A systematic method for analysing the protein hydration structure of T4 lysozyme

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water–protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. Copyright © 2013 John Wiley & Sons, Ltd.     

Hydrophobic hydration processes thermal and chemical denaturation of proteins

The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W₅)_I, clusters (W₄)_II and free water molecules W_III. The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(− ξ_wW_I → ξ_wW_II + ξ_wW_III + cavity) takes place, with expulsion from the bulk of ξ_w water molecules W_III, whereas in the processes of Class B the opposite transformation B(− ξ_wW_III − ξ_wW_II → ξ_wW_I − cavity) takes place, with condensation into the bulk of ξ_w water molecules W_III. The thermal equivalent dilution (TED) principle is exploited to determine the number ξ_w. The denaturation (unfolding) process belongs to Class A whereas folding (or renaturation) belongs to Class B. The enthalpy ΔH_den and entropy ΔS_den functions can be disaggregated in thermal and motive components, ΔH_den = ΔH_therm + ΔH_mot, and ΔS_den = ΔS_therm + ΔS_mot, respectively. The terms ΔH_therm and ΔS_therm are related to phase change of water molecules W_III, and give no contribution to free energy (ΔG_therm = 0). The motive functions refer to the process of cavity formation (Class A) or cavity reduction (Class B), respectively and are the only contributors to free energy ΔG_mot. The folded native protein is thermodynamically favoured (ΔG_fold ≡ ΔG_mot < 0) because of the outstanding contribution of the positive entropy term for cavity reduction, ΔS_red ? 0. The native protein can be brought to a stable denatured state (ΔG_den ≡ ΔG_mot < 0) by coupled reactions. Processes of protonation coupled to denaturation have been identified. In thermal denaturation by calorimetry, however, is the heat gradually supplied to the system that yields a change of phase of water W_III, with creation of cavity and negative entropy production, ΔS_for ? 0. The negative entropy change reduces and at last neutralises the positive entropy of folding. In molecular terms, this means the gradual disruption by cavity formation of the entropy-driven hydrophobic bonds that had been keeping the chains folded in the native protein. The action of the chemical denaturants is similar to that of heat, by modulating the equilibrium between W_I, W_II, and W_III toward cavity formation and negative entropy production. The salting-in effect produced by denaturants has been recognised as a hydrophobic hydration process belonging to Class A with cavity formation, whereas the salting-out effect produced by stabilisers belongs to Class B with cavity reduction.Some algorithms of denaturation thermodynamics are presented in the Appendices.     

Structural and thermodynamic insights into the assembly of the heteromeric pyridoxal phosphate synthase from Plasmodium falciparum

Pyridoxal 5′-phosphate (PLP) is required as a cofactor by many enzymes. The predominant de novo biosynthetic route is catalyzed by a heteromeric glutamine amidotransferase consisting of the synthase subunit Pdx1 and the glutaminase subunit Pdx2. Previously, Bacillus subtilis PLP synthase was studied by X-ray crystallography and complex assembly had been characterized by isothermal titration calorimetry. The fully assembled PLP synthase complex contains 12 individual Pdx1/Pdx2 glutamine amidotransferase heterodimers. These studies revealed the occurrence of an encounter complex that is tightened in the Michaelis complex when the substrate l-glutamine binds. In this study, we have characterized complex formation of PLP synthase from the malaria-causing human pathogen Plasmodium falciparum using isothermal titration calorimetry. The presence of l-glutamine increases the tightness of the interaction about 30-fold and alters the thermodynamic signature of complex formation. The thermodynamic data are integrated in a 3D homology model of P. falciparum PLP synthase. The negative experimental heat capacity (C_p) describes a protein interface that is dominated by hydrophobic interactions. In the absence of l-glutamine, the experimental C_p is less negative than in its presence, contrasting to the previously characterised bacterial PLP synthase. Thus, while the encounter complexes differ, the Michaelis complexes of plasmodial and bacterial systems have similar characteristics concerning the relative contribution of apolar/polar surface area. In addition, we have verified the role of the N-terminal region of PfPdx1 for complex formation. A “swap mutant” in which the complete αN-helix of plasmodial Pdx1 was exchanged with the corresponding segment from B. subtilis shows cross-binding to B. subtilis Pdx2. The swap mutant also partially elicits glutaminase activity in BsPdx2, demonstrating that formation of the protein complex interface via αN and catalytic activation of the glutaminase are linked processes.     

