Differential Scanning Calorimetry Transition Research Articles

Analytical Workflows to Unlock Predictive Power in Biotherapeutic Developability

PurposeForming accurate data models that assist the design of developability assays is one area that requires a deep and practical understanding of the problem domain. We aim to incorporate expert knowledge into the model building process by creating new metrics from instrument data and by guiding the choice of input parameters and Machine Learning (ML) techniques.MethodsWe generated datasets from the biophysical characterisation of 5 monoclonal antibodies (mAbs). We explored combinations of techniques and parameters to uncover the ones that better describe specific molecular liabilities, such as conformational and colloidal instability. We also employed ML algorithms to predict metrics from the dataset.ResultsWe found that the combination of Differential Scanning Calorimetry (DSC) and Light Scattering thermal ramps enabled us to identify domain-specific aggregation in mAbs that would be otherwise overlooked by common developability workflows. We also found that the response to different salt concentrations provided information about colloidal stability in agreement with charge distribution models. Finally, we predicted DSC transition temperatures from the dataset, and used the order of importance of different metrics to increase the explainability of the model.ConclusionsThe new analytical workflows enabled a better description of molecular behaviour and uncovered links between structural properties and molecular liabilities. In the future this new understanding will be coupled with ML algorithms to unlock their predictive power during developability assessment.

Crystallinity of Bitumen via WAXD and DSC and Its Effect on the Surface Microstructure

It is well documented that most bituminous binders contain crystallisable material. This crystallisable fraction, often referred to as paraffinic or natural wax, is associated with the bitumen’s origin and has an influence on its rheological performance. In the literature, Differential Scanning Calorimetry (DSC) is the primary technique used to determine the melting and crystallisation behaviour of this waxy fraction. However, in bitumen, thermal transitions in DSC are typically very broad and can be combined with recrystallisation effects upon reheating. This work explores the potential crystallisation and melting process of a waxy and a wax-free bitumen via three different approaches: DSC, Wide-Angle X-ray Diffraction (WAXD) and Confocal Laser Scanning Microscopy (CLSM). The findings reveal that the DSC transitions of the waxy bitumen are in good agreement with the corresponding occurrence of WAXD signals and to some extent with the formation and disappearance of the surface microstructures which were followed at two cooling and heating rates. WAXD results additionally demonstrate that the crystalline material in bitumen is organised in an orthorhombic unit cell, typical for straight chain aliphatic structures. On the other hand, DSC and WAXD support the lack of crystallinity for the wax-free bitumen which could explain its featureless CLSM surface. Overall, the originality of this work resides in the disclosure of connections between crystallographic properties, thermal transitions and the surface micromorphology of bitumen.

Biocomposites of PLA and Mango Seed Waste: Potential Material for Food Packaging and a Technological Alternative to Reduce Environmental Impact

AbstractMango seeds from agro‐industry represents an environmental problem due to the amounts of by‐products produced. Conversely, poly (lactic acid) (PLA) is a potential green alternative to conventional plastics. The goal of this study aimed to develop a biocomposite based on PLA and mango’s by‐product for rigid packaging. Six biocomposites are obtained by extrusion/injection processing using formulations with PLA as a matrix and up to 20% by weight of mango seed’s by‐products. The materials are characterized by chemical and physical analysis; scanning electron microscopy/energy dispersive X‐ray, X‐ray diffraction; Fourier transform infrared spectroscopy (FTIR); thermal gravimetric analysis/differential thermogravimetry; differential scanning calorimetry (DSC), and mechanical analysis. FTIR bands and DSC transitions related to starch are higher in the kernel, while more cellulose bands are found in the integument. Kernel presented thermal degradation in the biocomposites, the sample PLA+20 wt% kernel. For the other compositions, it is possible to observe that they could keep their morphology. Significant improvements in both mechanical and barrier properties are found in the formulation with 20 wt% integument (up to 38% in elastic modulus). Therefore, this study suggests that biocomposites developed from PLA / Integument / Kernel have potential as a new biomaterial for rigid food packaging systems.

