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Chemical Denaturation (chemical + denaturation)
Selected AbstractsPressure-exploration of the 33-kDa protein from the spinach photosystem II particleFEBS JOURNAL, Issue 9 2001Kangcheng Ruan The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 °C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (,Go) and in volume (,V) for the transition at pH 6.0 and 20 °C are ,14.6 kJ·mol,1 and ,120 mL·mol,1, respectively, in which the ,Go value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of ,Go (only a minor variation was observed for the ,V value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in ,V without any significant changes in ,Go. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 m NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of ,V is significantly reduced whilst that of ,Go remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s. [source] Quantification and characterization of subvisible proteinaceous particles in opalescent mAb Formulations Using Micro-Flow ImagingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2010Deepak K. Sharma Abstract Micro-flow imaging (MFIÔ) has been shown to be more sensitive than light obscuration (LO) methods for measuring subvisible proteinaceous particles in protein formulations. Given the potential challenges in detecting particulates in opalescent mAb formulations, the accuracy of MFI to size and count particles in opalescent solutions was investigated and compared to LO and membrane microscopy methods. Proteinaceous monoclonal antibody (mAb) particles, generated either by chemical denaturation or agitation stress, polystyrene and glass particles were used as model systems for measurements in opalescent mAb solutions. The sizing and counting accuracies of MFI were unaffected by the opalescence of the medium. Using glass particles as a model system for proteinaceous particles, MFI was able to detect relatively low particle concentrations (,10/mL) in opalescent solutions. MFI showed excellent linearity (R2,=,0.9969) for quantifying proteinaceous particles in opalescent solutions over a wide range of particle concentrations (,20,160,000/mL). Analyses of MFI particle image intensities revealed significant differences in the transparency of proteinaceous particles as a function of their size and mode of generation. LO method significantly underestimated proteinaceous particles, particularly those in the 2,10,µm size range. The less opaque proteinaceous particles were relatively more underestimated by the LO method in opalescent solutions. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2628,2642, 2010 [source] Molecular Recognition in Partially Folded States of a Transporter Protein: Temperature-dependent Specificity of Bovine Serum AlbuminPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2008Debapriya Banerjee The specificity of molecular recognition of a transporter protein bovine serum albumin (BSA) in its different partially folded states has been studied. In order to avoid complications due to chemical denaturation, we have prepared thermally induced partially folded states of the protein. The partially folded states have been structurally characterized by circular dichroism and differential thermal analysis techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering. Steady state, picosecond-resolved fluorescence and polarization gated spectroscopies on the ligands (DCM, LDS 750) in the protein reveal the dynamics of the binding sites and the specificity of ligand binding of BSA. Picosecond resolved Förster resonance energy transfer studies on the donor DCM and acceptor LDS 750 confirm that the specificity of ligand binding in the binding site is maintained up to 70°C. At 75°C, the protein loses its specificity of recognition at the aforesaid site. [source] Hyperstability and crystal structure of cytochrome c555 from hyperthermophilic Aquifex aeolicusACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Marii Obuchi In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c555 (AA c555) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c555 exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c552 (HT c552), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c555, which was determined using the multiple anomalous dispersion technique at 1.15,Å resolution, included a unique 14-residue extra helix, while the side-chain interactions of several amino-acid residues responsible for the stability of HT c552 were conserved in AA c555. The side chain of the Met61 residue in the extra helix was aligned towards the haem, forming a coordination bond between the Met S and haem Fe atoms. In other cytochromes c the corresponding regions always form , loops which also include the haem-liganding Met residue and are known to be involved in the initial step in cytochrome c denaturation. The formation of the extra helix in AA c555 results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c555 and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment. [source] Structural and thermodynamic encoding in the sequence of rat microsomal cytochrome b5,BIOPOLYMERS, Issue 5 2008Juliette T. J. Lecomte Abstract The water-soluble domain of rat microsomal cytochrome b5 is a convenient protein with which to inspect the connection between amino acid sequence and thermodynamic properties. In the absence of its single heme cofactor, cytochrome b5 contains a partially folded stretch of ,30 residues. This region is recognized as prone to disorder by programs that analyze primary structures for such intrinsic features. The cytochrome was subjected to amino acid replacements in the folded core (I12A), in the portion that refolds only when in contact with the heme group (N57P), and in both (F35H/H39A/L46Y). Despite the difficulties associated with measuring thermodynamic quantities for the heme-bound species, it was possible to rationalize the energetic consequences of both types of replacements and test a simple equation relating apoprotein and holoprotein stability. In addition, a phenomenological relationship between the change in Tm (the temperature at the midpoint of the thermal transition) and the change in thermodynamic stability determined by chemical denaturation was observed that could be used to extend the interpretation of incomplete holoprotein stability data. Structural information was obtained by nuclear magnetic resonance spectroscopy toward an atomic-level analysis of the effects. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 428,442, 2008. 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] Contributions of folding cores to the thermostabilities of two ribonucleases HPROTEIN SCIENCE, Issue 2 2002Srebrenka 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] | ||||||||||