear. Most of what is known to date of these interactions comes from a series of organ culture experiments done in the late 1960s. Nevertheless, these important experiments may have been confounded by less-defined culture media and organ dissection techniques, because the results are not reproducible in our hands. The authors undertook a study to reexplore these basic epithelio-mesenchymal interactions. METHODS: Using previously described organ dissection and culture techniques the authors examined the basic interactions between the embryonic pancreatic epithelium and its mesenchyme with histological and immunohistological techniques. RESULTS: The authors found that, contrary to previous reports, the earliest pancreatic anlage did not possess the intrinsic signaling necessary to support normal growth and differentiation in vitro. Intimate contact between the epithelium and the mesenchyme may be necessary until E11.5 for normal growth and differentiation. The age of the mesenchyme seemed to correlate with the degree of acinar differentiation, and proximity of mesenchyme was important for acinar differentiation. CONCLUSIONS: Previous investigations into the basic epithelio-mesenchymal interactions in the developing mammalian pancreas may have had confounding factors. Extrinsic signals seem necessary for complete pancreatic differentiation, and mesenchymal factors appear important for acinar differentiation.

Vascular development in the mouse embryonic pancreas and lung.

PURPOSE: The purpose of this study was to analyze the formation of blood vessels in the developing mouse pancreas and lung by studying two ligands, angiopoietin-1 (ang1) and angiopoietin-2 (ang2), which are thought to play a role as angiogenesis-activating factors in development. Understanding the role of vasculogenic peptides in normal embryonic development also may have important implications for common clinical problems regarding neonatal pulmonary vasculature. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Southern blotting was used to determine the ontogeny of angiopoietin-1 and angiopoietin-2 gene expression in the embryonic mouse pancreas and lung. Immunohistochemistry was performed for von Willebrand factor, a known marker of endothelial cells, to chronicle the development of the vasculature in these organs. RESULTS: The authors determined the temporal expression of angiopoietin-1 and angiopoietin-2 as a function of gestational age. RT-PCR data demonstrated expression of ang1 and ang2 in the developing mouse lung between gestational day E9.5 and postnatal day 1, and in the developing pancreas between gestational days E12.5 and E18.5. Southern blot analysis confirmed PCR data for ang2 expression in both the lung and pancreas. The authors also traced the spatial development of the vascular system by von Willebrand factor staining. For both lung and pancreas specimens, no blood vessels were identifiable by immunohistochemistry until embryonic day 12.5. With increased gestational age, the blood vessel networks grew larger. CONCLUSION: The authors have demonstrated that ang1 and ang2 may be involved in the mechanisms of vascular development in the embryonic mouse lung and pancreas.

Esophageal atresia with tracheoesophageal fistula: suggested mechanism in faulty organogenesis.

BACKGROUND/PURPOSE: The organogenesis of esophageal atresia with tracheoesophageal fistula (EA-TEF) is unknown. Using an established model for EA-TEF in rats, the authors proposed to study this aberrancy of development in the hope of gaining insight into its mechanism of formation. METHODS: Pregnant Sprague-Dawley rats were injected with 2.2 mg/kg of Adriamycin intraperitoneally on days 6 through 9 of gestation. Using microdissection, the trachea, blind-ending esophagus, TEF, and stomach were isolated from embryos of various gestional ages. The specimens were analyzed histologically with routine H&E staining. RESULTS: The classic EA-TEF developed in the embryos, with proximal EA and distal TEF. As expected, the atresia formed as a blind-ending pouch, but the distal fistula began as an apparent equal trifurcation of the tracheal anlage into two mainstem bronchi and the fistula tract leading to the stomach. Histological analysis of the fistula tract showed respiratorylike pseudostratified columnar epithelium. CONCLUSIONS: TEF develops as the middle branch of a tracheal trifurcation. EA-TEF occurs by a primary atresia of the esophagus. As a secondary phenomenon, the distal foregut anlage is switched toward the pulmonary phenotype. It trifurcates, and its middle branch grows caudally to fistulize into the stomach.

In vitro prefabrication of human cartilage shapes using fibrin glue and human chondrocytes.