Interactions and cooperativity between P-glycoprotein structural domains determined by thermal unfolding provides insights into its solution structure and function

Structural changes in mouse P-glycoprotein (Pgp) induced by thermal unfolding were studied by differential scanning calorimetry (DSC), circular dichroism and fluorescence spectroscopy to gain insight into the solution conformation(s) of this ABC transporter that may not be apparent from current crystal structures. DSC of reconstituted Pgp showed two thermal unfolding transitions in the absence of MgATP, suggesting that each transition involved the cooperative unfolding of two or more interacting structural domains. A low calorimetric unfolding enthalpy and minimal structural changes were observed, which are hallmarks of the thermal unfolding of α-helical membrane proteins, because generally only the extramembranous regions undergo significant unfolding. Nucleotide binding increased the unfolding temperature of both transitions to the same extent, suggesting that one nucleotide binding domain (NBD) unfolds with each transition. Combined with the results from the two isolated NBDs, we propose that each DSC transition represents the cooperative unfolding of one NBD and the two contacting intracellular loops. Further, the presence of two transitions in both apo and MgATP bound wild-type Pgp suggests the NBD-dimeric conformation is transient, and that Pgp resides predominantly in the crystallographically observed inward-facing conformation with NBDs separated, even under conditions supporting continuous MgATP hydrolysis. In contrast, DSC of the vanadate-trapped MgADP·Pgp complex and the MgATP-bound catalytically inactive mutant, E552A/E1197A, show an additional transition at much higher temperature, corresponding to the unfolding of the nucleotide-trapped NBD-dimeric outward-facing conformation. The collective results indicate a strong preference for an NBD dissociated, inward-facing conformation of Pgp.

Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding.

Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm-Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm-Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm-Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm-Bragg theory by introducing the concept of 'folded' and 'unfolded' peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, , but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm-Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.

Thermal unfolding of apolipoprotein A-1. Evaluation of methods and models.

Human apolipoprotein A-1 (Apo A-1) was used as a model protein to compare experimental methods and theoretical models for protein unfolding. Thermal unfolding was investigated in aqueous buffer, in β-octylglucoside solution, and with phospholipid bilayer vesicles. The α-helix content of Apo A-1 increased from 50% in aqueous buffer to 75% in the presence of lipid vesicles, but remained constant in solutions of β-octyl glucoside. Differential scanning calorimetry (DSC) measured the thermodynamic properties of the unfolding process and was our reference method. The increased heat capacity of the unfolded protein made an important contribution to the total enthalpy of unfolding. The structural properties of Apo A-1 were studied with circular dichroism (CD) spectroscopy. The CD-recorded unfolding transitions were broader than the corresponding DSC transitions and were shifted toward higher temperatures. DSC and CD data were analyzed with the two-state model and the Zimm-Bragg theory. The two-state model assumes just two species in solution, native (N) and unfolded (U) Apo A-1. However, Apo A-1 unfolding is a highly cooperative event with helical amino acid residues unfolding and refolding rapidly. For such a sequential process, the Zimm-Bragg theory provides an alternative and physically more realistic model. The Zimm-Bragg theory allowed perfect simulations of the DSC and CD experiments. In contrast, incorrect thermodynamic results were obtained with the two-state model. The Zimm-Bragg theory also provided a physically well-defined analysis of the cooperativity of the folding ⇄ unfolding equilibrium. The cooperative unfolding of Apo A-1 increased upon addition of lipids and decreased in detergent solution.

PH stability study of lectin from black turtle bean (Phaseolus vulgaris) as influenced by guanidinium-HCl and thermal treatment.

