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- McGuire, J, et al.
(författare)
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Comparative Adsorption Studies with Synthetic, Structural Stability and Charge Mutants of Bacteriophage T4 Lysozyme.
- 1995
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Ingår i: Proteins at Interfaces II. - Washington, DC : American Chemical Society. - 9780841233041 - 9780841215276 ; 602, s. 52-65
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Bokkapitel (refereegranskat)abstract
- We have purified wild type, three structural stability mutants and four charge mutants of bacteriophage T4 lysozyme from E. coli strains harboring desired expression vectors. Structural stability mutants were produced by substitution of the isoleucine at amino acid position three, yielding a set of proteins with stabilities ranging from 1.2 kcal/mol greater, to 2.8 kcal/mol less, than that of the wild type. Charge mutants were produced by replacement of positively charged lysine residues with glutamic acid, yielding a set of molecules with formal charges ranging from +5 to +9 units. Adsorption kinetic data, along with the dodecyltrimethylammonium bromide-mediated elutability of each protein, has been monitored with in situ ellipsometry at hydrophobic and hydrophilic silica surfaces. A simple mechanism that allows adsorbing protein to adopt one of two states, each associated with a different resistance to elution and a different interfacial area occupied per molecule, has been used to assist interpretation of the adsorption data. Conditions implicit in the model have been used to estimate the fraction of molecules present on the surface just prior to surfactant addition that had adopted the more resistant state, and this fraction has been observed to correlate positively with resistance to elution. For the stability mutants, these properties were clearly related to protein stability as well. Concerning the charge mutants, results have not been clearly explainable in terms of protein net charge, but rather in terms of the probable influence of the location of each substitution relative to other mobile, solvent-exposed, charged side chains of the molecule.
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- McGuire, J, et al.
(författare)
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Structural Stability Effects on Adsorption and Dodecyltrimethylammonium Bromide-Mediated Elutability of Bacteriophage T4 Lysozyme at Silica Surfaces
- 1995
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 170, s. 182-192
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Tidskriftsartikel (refereegranskat)abstract
- The effect of structural stability on the adsorption and dodecyltrimethylammonium bromide (DTAB)-mediated elutability of bacteriophage T4 lysozyme was monitored at hydrophilic and hydrophobic silica surfaces with in situ ellipsometry. Mutant lysozymes were produced by substitution of the isoleucine at amino acid position three, yielding a set of proteins with values of ΔGunfolding ranging from 1.2 kcal/mol greater to 2.8 kcal/mol less than that of the wild type. Three structural stability mutants along with the wild type protein were purified from Escherichia coli strains harboring the desired expression vectors. Differences in interfacial behavior among the proteins were observed to be substantial with respect to both the adsorption kinetic behavior and the DTAB-mediated elutability exhibited by each. A positive correlation was observed to exist between elutability and protein stability. A simple mechanism that allows absorbing protein to adopt one of two states, each associated with a different resistance to elution and a different interfacial area occupied per molecule, was used to assist interpretation of the adsorption data recorded prior to surfactant addition. Conditions implicit in the model allowed estimation of the mass of molecules present on the surface just prior to surfactant addition, that were in the more resistant state in each test. The calculated fraction of adsorbed protein in the more tightly bound state correlated positively with resistance to elution.
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- McGuire, J, et al.
(författare)
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The Influence of Net Charge and Charge Location on Adsorption and Dodecyltrimethylammonium Bromide-Mediated Elutability of Bacteriophage T4 Lysozyme at Silica Surfaces
- 1995
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 1095-7103 .- 0021-9797. ; 170:1, s. 193-202
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Tidskriftsartikel (refereegranskat)abstract
- The effect of net charge and charge location on the adsorption and dodecyltrimethylammonium bromide (DTAB)-mediated elutability of bacteriophage T4 lysozyme was monitored at hydrophilic and hydrophobic silica surfaces with in situ ellipsometry. Mutant lysozymes were produced by substitution of selected lysine residues with glutamic acid, each substitution thus decreasing the net charge of the protein by 2 units. The wild-type protein (net charge +9) and four mutant proteins, each of net charge +7 or +5, were purified from Escherichia coli strains harboring the desired expression vectors. Differences in interfacial behavior among the proteins were observed with respect to both the adsorption kinetics and the DTAB-mediated elutability exhibited by each. No simple relationship between protein net charge and surface behavior was observed, indicating that the location of the charge replacements had the major effect on surface behavior. At hydrophilic surfaces, mutations allowing the most mobile regions of positive charge to more readily orient toward the interface increased that protein's resistance to elutability; at hydrophobic surfaces, mutations favoring or otherwise not inhibiting hydrophobic association between the protein and the surface increased the resistance to elutability. This was not related to protein net charge, but to the probable influence of the location of each substitution relative to the other mobile, solvent-exposed, charged side chains of the molecule.
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