We report the first generation of human cartilage from fibrin glue using a technique of molding chondrocytes in fibrin glue developed in our laboratory. Human costal chondrocytes were suspended in cryoprecipitate and polymerized into a human nasal shape with bovine thrombin. After culture in vitro for 4 weeks, this construct was implanted subcutaneously into a nude mouse. The final construct harvested after 4 weeks in vivo demonstrated some preservation of its original features. Histological analysis showed features of native cartilage, including matrix synthesis and viable chondrocytes by nuclear staining. Biochemical analysis demonstrated active matrix production. Biomechanical testing was performed. To our knowledge this is the first reported creation of human cartilage from fibrin glue, and the first creation of human cartilage in vitro. This technique may become a promising means of engineering precisely designed autogenous cartilage for human reconstruction.

Allosteric properties of inosine monophosphate dehydrogenase revealed through the thermodynamics of binding of inosine 5'-monophosphate and mycophenolic acid. Temperature dependent heat capacity of binding as a signature of ligand-coupled conformational equilibria.

The thermodynamic properties of binding of the substrate, inosine monophosphate (IMP), and the uncompetitive inhibitor, mycophenolic acid, to inosine monophosphate dehydrogenase (IMPDH) were measured. Specifically, the free energy, enthalpy, entropy, and heat capacity changes were determined for each ligation state of the tetrameric enzyme, over a temperature range from 2.5 to 37 degrees C by high-precision titration microcalorimetry. It was discovered that IMP binds to IMPDH in a negatively cooperative fashion and that mycophenolic acid binding is critically dependent on the presence of IMP. Moreover, the binding of IMP is entropically driven at low temperatures and enthalpically driven at high temperatures, with an unusually large, temperature dependent heat capacity change. A thermodynamic argument, based on the general nature of the heat capacity function for a binding reaction and its temperature dependence, is used to infer the existence of an equilibrium mixture of at least two structural forms of apo-IMPDH. The equilibrium is perturbed in the presence of IMP and mycophenolic acid, suggesting a mechanism for the ligand-linked conformational changes. An allosteric model, incorporating subunit-subunit interactions nested within a concerted conformational change involving the entire tetrameric macromolecule, is proposed to account for the observed binding behavior. The implications of these findings for the design of novel "allosteric-effector" inhibitors of IMPDH, to be used for the purpose of immunosuppression, are discussed.

Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis.

The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.

Determination of the differential effects of hydrogen bonding and water release on the binding of FK506 to native and Tyr82-->Phe82 FKBP-12 proteins using free energy simulations.

We use the thermodynamic integration technique to calculate the free energy associated with the Tyr82-->Phe82 mutation (Y82F) in the protein FKBP-12, both free and bound to known inhibitor FK506 (tacrolimis). We find that the net difference in free energy for the two changes is 0.85 kcal/mol, with the binding of FK506 relatively more favorable for the native protein than the mutant. This net energy compares very favorably with the experimentally measured value of 0.60 kcal/mol. The results indicate that the relatively better binding of FK506 to the native protein is driven by the favorable entropy associated with the release of water molecules from the protein when the ligand binds. For a variety of reasons, modest size of the system, smallness of the change being examined, rapid convergence of the ensemble that needs to be determined and use of statistical estimates to control sampling, we have been able to carry out atypically reliable and reproducible free energy calculations for this protein system. Free energy changes for the two simulations (Y82F FKBP-12/FK506 and Y82F FKBP-12) have been calculated a total of eight times each, to compare a variety of different methodological choices and to ensure that the results are statistically significant. Detailed analysis of the free energy results has been carried out, and indicates that even when applicable, deconvolution of the total free energy into components can be very difficult, that the statistical error estimates can give a reasonable bound on the error in a simulation, and that one must be careful to use the same simulation protocol in all simulations being compared.

Acquisition and use of calorimetric data for prediction of the thermodynamics of ligand-binding and folding reactions of proteins.

The commercialization of high-precision microcalorimeters has led to a sharp increase in the acquisition and utilization of calorimetric data on the binding and folding reactions of proteins. This thermodynamic database, together with high-resolution structures of proteins and their complexes with ligands, has facilitated the development, calibration, refinement and testing of empirically based and computational methods for predicting the thermodynamics of processes involving macromolecules. Even so, a generally accepted method for assigning structural parameters to the various energetic contributions of protein folding and ligand-binding reactions, much less a method for predicting the changes in thermodynamic parameters of these reactions, has not yet been firmly established.

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

Probing hydration contributions to the thermodynamics of ligand binding by proteins. Enthalpy and heat capacity changes of tacrolimus and rapamycin binding to FK506 binding protein in D2O and H2O.