The pH stability of lectin from black turtle bean was characterized by a variety of biophysical techniques over the pH range from 2.0 to 10.0. The zero-order and second-derivative UV spectra at different pH values indicated that the tertiary structure of the protein does not change significantly with changes in pH. The maxima emission fluorescence of the lectin upon excitation at 280 nm and 295 nm were found to be blue shifted 1.2 nm at pH 2.0, and with a red-shift of 1.0 nm at pH 10.0. The chemical denaturation results obtained by monitoring the intrinsic fluorescence of the lectin indicated that unfolding of the protein induced by guanidine hydrochloride (GdnHCl) could be described using a three-state model. Complete unfolding of the protein was observed at pH 7.2 in the presence of 6.5 M GdnHCl after 2 weeks, which suggested that the lectin was stable at various pHs. Irreversible thermal denaturation of lectin was also investigated at various pHs by differential scanning calorimetry (DSC). The first-order two-state kinetic model was applied to explain the scan-rate dependent DSC transitions. Both the higher activation energy (Ea) computed from the irreversible thermal denaturation and the three-state chemical denaturation process suggested that the lectin structure remained significantly unchanged from pH 2.0 to 10.0, which indicated that the structure of the lectin was stable over a wide pH range.

Phenylene bolaamphiphiles: Influence of the substitution pattern on the aggregation behavior and the miscibility with classical phospholipids

The synthesis of two symmetric single‐chain phenylene‐modified bolaamphiphiles with meta and ortho phenyl substitution pattern is described. The aggregation behavior of both bolaamphiphiles in aqueous suspension was investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM), Fourier‐transform IR (FTIR) spectroscopy, and small angle neutron scattering (SANS). We could show that a change in the substitution pattern from para to meta or ortho leads to a change in the aggregation behavior so that small micelles instead of nanofibers are formed. Furthermore, the mixing behavior of these bolaamphiphiles with classical bilayer forming phospholipids, such as 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine (DPPC) and 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) was studied by means of DSC, TEM, cryo‐TEM, and small angle X‐ray scattering (SAXS). The aim was to stabilize bilayer membranes formed by classical phospholipids by incorporation of bolaamphiphiles to obtain liposomes with improved stability suitable for drug delivery purposes. We could show that the phenylene‐modified bolaamphiphiles are indeed miscible with DPPC and DSPC; however, closed vesicles as observed for pure DPPC and DSPC were not found. Instead, small disk‐like aggregates are formed. In the case of mixtures of phenylene‐modified bolaamphiphiles with DPPC, these bilayer disks have a higher DSC transition temperature compared to pure DPPC indicating an increased stability of the ordered gel phase inside the disks.Practical applications: Vesicles composed of classical phospholipids are widely used as drug delivery systems for pharmaceutical applications. The stability of these vesicles in, e.g., body fluids is an important prerequisite for long‐term applications and it can possibly be enhanced by the incorporation of bipolar amphiphiles (bolaamphiphiles) into phospholipid bilayers. With the use of novel single‐chain phenylene‐modified bolaamphiphiles, synthesized in this work, we are able to generate small lipid‐disks in mixtures with classical phospholipids that have an increased stability of the ordered gel phase inside these disks compared to pure phospholipids. These disks might be further used as nano‐dimensioned drug carrier systems.The incorporation of ortho‐substituted phenylene‐modified bolaamphiphiles (PC‐C17oPhC17‐PC) into DPPC bilayer leads to the formation of small disk‐like aggregates with an increased stability compared to pure DPPC vesicles.

DSC serum profiles of sportsmen

The blood serum of young athletes has been investigated by differential scanning calorimetry (DSC) method. The component peaks of complex endothermic transition corresponding to serum denaturation may be correlated with the main protein fractions of serum: albumin, alpha-1, alpha-2, beta- and gamma-globulins. The averaged shape of thermal transition observed for serum of sportsmen participating in our pilot study differs from that reported previously for healthy subjects. One of the reason for the difference may be the lower than normal value of albumins to globulins ratio found for individuals in our group of athletes. Modifications of serum DSC transition due to the exercise test and 1 and 2 h rest have been observed. Statistically based conclusions are not currently possible because of the low number of participants in the study. The averaged responses to the effort and to the rest have been shown clearly different for athletes classified as the best and typical. The first moment of the transition with respect to the temperature axis has been found significantly lower for the group of athletes considered as the best.

