Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration

Richard, Allison J.; Liu, Chin‐Chi; Klinger, Alexandra L.; Todd, Matthew J.; Mezzasalma, Tara M.; LiCata, Vince J.

doi:10.1016/j.bbapap.2006.08.011

Cited by 21 publications

(18 citation statements)

References 35 publications

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“…There is a body of work which studies the role salts play during the unfolding of proteins induced by urea, guanidinium HCl, low pH, and heat . The role of salts during heat‐induced unfolding is not clear from the literature.…”

Section: Introductionmentioning

confidence: 99%

“…There is a body of work which studies the role salts play during the unfolding of proteins induced by urea, [11][12][13] guanidinium HCl, 14 low pH, 15,16 and heat. [17][18][19][20][21][22] The role of salts during heat-induced unfolding is not clear from the literature. Research on the effect of the anions on unfolding of the B1 domain of protein L demonstrated that sulfate, phosphate and fluoride stabilized, while chloride was neutral and nitrate, perchlorate and thiocyanide destabilized the protein.…”

Section: Introductionmentioning

confidence: 99%

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Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Bye

Falconer

2013

Protein Science

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Anion and cation effects on the structural stability of lysozyme were investigated using differential scanning calorimetry. At low concentrations (<5 mM) anions and cations alter the stability of lysozyme but they do not follow the Hofmeister (or inverse Hofmeister) series. At higher concentrations protein stabilization follows the well-established Hofmeister series. Our hypothesis is that there are three mechanisms at work. At low concentrations the anions interact with charged side chains where the presence of the ion can alter the structural stability of the protein. At higher concentrations the low charge density anions perchlorate and iodide interact weakly with the protein. Their presence however reduces the Gibbs free energy required to hydrate the core of the protein that is exposed during unfolding therefore destabilizing the structure. At higher concentrations the high charge density anions phosphate and sulfate compete for water with the protein as it unfolds increasing the Gibbs free energy required to hydrate the newly exposed core of the protein therefore stabilizing the structure.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Bye

Falconer

2013

Protein Science

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“…Both experimental measurements and theoretical results have shown that subtle and small changes in salt concentration can have a strong effect on a broad spectrum of protein properties. For instance, the stability of proteins as well the protein's association with molecules ranging from small charged peptides and drugs to larger proteins and polysaccharides at both the kinetic and thermodynamic level, can be altered by changing the salt type and concentration 1–6. We still lack a complete theoretical understanding of why increasing the salt concentration and changing the salt type can either enhance or diminish protein stability and binding to charged ligands ( e.g.…”

Section: Introductionmentioning

confidence: 99%

Using Correlated Monte Carlo Sampling for Efficiently Solving the Linearized Poisson−Boltzmann Equation Over a Broad Range of Salt Concentration

Fenley

Mascagni

McClain

et al. 2009

J. Chem. Theory Comput.

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Dielectric continuum or implicit solvent models provide a significant reduction in computational cost when accounting for the salt-mediated electrostatic interactions of biomolecules immersed in an ionic environment. These models, in which the solvent and ions are replaced by a dielectric continuum, seek to capture the average statistical effects of the ionic solvent, while the solute is treated at the atomic level of detail. For decades, the solution of the three-dimensional Poisson-Boltzmann equation (PBE), which has become a standard implicit-solvent tool for assessing electrostatic effects in biomolecular systems, has been based on various deterministic numerical methods. Some deterministic PBE algorithms have drawbacks, which include a lack of properly assessing their accuracy, geometrical difficulties caused by discretization, and for some problems their cost in both memory and computation time. Our original stochastic method resolves some of these difficulties by solving the PBE using the Monte Carlo method (MCM). This new approach to the PBE is capable of efficiently solving complex, multi-domain and salt-dependent problems in biomolecular continuum electrostatics to high precision. Here we improve upon our novel stochastic approach by simultaneouly computating of electrostatic potential and solvation free energies at different ionic concentrations through correlated Monte Carlo (MC) sampling. By using carefully constructed correlated random walks in our algorithm, we can actually compute the solution to a standard system including the linearized PBE (LPBE) at all salt concentrations of interest, simultaneously. This approach not only accelerates our MCPBE algorithm, but seems to have cost and accuracy advantages over deterministic methods as well. We verify the effectiveness of this technique by applying it to two common electrostatic computations: the electrostatic potential and polar solvation free energy for calcium binding proteins that are compared with similar results obtained using mature deterministic PBE methods.

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“…It can also identify other conditions (such as variations in buffer, pH, salt and glycerol concentration, types of cations, etc.) that stabilize proteins and thus make them more amenable to purification and crystallization 12–15. It is applicable to a wide variety of eukaryotic and prokaryotic proteins and yields results comparable to static light scattering (SLS) measurements of protein aggregation 4,11,16…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies of factors affecting proteins' melting curves have focused almost exclusively on identifying factors that increase T m 12–15. However, in setting up a thermal melt-based high-throughput screen (HTS) designed to identify inhibitors or other ligands of a particular protein, the absolute value of the T m is less important than the variability of T m measurements.…”

Section: Introductionmentioning

confidence: 99%

Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

et al. 2009

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In the last decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. Here we present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES, pH 7.5, 150 mM NaCl) permitted estimation of Tm for 58 of 61 Plasmodium proteins tested. However, with several proteins, Tm could not be measured with a consistency suitable for high-throughput screening unless alternative protein-specific buffers were employed. We conclude that buffer optimization to minimize variability in Tm measurements increases the success of thermal melt screens involving proteins for which a standard buffer is suboptimal.

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Thermal stability landscape for Klenow DNA polymerase as a function of pH and salt concentration

Cited by 21 publications

References 35 publications

Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Thermal stability of lysozyme as a function of ion concentration: A reappraisal of the relationship between the Hofmeister series and protein stability

Using Correlated Monte Carlo Sampling for Efficiently Solving the Linearized Poisson−Boltzmann Equation Over a Broad Range of Salt Concentration

Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands

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