Denatured State (denatured + state)

Distribution by Scientific Domains


Selected Abstracts


Thermally induced conformational changes in horseradish peroxidase

FEBS JOURNAL, Issue 1 2001
David G. Pina
Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated. [source]


Stabilization of PbS Nanocrystals by Bovine Serum Albumin in its Native and Denatured States

ADVANCED FUNCTIONAL MATERIALS, Issue 9 2009
Mandeep Singh Bakshi
Abstract PbS nanocrystals (NCs) are synthesized in aqueous phase within a temperature range of 40,80,°C in the presence of native and denatured states of bovine serum albumen (BSA) as the capping/stabilizing agent. The NCs are characterized with the help of field-emission scanning electron microscopy, high-resolution transmission electron microscopy, X-ray diffraction, and energy-dispersive X-ray analysis. At 40,°C, large ball-shaped NCs (145,±,37,nm) with small surface protrusions are formed when 1,×,10,4,g mL,1 BSA is used. As the reaction temperature is increased towards 80,°C, the size of NCs decreases and they acquire somewhat cubic geometries (49.1,±,7.0,nm) due to a change in the capping behavior of BSA between its native and denatured states. The native and denatured states of BSA are simultaneously studied by fluorescence spectroscopy using tryptophan emission, and pH measurements with respect to time and temperature. Gel electrophoresis is used to determine the polarity of the BSA capped NCs. Only the small sized NCs conjugated with relatively larger amounts of BSA show a displacement towards the positively charged electrode in comparison to larger NCs with lower amounts of BSA capping. It was concluded that the denatured state of BSA is more effective in controlling the crystal growth of PbS than its native state especially in the low concentration range. [source]


Applications of time-resolved resonance energy transfer measurements in studies of the molecular crowding effect,

JOURNAL OF MOLECULAR RECOGNITION, Issue 5 2004
Varda Ittah
Abstract The native structures of many globular proteins are only weakly stabilized and form in solution ensembles of multiple conformers. The energy differences between the conformers are assumed to be small. This is the case of flexible multidomain proteins where domain motions were observed. High concentrations of inert macrosolute, which create a crowded or confined environment, can cause shifts of the distribution of the conformers of such proteins towards the more compact structures. This effect may also promote compact structures in partially folded proteins. Time-resolved dynamic non-radiative excitation energy transfer (tr-RET) is suitable for detection of either subtle or major changes in distributions of intramolecular distances in protein molecules in solutions. Two experiments were performed which demonstrated the applicability of tr-RET for detection of the effect of macrosolutes on the conformational ensembles of flexible states of protein molecules. The distribution of distances between residues 203 and 169 in the CORE domain of E. coli adenylate kinase (AK) in the denatured state was determined in the presence of high concentrations of dextran 40. A significant shift of the mean of the distribution was observed without reduction of its width. This was interpreted as a shift to compact structure without change of the degree of disorder of the chain. In a second experiment the distribution of the distance between residues 55 and 169 in AK, which spans the cleft between the CORE and the AMPbind domains, was monitored. No clear effect of high concentrations of dextran 40 was found. These experiments show the strength of the application of tr-RET in investigation of changes in the sub-states of flexible conformations of globular protein. Networks of pairs of labeled sites can be prepared and tr-RET experiments can be performed in order to search for the segments of the protein molecules, which respond to the presence of inert macromolecules in their environment. Copyright © 2004 John Wiley & Sons, Ltd. [source]


Structural and ligand-binding properties of serum albumin species interacting with a biomembrane interface

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2007
Takamitsu Kosa
Abstract In the process of drug development, preclinical testing using experimental animals is an important aspect, for verification of the efficacy and safety of a drug. Serum albumin is a major binding protein for endogenous and exogenous ligands and regulates their distribution in various tissues. In this study, the structural and drug-binding properties of albumins on a biomembrane surface were investigated using reverse micelles as a model membrane. In reverse micelles, the secondary structures of all albumins were found, to varying degrees, to be intermediate between the native and denatured states. The tertiary structures of human and bovine albumin were similar to those of the native and intermediate states, respectively, whereas those of the dog, rabbit, and rat were in a denatured state. Thus, bovine albumin is an appropriate model for studying structural changes in human albumin in a membrane-water phase. Binding studies also showed the presence of species difference in the change in binding capacity of albumins during their interaction with reverse micelles. Among the albumins, rat albumin appears to be a good model for the protein-mediated drug uptake of human albumin in a biomembrane environment. These findings are significant in terms of the appropriate extrapolation of pharmacokinetics and pharmacodynamics data in various animals to humans. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 3117,3124, 2007 [source]


Temperature and urea induced denaturation of the TRP-cage mini protein TC5b: A simulation study consistent with experimental observations

PROTEIN SCIENCE, Issue 10 2009
Z. Gattin
Abstract The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20-residue Trp-cage mini-protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force-field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR-NOE atom,atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40,90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force-field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side-chain side-chain contact (Trp6-Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced. [source]


Structure of the 21,30 fragment of amyloid ,-protein

PROTEIN SCIENCE, Issue 6 2006
Andrij Baumketner
Abstract Folding and self-assembly of the 42-residue amyloid ,-protein (A,) are linked to Alzheimer's disease (AD). The 21,30 region of A,, A,(21,30), is resistant to proteolysis and is believed to nucleate the folding of full-length A,. The conformational space accessible to the A,(21,30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24,K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures. [source]


Water and urea interactions with the native and unfolded forms of a ,-barrel protein