Heat-Induced Conformational Unfolding of Fatty Acid-Free and Fatted Camel Serum Albumin: A Comparative Study

Albumin is a multifunctional non-glycosylated, negatively charged plasma protein, with extraordinary ligand-binding and transport properties, antioxidant functions, and enzymatic activities. Physiologically, albumin transports free fatty acids in plasma and contributes in maintaining colloid osmotic pressure. Recent progresses in using albumin as a versatile protein carrier for drug targeting and for improving the pharmacokinetic profile of peptide or protein-based drugs, increased the attempts for improving albumin stability. Studying the thermal stability of camel albumin may provide us not only new clues for designing recombinant albumins, but also molecular insights on camel physiology. This study aims to determine the thermal stability of camel albumin. Fatted camel serum albumin (FCSA) was purified from blood via combination of Cohn's method and anion-exchange chromatography. Activated charcoal treatment was used to obtain defatted camel serum albumin (CSA). Fluorescence spectroscopy and differential scanning calorimetry (DSC) were used to study thermal denaturation of this protein. The set of fluorescence spectra were deconvoluted using the convex constraint analysis method (CCA). The results from deconvolution of fluorescence spectroscopy and DSC showed three and two components for CSA and FCSA, respectively. The bimodal DSC transition can be attributed to a crevice between domains I and II and formation of two independent thermodynamic domains. The crevice formation can be prevented by fatty acid binding between domains I and II. The calculated values of ∆H v/∆H cal, approximately 0.4 for CSA and near 1 for FCSA, confirmed the presence of at least one intermediate in thermal unfolding of CSA and the absence of the intermediate for FCSA. The obtained midpoint transition temperature (T m) of FCSA was about 20°C higher than that of CSA. Such enormous stabilizing effect may be attributed to the fact that fatty acid serves as glue which preserves different domains beside each other and prevents formation of the mentioned intermediate.

Application of Differential Scanning Calorimetry-Chemometric Coupled Procedure to the Evaluation of Thermo-Oxidation on Extra Virgin Olive Oil

In this work, the opportunity of adopting a differential scanning calorimetry (DSC)-principal component analysis (PCA) coupled procedure to measure the degree of thermal stress for extra virgin olive oil (EVOO) was presented. Oil was subjected to thermal stress under convectional or microwave heating treatments at different heating times up to 1,440 and 15 min, respectively, and p-anisidine values (PAV) were obtained on all samples to measure their oxidative degradation. The entire DSC profiles obtained on the oil upon cooling in the range from 30 °C to –80 °C and subsequent re-heating to 30 °C at different times and under cooking procedures have been subjected to PCA data analysis. PCA discriminated samples by means of profile changes in DSC transition both upon cooling and heating not only according to treatment times (which accounted for the degree of thermo-oxidation) but also considering different heating process. The proposed procedure may be useful to measure oil thermal stress and to select appropriate heating condition to be applied for EVOO both in industrial and/or in food-catering sectors.

Ensemble Perspective for Catalytic Promiscuity

Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. However, catalytic promiscuity is a poorly understood property, and no thermodynamic treatment has described the conformational landscape of promiscuous versus substrate-specific enzymes. Here, two structurally similar glutathione transferase (GST, glutathione S-transferase) isoforms with high specificity or high promiscuity are compared. Differential scanning calorimetry (DSC) indicates a reversible low temperature transition for the promiscuous GSTA1-1 that is not observed with substrate-specific GSTA4-4. This transition is assigned to rearrangement of the C terminus at the active site of GSTA1-1 based on the effects of ligands and mutations. Near-UV and far-UV circular dichroism indicate that this transition is due to repacking of tertiary contacts with the remainder of the subunit, rather than "unfolding" of the C terminus per se. Analysis of the DSC data using a modified Landau theory indicates that the local conformational landscape of the active site of GSTA1-1 is smooth, with barrierless transitions between states. The partition function of the C-terminal states is a broad unimodal distribution at all temperatures within this DSC transition. In contrast, the remainder of the GSTA1-1 subunit and the GSTA4-4 protein exhibit folded and unfolded macrostates with a significant energy barrier separating them. Their partition function includes a sharp unimodal distribution of states only at temperatures that yield either folded or unfolded macrostates. At intermediate temperatures the partition function includes a bimodal distribution. The barrierless rearrangement of the GSTA1-1 active site within a local smooth energy landscape suggests a thermodynamic basis for catalytic promiscuity.

