Partially folded intermediates during trypsinogen denaturation

The equilibrium unfolding of bovine trypsinogen was studied by circular dichroism, differential spectra and size exclusion HPLC. The change in free energy of denaturation was = 6.99 ± 1.40 kcal/mol for guanidine hydrochloride and = 6.37 ± 0.57 kcal/mol for urea. Satisfactory fits of equilibrium unfolding transitions required a three-state model involving an intermediate in addition to the native and unfolded forms. Size exclusion HPLC allowed the detection of an intermediate population of trypsinogen whose Stokes radii varied from 24.1 ± 0.4 Å to 26.0 ± 0.3 Å for 1.5 M and 2.5 M guanidine hydrochloride, respectively. During urea denaturation, the range of Stokes radii varied from 23.9 ± 0.3 Å to 25.7 ± 0.6 Å for 4.0 M and 6.0 M urea, respectively. Maximal intrinsic fluorescence was observed at about 3.8 M urea with 8-aniline-1-naphthalene sulfonate (ANS) binding. These experimental data indicate that the unfolding of bovine trypsinogen is not a simple transition and suggest that the equilibrium intermediate population comprises one intermediate that may be characterized as a molten globule. To obtain further insight by studying intermediates representing different stages of unfolding, we hope to gain a better understanding of the complex interrelations between protein conformation and energetics.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Whey protein mouth drying influenced by thermal denaturation

Whey proteins are becoming an increasingly popular functional food ingredient. There are, however, sensory properties associated with whey protein beverages that may hinder the consumption of quantities sufficient to gain the desired nutritional benefits. One such property is mouth drying. The influence of protein structure on the mouthfeel properties of milk proteins has been previously reported. This paper investigates the effect of thermal denaturation of whey proteins on physicochemical properties (viscosity, particle size, zeta-potential, pH), and relates this to the observed sensory properties measured by qualitative descriptive analysis and sequential profiling. Mouthcoating, drying and chalky attributes built up over repeated consumption, with higher intensities for samples subjected to longer heating times (p < 0.05). Viscosity, pH, and zeta-potential were found to be similar for all samples, however particle size increased with longer heating times. As the pH of all samples was close to neutral, this implies that neither the precipitation of whey proteins at low pH, nor their acidity, as reported in previous literature, can be the drying mechanisms in this case. The increase in mouth drying with increased heating time suggests that protein denaturation is a contributing factor and a possible mucoadhesive mechanism is discussed.

On the Denaturation Mechanisms of the Ligand Binding Domain of Thyroid Hormone Receptors

The ligand binding domain (LBD) of nuclear hormone receptors adopts a very compact, mostly alpha-helical structure that binds specific ligands with very high affinity. We use circular dichroism spectroscopy and high-temperature molecular dynamics Simulations to investigate unfolding of the LBDs of thyroid hormone receptors (TRs). A molecular description of the denaturation mechanisms is obtained by molecular dynamics Simulations of the TR alpha and TR beta LBDs in the absence and in the presence of the natural ligand Triac. The Simulations Show that the thermal unfolding of the LBD starts with the loss of native contacts and secondary Structure elements, while the Structure remains essentially compact, resembling a molten globule state. This differs From most protein denaturation simulations reported to date and suggests that the folding mechanism may start with the hydrophobic collapse of the TR LBDs. Our results reveal that the stabilities of the LBDs of the TR alpha and TR beta Subtypes are affected to different degrees by the binding of the isoform selective ligand Triac and that ligand binding confers protection against thermal denaturation and unfolding in a subtype specific manner. Our Simulations indicate two mechanisms by which the ligand stabilizes the LBD: (1) by enhancing the interactions between H8 and H 11, and the interaction of the region between H I and the Omega-loop with the core of the LBD, and (2) by shielding the hydrophobic H6 from hydration.

DMSO-Induced Denaturation of Hen Egg White Lysozyme

We report on the size, shape, structure, and interactions of lysozyme in the ternary system lysozyme/DMSO/water at low protein concentrations. Three structural regimes have been identified, which we term the ""folded"" (0 < phi(DMSO) < 0.7), ""unfolded"" (0.7 <= phi(DMSO) < 0.9), and ""partially collapsed"" (0.9 <= phi(DMSO) < 1.0) regime. Lysozyme resides in a folded conformation with an average radius of gyration of 1.3 +/- 0.1 nm for phi(DMSO) < 0.7 and unfolds (average R(g) of 2.4 +/- 0.1 nm) above phi(DMSO) > 0.7. This drastic change in the protein`s size coincides with a loss of the characteristic tertiary structure. It is preceded by a compaction of the local environment of the tryptophan residues and accompanied by a large increase in the protein`s overall flexibility. In terms of secondary structure, there is a gradual loss of alpha-helix and concomitant increase of beta-sheet structural elements toward phi(DMSO) = 0.7, while an increase in phi(DMSO) at even higher DMSO volume fractions reduces the presence of both a-helix and beta-sheet secondary structural elements. Protein-protein interactions remain overall repulsive for all values of phi(DMSO) An attempt is made to relate these structural changes to the three most important physical mechanisms that underlie them: the DMSO/water microstructure is strongly dependent on the DMSO volume fraction, DMSO acts as a strong H-bond acceptor, and DMSO is a bad solvent for the protein backbone and a number of relatively polar side groups, but a good solvent for relatively apolar side groups, such as tryptophan.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with Tc value of 333.4±0.2K for oxy-HbGp, while two peaks with Tc values of 332.2±0.1 and 338.4±0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher Tc values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323K, presenting oligomeric dissociation at 333 (12±3% of tetramer and 88±5% of whole HbGp) and 343K (70±5% of monomer and 30±2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar Tc values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure. © 2012 Elsevier B.V.

Analysis of Lattice Size, Energy Density and Denaturation for a One-Dimensional DNA Model

There are several mechanical models to describe the DNA phenomenology. In this work the DNA denaturation is stu- died under thermodynamical and dynamical point of view using the well known Peyrard-Bishop model. The thermody-namics analysis using the transfer integral operator method is briefly reviewed. In particular, the lattice size is discussed and a conjecture about the minimum energy to denaturation is proposed. In terms of the dynamical aspects of the model, the equations of motion for the system are integrated and the results determine the energy density where the denatura- tion occurs. The behavior of the lattice near the phase transition is analyzed. The relation between the thermodynamical and dynamical results is discussed.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Light scattering study of human serum albumin in pre-denaturation:relation to dynamic transition in water at 42°C

Protein functional motions are ultimately connected to water dynamics. The goal of this study is to link the conformational dynamics of albumin to a dynamic transition taking place at ∼ 42°C in water. We report the results of dynamic light scattering measurements of albumin aqueous solution in the temperature interval 20-65°C. The processing of the experimental data produced the temperature dependence of the macromolecular hydrodynamic radius. We demonstrate that the growth of the macromolecular size in this temperature range can be divided into two stages that are connected to the dynamical properties of water. © 2012 Elsevier B.V. All rights reserved.