PROTEIN SCIENCE, Issue 12 2003
Kristofer Modig
CD, circular dichroism; I-FABP, intestinal fatty acid-binding protein; MRD, magnetic relaxation dispersion; NOE, nuclear Overhauser effect Abstract A fundamental understanding of protein stability and the mechanism of denaturant action must ultimately rest on detailed knowledge about the structure, solvation, and energetics of the denatured state. Here, we use 17O and 2H magnetic relaxation dispersion (MRD) to study urea-induced denaturation of intestinal fatty acid-binding protein (I-FABP). MRD is among the few methods that can provide molecular-level information about protein solvation in native as well as denatured states, and it is used here to simultaneously monitor the interactions of urea and water with the unfolding protein. Whereas CD shows an apparently two-state transition, MRD reveals a more complex process involving at least two intermediates. At least one water molecule binds persistently (with residence time >10 nsec) to the protein even in 7.5 M urea, where the large internal binding cavity is disrupted and CD indicates a fully denatured protein. This may be the water molecule buried near the small hydrophobic folding core at the D,E turn in the native protein. The MRD data also provide insights about transient (residence time <1 nsec) interactions of urea and water with the native and denatured protein. In the denatured state, both water and urea rotation is much more retarded than for a fully solvated polypeptide. The MRD results support a picture of the denatured state where solvent penetrates relatively compact clusters of polypeptide segments. [source]


Contributions of folding cores to the thermostabilities of two ribonucleases H

PROTEIN SCIENCE, Issue 2 2002
Srebrenka Robic
Abstract To investigate the contribution of the folding cores to the thermodynamic stability of RNases H, we used rational design to create two chimeras composed of parts of a thermophilic and a mesophilic RNase H. Each chimera combines the folding core from one parent protein and the remaining parts of the other. Both chimeras form active, well-folded RNases H. Stability curves, based on CD-monitored chemical denaturations, show that the chimera with the thermophilic core is more stable, has a higher midpoint of thermal denaturation, and a lower change in heat capacity (,Cp) upon unfolding than the chimera with the mesophilic core. A possible explanation for the low ,Cp of both the parent thermophilic RNase H and the chimera with the thermophilic core is the residual structure in the denatured state. On the basis of the studied parameters, the chimera with the thermophilic core resembles a true thermophilic protein. Our results suggest that the folding core plays an essential role in conferring thermodynamic parameters to RNases H. [source]


NMR and SAXS characterization of the denatured state of the chemotactic protein Che Y: Implications for protein folding initiation

PROTEIN SCIENCE, Issue 6 2001
Pascal Garcia
Abstract The denatured state of a double mutant of the chemotactic protein CheY (F14N/V83T) has been analyzed in the presence of 5 M urea, using small angle X-ray scattering (SAXS) and heteronuclear magnetic resonance. SAXS studies show that the denatured protein follows a wormlike chain model. Its backbone can be described as a chain composed of rigid elements connected by flexible links. A comparison of the contour length obtained for the chain at 5 M urea with the one expected for a fully expanded chain suggests that ,25% of the residues are involved in residual structures. Conformational shifts of the ,-protons, heteronuclear 15N-{1H} NOEs and 15N relaxation properties have been used to identify some regions in the protein that deviate from a random coil behavior. According to these NMR data, the protein can be divided into two subdomains, which largely coincide with the two folding subunits identified in a previous kinetic study of the folding of the protein. The first of these subdomains, spanning residues 1,70, is shown here to exhibit a restricted mobility as compared to the rest of the protein. Two regions, one in each subdomain, were identified as deviating from the random coil chemical shifts. Peptides corresponding to these sequences were characterized by NMR and their backbone 1H chemical shifts were compared to those in the intact protein under identical denaturing conditions. For the region located in the first subdomain, this comparison shows that the observed deviation from random coil parameters is caused by interactions with the rest of the molecule. The restricted flexibility of the first subdomain and the transient collapse detected in that subunit are consistent with the conclusions obtained by applying the protein engineering method to the characterization of the folding reaction transition state. [source]


The effects of disulfide bonds on the denatured state of barnase

PROTEIN SCIENCE, Issue 12 2000
Jane Clarke
Abstract The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended ,-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure. [source]


Characterization of the polyanion-induced molten globule-like state of cytochrome c

BIOPOLYMERS, Issue 2 2007
Erik Sedlák
Abstract Cytochrome c (cyt c) undergoes a poly(vinylsulphate) (PVS)-induced transition at slightly acidic pH into a molten globule-like state that resembles the effect that negatively charged membrane surfaces have on this protein. In this work, the thermodynamic properties of the molten globule-like state of cyt c in complex with PVS are studied using differential scanning calorimetry, circular dichroism, fluorescence, and absorbance spectroscopy. The temperature-induced transition of the molten globule-like state of cyt c in the complex with PVS is characterized by a significantly lower calorimetric enthalpy than in the "typical" molten globule state of cyt c, i.e. free protein at pH 2.0 in high ionic strength. Moreover, the thermally-denatured state of cyt c in the complex at pH < 6 contains nearly 50% of the native secondary structure. The dependence of the transition temperature on the pH indicates a role for histidine residues in the destabilization of the cyt c structure in the PVS complex and in stabilization of the denatured state with the residual secondary structure. A comparison of the effects of small anions and polyanions demonstrates the importance of cooperativity among the anions in the destabilization of cyt c. Predictably, other hydrophilic flexible polyanions such as heparin, polyglutamate, and polyadenylate also have a destabilizing effect on the structure of cyt c. However, a correlation between the properties of the polyanions and their effect on the protein stability is still unclear. © 2007 Wiley Periodicals, Inc. Biopolymers 86: 119,126, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source]