Gelation process in two different squid (Dosidicus gigas) surimis throughout frozen storage as affected by several cryoprotectants: Thermal, mechanical and dynamic rheological properties

Differential scanning calorimetry (DSC) was used to follow the thermal denaturation of two differently processed surimis from giant squid (Dosidicus gigas), A (isoelectric precipitation) and B (acid washing). Both surimis were added with several cryoprotectants (sucrose, sorbitol and trehalose), and kept in frozen storage at −15°C for 6months. DSC profiles and transition enthalpies showed that protein quality was better in B than in A surimis whether fresh or frozen. It was also demonstrated that the sucrose-plus-sorbitol mixture and trehalose were the most efficient for preserving B and A type formulations respectively after frozen storage. Mechanical (puncture), small amplitude oscillatory (SAOS) and dynamic-mechanical thermoanalyses were performed on both gels from A and B series surimis throughout frozen storage. Gels from surimi A (GA) were considerably brittler than gels from surimi B (GB) and presented significantly lower strain amplitudes. Several correlations among different experimental data have been developed.

Thermomechanical properties of cured isophtalic polyester resin modified with poly(ε-caprolactone)

The effect of a low profile additive, poly(e-caprolactone) (PCL), on the thermal and mechanical properties of unsaturated polyester resins (UP) was investigated by differential scanning calorimetry (DSC), dynamic mechanical thermal analysis (DMTA), and tensile tests. The morphology of the systems has been studied by scanning electronic microscopy (SEM). Two PCL molecular mass were selected (PCL2: M n = 2000 g mol−1 and PCL50: M n = 50000 g mol−1) to analyze the influence of the molecular mass and the content of PCL on the UP resins and to establish the relation between thermomechanical behavior and morphology. DSC and DMTA glass transition temperatures (T g) of the UP cured samples containing PCL indicate that PCL2 is miscible with UP whereas for UP + PCL50 system, T g values are very close to the ones corresponding to neat UP. Besides in UP + PCL2 systems, one phase morphology is observed in which PCL2 would act as solvent of the reacting mixture along curing process; however, UP + PCL50 systems present phase-separated morphology. The presence of PCL2 and PCL50 in UP resin leads to a decrease of the tensile strength and the Young′s modulus as much notorious as the PCL concentration increases. For UP + PCL2 system the elongation at fracture increases in relation to neat UP, increasing as well with the PCL content.

Effect of Lignosulfonate on the Thermal and Morphological Behavior of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The effect of lignosulfonate on poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV, was studied by scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The PHBV/lignosulfonate samples were prepared by melt mixing in an internal mixer. SEM showed that PHBV/lignosulfonate samples present a cracked surface that is more intense in mixtures with high lignosulfonate proportions. According to DSC, melting and glass transition temperatures of the PHBV matrix decrease with lignosulfonate addition. The same effect was observed for melting enthalpies (ΔHm), which indicates a decrease of crystallinity. TGA showed that thermal stability of PHBV/lignosulfonate samples was shifted to lower temperatures, which indicates the existence of an interaction between the thermal decomposition processes of PHBV and lignosulfonate.

