Denaturing simulations

Thermal denaturation simulations "" were designed to simulate the unfolding pathway and provide explicit structural models for the transition state. Four thermal denaturation simulations were run in water at 498 K, using the correct density, for at least 1 ns to ensure consistency of the simulation results. The transition states were characterized in terms of the rate of change of the rmsd(Ca) for every structure against every other structure from the trajectory. Since one expects a rapid change in the protein structure once the transition state has been passed, Ae transition state was identified as... 

Bond C ], K-B Wong, ] Clarke, A R Ferscht and V Daggett 1997. Characterisation of Residual Structure in the Tliermally Denatured State of Barnase by Simulation and Experiment Description of the Folding Pathway. Proceedings of the National Academy of Sciences USA 94 13409-13413. 

Although additional experiments and simulations are needed to determine how much of reality is captured in this model, it does explain one important property of proteins their rapid rate of refolding, which is independent of denaturation conditions. If the polypeptide chain is unable to escape from this steric trap and access conformations with the wrong topology, it could never wander far from the folded conformation and thereby avoid incorrect side chain/side chain interactions. 

Pan, Y. P. Daggett, V., Direct comparison of experimental and calculated folding free energies for hydrophobic deletion mutants of chymotrypsin inhibitor. 2 Free energy perturbation calculations using transition and denatured states from molecular dynamics simulations of unfolding, Biochemistry 2001,40, 2723-2731. 

We used outdated human CPD (citrate-phosphate-dextrose) blood from the Dayton Community Blood Center. At 21 days, CPD blood still retains 78% survival of the red blood cells and would fairly well simulate in vivo physiological conditions. During these tests, many enzymes and proteins may denature and/or precipitate. Even after suffering that trauma, the resulting fluid is more suitable for material testing than other pseudo-physiological fluids, since it still contains most of the salts, lipids, hormones, oligomers, nucleotides, saccharides, etc., found in whole blood in vivo. 

Lepock, J.R., K.P. Ritchie, M.C. Kolios, A.M. Rodahl, K.A. Heinz, and J. Kruuv. 1992. Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation. Biochemistry 31 12706-12712. 

Lerman LS, Silverstein (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 155 482-501... 

Whey protein concentrates (WPC), which are relatively new forms of milk protein products available for emulsification uses, have also been studied (4,28,29). WPC products prepared by gel filtration, ultrafiltration, metaphosphate precipitation and carboxymethyl cellulose precipitation all exhibited inferior emulsification properties compared to caseinate, both in model systems and in a simulated whipped topping formulation (2. However, additional work is proceeding on this topic and it is expected that WPC will be found to be capable of providing reasonable functionality in the emulsification area, especially if proper processing conditions are followed to minimize protein denaturation during their production. Such adverse effects on the functionality of WPC are undoubtedly due to their Irreversible interaction during heating processes which impair their ability to dissociate and unfold at the emulsion interface in order to function as an emulsifier (22). 

N. D. Socci, Z. Luthey Schulten, and P. G. Wolynes, Folding and Design 1,441 (1996).] Bottom Structures of the denatured, intermediate, major transition, and native states for folding of bamase from molecular dynamics simulations that were benchmarked by < > values and NMR experiments. [Data from C. J. Bond, K. B. Wong, J. Clarke,... 

In their natural habitat, proteins are usually surrounded by other proteins and organic factors. When these are removed or diluted as during purification, the protein becomes surrounded by water on all sides. Proteins react differently to a pure aqueous environment many are destabilized and rapidly denatured. A common remedial measure is to add 5%-20% glycerol to the purification buffer. The organic surface of the glycerol is believed to simulate the environment of the protein in the intact cell. Two other ingredients that are most frequently added to purification buffers are mercaptoethanol and ethylenedia-minetetraacetate (EDTA). The mercaptoethanol inhibits the oxidation of protein —SH groups, and the EDTA chelates... 

Falchini, L., Naumova, N., Kuikman, P. J., Bloemc, J., and Nannipieri, P. (2003). C02 evolution and denaturing gradient gel electrophoresis profiles of bacterial communities in soil following addition of low molecular weight substrates to simulate root exudation. Soil Biol. Biochem. 35,775-782. 

The thermal stability of metmyoglobin and apomyoglobin has been extensively studied under different solvent conditions (Privalov et al., 1986). In particular, it was shown that at low pH values both the heat and cold denaturation peaks are clearly visible in the calorimetric scans. Figure 10 shows the excess heat capacity function for apomyoglobin predicted by the hierarchical partition function and the thermodynamic parameters described above. In order to simulate the experimental curve obtained at pH 3.83 (Privalov et al., 1986), the protonation of five specific histidine residues on unfolding was... 

Fig. 10. Predicted excess heat capacity function versus temperature for myoglobin. The curve simulates the experimental curve obtained at pH 3.83 by Privalov et al. (1986). Under those conditions both the cold and heat denaturation curves can be studied experimentally. The predicted values are Tm,cold = 4°C Tm>heat = 58°C A// = 59 kcal mol-1 ACp = 2.45 kcal K-1 mol-1. The experimental values are Tm>coid = 3°C Tm>heat = 57.5°C AH = 53 kcal mol-1 ACP = 2.5 kcal K-1 mol-1 (Privalov et al., 1986). [Reprinted from Freire and Murphy (1991).]...

Surimi is fish paste from deboned fish used to make simulated crab legs and other seafood. For preservation the paste is blended with cryoprotectants, such as sucrose, sorbitol and phosphates, and frozen. To make the final product, the frozen paste is thawed, blended with starch and extruded as a film onto a belt. The belt takes the film into an oven that heat-denatures the fish protein and cooks the starch. The film is then rolled to form striations, shaped, colored and cut. Depending on the required distribution, the product is frozen or refrigerated. Potato and tapioca starch were used in surimi products 400 years ago, since they provided a cohesive, elastic matrix consistent with seafood. Frozen distribution has made the use of highly-stabilized, moderately crosslinked tapioca starch popular, alone or with native tapioca starch. Modified waxy maize products are used, as is unmodified com starch, for increased cuttability. Kim188 reported that the gel strengthening ability of starch correlates with starch paste viscosity. 

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