Survey of the year 2005: literature on applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of an interaction. Its usage does not suffer from constraints of molecular size, shape or chemical constitution. Neither is there any need for chemical modification or attachment to solid support. This ease of use has made it an invaluable instrumental resource and led to its appearance in many laboratories. Despite this, the value of the thermodynamic parameterization has, only quite recently, become widely appreciated. Although our understanding of the correlation between thermodynamic data and structural details continues to be somewhat naïve, a large number of publications have begun to improve the situation. In this overview of the literature for 2005, we have attempted to highlight works of interest and novelty. Furthermore, we draw attention to those works which we feel have provided a route to better analysis and increased our ability to understand the meaning of thermodynamic change on binding. Copyright © 2006 John Wiley & Sons, Ltd.     

Calibration in isothermal titration calorimetry: heat and cell volume from heat of dilution of NaCl(aq)

An isothermal titration calorimeter of the perfusion type (MicroCal model VP-ITC) is calibrated using the heat of dilution of NaCl in water. The relative apparent molar enthalpy function (L(phi)) for NaCl(aq) varies strongly and nonlinearly with concentration in the low-concentration region (<0.2M) that is sampled easily and extensively in a single program of injections of NaCl solution into water. This nonlinearity makes it possible to calibrate with respect to two quantities: the measured heat and the active cell volume. The heat factor is determined with typical standard error 0.003; its value in the current case is 0.987. The cell volume factor is 0.93 but is quite sensitive to possible systematic errors in the temperature and in the literature values for L(phi). Both correction factors are closely tied to the delivered volume from the injection syringe, which required a correction factor of 0.973, attributed to an instrumental gear ratio error. Temperature calibration of the instrument showed a small offset of 0.12K at the temperature 25 degrees C of the experiments, but the error increased to more than 1K at 46 degrees C. The experiments were not able to distinguish clearly between mixing algorithms that assume instantaneous mixing on injection and those that assume instantaneous injection followed by mixing; however, examination of these algorithms has revealed an error in a program widely used to analyze isothermal titration calorimetry data.     

Designing allosteric peptide ligands targeting a globular protein

Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein-targeted, L or D retro-inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G-protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, beta-galactosidase, betaGAL. Three of the eight 14mer peptides allosterically activated betaGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 x 10(-9) by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their betaGAL binding-mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the betaGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy-entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein-targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins.     

Thermodynamic and structural characteristics of collagen fibrils formed in vitro at different temperatures and concentrations

Analysis of the results of calorimetric study of reconstituted collagen (type I) fibrils, in particular, the half-width of the temperature transition, shows that the collagen packing density in the fibrils and the size of cooperative blocks therein depend on the assembly temperature and on the initial collagen concentration. The least dense fibrils are formed at subphysiological temperatures (25° or 30°C) and low concentration (0.3 mg/ml). The extent of ordering does not change upon doubling the concentration but increases upon quadrupling it. At physiological temperature (35°C) the fibrils are densely packed regardless of collagen concentration. The enthalpy of fibril assembly is minimal at 35°C, 1.2 mg/ml, and ionic strength of 0.17 M. The influence of temperature on particular steps of fibrillogenesis and the role of water in these processes are discussed.     

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH = 6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N₂) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N₂ ? 2N ? 2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor

The disulfide bond between Cys14 and Cys38 of bovine pancreatic trypsin inhibitor lies on the surface of the inhibitor and forms part of the protease-binding region. The functional properties of three variants lacking this disulfide, with one or both of the Cys residues replaced with Ser, were examined, and X-ray crystal structures of the complexes with bovine trypsin were determined and refined to the 1.58-Å resolution limit. The crystal structure of the complex formed with the mutant with both Cys residues replaced was nearly identical with that of the complex containing the wild-type protein, with the Ser oxygen atoms positioned to replace the disulfide bond with a hydrogen bond. The two structures of the complexes with single replacements displayed small local perturbations with alternate conformations of the Ser side chains. Despite the absence of the disulfide bond, the crystallographic temperature factors show no evidence of increased flexibility in the complexes with the mutant inhibitors. All three of the variants were cleaved by trypsin more rapidly than the wild-type inhibitor, by as much as 10,000-fold, indicating that the covalent constraint normally imposed by the disulfide contributes to the remarkable resistance to hydrolysis displayed by the wild-type protein. The rates of hydrolysis display an unusual dependence on pH over the range of 3.5-8.0, decreasing at the more alkaline values, as compared with the increased hydrolysis rates for normal substrates under these conditions. These observations can be accounted for by a model for inhibition in which an acyl-enzyme intermediate forms at a significant rate but is rapidly converted back to the enzyme-inhibitor complex by nucleophilic attack by the newly created amino group. The model suggests that a lack of flexibility in the acyl-enzyme intermediate, rather than the enzyme-inhibitor complex, may be a key factor in the ability of bovine pancreatic trypsin inhibitor and similar inhibitors to resist hydrolysis.