The glass transition in athermal poly(α‐methyl styrene)/oligomer blends

AbstractThe glass transition behavior in athermal blends of poly(α‐methyl styrene) (PaMS) and its hexamer is investigated using differential scanning calorimetry (DSC). The results, along with previous data on similar blends of PaMS/pentamer, are analyzed in the context of the Lodge–McLeish self‐concentration model. A methodology is described to partition the calorimetric transition to obtain effective Tgs for each component of the blend. The dependences of these effective Tgs on overall blend composition are described by the Lodge–McLeish model, although the self‐concentration effect is less than expected based on the Kuhn length. The length scales of the cooperatively rearranging regions for the two components in the blends are also calculated adapting Donth's fluctuation model to the partitioned DSC transitions and are found to be similar for the two components and show a slight decrease at intermediate concentrations. The kinetics associated with the glass temperature, Tg, is examined by studying the cooling rate dependence of Tg for the pure components and the blends, as well as by examining the enthalpy overshoots in the heating DSC scans. It is observed that the cooling rate dependence of Tg in PaMS/hexamer blends at intermediate concentrations is similar to that of the hexamer, indicating that the kinetics of the glass transition for blends is dominated by the high mobility oligomeric component. Moreover, compared to the pure materials, the PaMS/hexamer blends exhibit a considerably depressed enthalpy overshoot, presumably resulting from their broader relaxation time distribution. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 418–430, 2008

Melting and freezing of water in cylindrical silica nanopores

Freezing and melting of H(2)O and D(2)O in the cylindrical pores of well-characterized MCM-41 silica materials (pore diameters from 2.5 to 4.4 nm) was studied by differential scanning calorimetry (DSC) and (1)H NMR cryoporometry. Well-resolved DSC melting and freezing peaks were obtained for pore diameters down to 3.0 nm, but not in 2.5 nm pores. The pore size dependence of the melting point depression DeltaT(m) can be represented by the Gibbs-Thomson equation when the existence of a layer of nonfreezing water at the pore walls is taken into account. The DSC measurements also show that the hysteresis connected with the phase transition, and the melting enthalpy of water in the pores, both vanish near a pore diameter D* approximately equal to 2.8 nm. It is concluded that D* represents a lower limit for first-order melting/freezing in the pores. The NMR spin echo measurements show that a transition from low to high mobility of water molecules takes place in all MCM-41 materials, including the one with 2.5 nm pores, but the transition revealed by NMR occurs at a higher temperature than indicated by the DSC melting peaks. The disagreement between the NMR and DSC transition temperatures becomes more pronounced as the pore size decreases. This is attributed to the fact that with decreasing pore size an increasing fraction of the water molecules is situated in the first and second molecular layers next to the pore wall, and these molecules have slower dynamics than the molecules in the core of the pore.

The influence of the tryptophan point mutation on the stability of tetracycline repressor (TetR)

The influence of a point mutation on the thermal stability of the tetracycline repressor (TetR) was studied with differential scanning calorimetry (DSC) and circular dichroism (CD) measurements, performed at pH 8.0. Single tryptophan TetR mutants, namely TetR W43 and TetR W75, were chosen because they had been used as fluorescence probes in structural investigations of TetR. The DSC results show that in the absence of tetracycline, the thermally induced transitions of all proteins can be described as irreversible process, strongly dependent upon scan rate. The observed scan dependence indicates that the protein denaturation is under kinetic control described by simple kinetic scheme: N 2 ⟶ k F 2 , where k is first-order kinetic constant, N 2 the native state and F 2 is the final, denatured state. The replacement of tryptophan by phenylalanine at position 43 located on the solvent accessible surface of TetR WT (mutant TetR W75) results in a slight increase in the denaturation temperature ( T max), whereas Trp75Phe substitution located in the core of TetR WT (mutant TetR W43) induces a strong decrease of T max. These opposing effects are discussed in the frame of structural changes of modified proteins. The effect of tetracycline (Tc) binding on the DSC transitions of TetR tryptophan mutants is also shown. Based on the observed increase of the T max, it is concluded that the binding of Tc to the mutants causes strong enhancement of their stability. Moreover, in case of TetR W43 the Tc binding compensates the stability loss due to introduced mutation.

Conformational Features of a Natural Break in the Type IV Collagen Gly-X-Y Repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.

L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study.

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.