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atoms within the proteins and ligands and of these atoms with water. Delineation of the various energetic contributions of the stabilities of proteins or protein-ligand complexes in aqueous solution, and an evaluation of their structural basis, requires a direct account of the changes, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamycin, each bind with high affinity to a common site on a small FK506 binding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isomerase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the enthalpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solutions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titration calorimetry in the temperature range 5-30 degrees C. For both tacrolimus and rapamycin binding, a large enthalpic destabilization of binding is observed in D2O relative to H2O, in the temperature range examined. Additionally, large negative constant pressure heat capacity changes are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determinants of the differences in the energetics of binding in light and heavy water. The analysis suggests that a chief contributor to the observed enthalpic destabilization is the differential hydration, of protein and ligand atoms, by light and heavy water.

Heat capacity changes and hydrophobic interactions in the binding of FK506 and rapamycin to the FK506 binding protein.

Differential interactions among nonpolar moieties at protein/ligand interfaces, and of these nonpolar groups with water, collectively termed hydrophobic interactions, are widely believed to make important energetic contributions to the stability of protein/ligand complexes. Quantitative estimates of hydrophobic interactions, and an evaluation of their structural basis, are essential for obtaining structure-based predictions of the free energies of binding for the purpose of drug design. Two largely nonpolar, immunosuppressive agents, FK506 and rapamycin, each bind with high affinity to a common hydrophobic pocket on a small peptidylproline cis-trans isomerase known as FK506 binding protein (FKBP-12) and inhibit its activity. In an effort to elucidate the structural features of these ligands responsible for the observed energetics, we have undertaken an investigation of the thermodynamics of binding of FK506 and rapamycin to FKBP-12. Enthalpies of binding have been determined by high-precision titration calorimetry over a range of temperature, allowing estimates of heat capacity changes. By analyzing the distribution of changes in solvent-accessible surface area upon binding of FK506 to FKBP-12 from crystallographic data, it is found that 99% of the net surface buried upon binding involves nonpolar groups. This leads to a heat capacity change of FK506 binding, normalized to the amount of nonpolar surface, of -0.40 +/- 0.02 cal.K-1.mol-1.A-2 (1 cal = 4.18 J), a value similar to that obtained for the aqueous dissolution of hydrophobic substances. Our observations are discussed in view of the general nature of hydrophobic interaction processes.

Heat capacity changes for protein-peptide interactions in the ribonuclease S system.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. We have substituted the wild-type residue Met-13 with six other hydrophobic residues ranging in size from alanine to phenylalanine and have determined the thermodynamic parameters associated with binding of these analogues to S-protein by titration calorimetry in the temperature range 5-25 degrees C. The heat capacity change (delta Cp) associated with binding was obtained from a global analysis of the temperature dependences of the free energies and enthalpies of binding. The delta Cp's were not correlated in any simple fashion with the nonpolar surface area (delta Anp) buried upon binding.

Thermodynamics of protein-peptide interactions in the ribonuclease S system studied by titration calorimetry.

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex designated ribonuclease S. Residue 13 in the peptide is methionine. According to the X-ray structure of the complex of S-protein and S-peptide (1-20), this residue is almost fully buried. We have substituted Met-13 with seven other hydrophobic residues ranging in size from glycine to phenylalanine and have determined the thermodynamic parameters associated with the binding of these analogues to S-protein by titration calorimetry at 25 degrees C. These data should provide useful quantitative information for evaluating the contribution of hydrophobic interactions in the stabilization of protein structures.

Binding of oxygen and carbon monoxide to the hemocyanin from the spiny lobster.

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O2 ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechanism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning "nested" allosteric interactions.

Identical linkage and cooperativity of oxygen and carbon monoxide binding to Octopus dofleini hemocyanin.

Employment of high-precision thin-layer methods has enabled detailed functional characterization of oxygen and carbon monoxide binding for (1) the fully assembled form with 70 binding sites and (2) the isolated chains with 7 binding sites of Octopus dofleini hemocyanin. The striking difference in the cooperativities of the two ligands for the assembled decamer is revealed through an examination of the binding capacities and the partition coefficient, determined as functions of the activities of both ligands. A global analysis of the data sets supported a two-state allosteric model assuming an allosteric unit of 7. Higher level allosteric interactions were not indicated. This contrasts to results obtained for arthropod hemocyanins. Oxygen and carbon monoxide experiments performed on the isolated subunit chain confirmed the presence of functional heterogeneity reported previously [Miller, K. (1985) Biochemistry 24, 4582-4586]. The analysis shows two types of binding sites in the ratio of 4:3.