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J A Schellman

Publications and source records attributed to J A Schellman.

At least 19 recordsLinked to original sources
PubMed1992

Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C.

The kinetics of unfolding and refolding of T4 lysozyme and nine of its mutants have been investigated as a function of guanidinium chloride concentration at 12 degrees C. All show simple two-state, first-order kinetics. Two types of mutants were studied: proline-alanine interchanges and substitutions at position 3 with side chains of varying hydrophobicity. Crystal structures are available for seven of the ten proteins. The effect of mutations on the folding kinetics is more pronounced and complex than on equilibrium thermodynamics. The proteins fall into two broad kinetic classes with one class rather close to the wild type. P86A is a mutant with marked changes in kinetics but only a very small change in stability. Since the 86 position is in the middle of an alpha-helix, the indications are that the helix containing an A residue is more stable in the transition state than one containing a P residue. The other mutants are more complicated, with the refolding and unfolding rates unequally affected by the mutations. On the basis of comparisons with other investigations, we conclude that the rate-determining step in the presence of guanidinium chloride is not the same as in aqueous solution and that it most likely precedes it. The indications are that we are studying the formation of a transition intermediate which is destabilized by the denaturant and which resembles the A intermediate of the framework or molten globule models for protein folding.

Amino Acid Sequence↗
PubMed1991

Large differences in the helix propensities of alanine and glycine.

The standard view of alpha helix formation in water, based on helix propensities determined by the host-guest method, is that differences in helix propensity among the amino acids are small, except for proline, and that the average value of the helix propagation parameter s is near 1. A contradictory view of alpha helix formation in water is emerging from substitution experiments with short, unique-sequence peptides that contain only naturally occurring amino acids. Short peptides that contain only alanine and lysine, or alanine and glutamate, form surprisingly stable monomeric helices in water and substitution of a single alanine residue by another amino acid in these or related peptides produces a wide range of changes in helix content, depending on which amino acid is substituted for alanine. We show here that the ratio of the helix propensities of alanine to glycine is large, about 100, in substitution experiments with a 17-residue reference peptide containing alanine and lysine. The helix propensity is identified with s, the helix propagation parameter of the statistical mechanics model for alpha helix formation, and the results are interpreted by the Lifson-Roig theory. Single alanine----glycine substitutions have been made at a series of positions in individual peptides. The helix-destabilizing effect of an Ala----Gly substitution depends strongly on its position in the helix, as predicted by the Lifson-Roig theory if the ratio of s values for Ala:Gly is large.

Alanine↗
PubMed1990

A simple model for solvation in mixed solvents. Applications to the stabilization and destabilization of macromolecular structures.

The properties of a simple model for solvation in mixed solvents are explored in this paper. The model is based on the supposition that solvent replacement is a simple one-for-one substitution reaction at macromolecular sites which are independent of one another. This leads to a new form for the binding polynomial in which all terms are associated with ligand interchange rather than ligand addition. The principal solvent acts as one of the ligands. Thermodynamic analysis then shows that thermodynamic binding (i.e., selective interaction) depends on the properties of K'-1, whereas stoichiometric binding (site occupation) depends on K'. K' is a 'practical' interchange equilibrium constant given by (f3/f1)K, where K is the true equilibrium constant for the interchange of components 3 and 1 on the site and f3 and f4 denote their respective activity coefficients on the mole fraction scale. Values of K' less than unity lead to negative selective interaction. It is selective interaction and not occupation number which determines the thermodynamic effects of solvation. When K' greater than 100 on the mole fraction scale or K' greater than 2 on the molality scale (in water), the differences between stoichiometric binding and selective interaction become less than 1%. The theory of this paper is therefore necessary only for very weak binding constants. When K'-1 is small, large concentrations of the added solvent component are required to produce a thermodynamic effect. Under these circumstances the isotherms for the selective interaction and for the excess (or transfer) free energy are strongly dependent on the behavior of the activity coefficients of both solvent components. Two classes of behavior are described depending on whether the components display positive or negative deviations from Raoult's law. Examples which are discussed are aqueous solutions of urea and guanidinium chloride for positive deviations and of sucrose and glucose for negative deviations. Examination of the few studies which have been reported in the literature shows that most of the qualitative features of the stabilization of proteins by sugars and their destabilization by urea and guanidinium chloride are faithfully represented with the model. This includes maxima in the free energy of stabilization and destabilization, decreased and zero selective interaction at high concentrations, etc. These phenomena had no prior explanation. Deficiencies in the model as a representation of solvation in aqueous solution are discussed in the appendix.

Binding Sites↗
PubMed1990

Fluctuation and linkage relations in macromolecular solution.

It is shown in the appendix that the derivatives of the excess free energy of a macromolecule in solution, with respect to the activities of other solution components, lead to fluctuation and linkage relations among these other components. Solution fluctuation theory is used, but it is specialized to the fluctuations and correlations associated with the presence of a macromolecule, and is developed with a modified ensemble. The relations of the appendix are used to analyze the interaction of two solution components, A and B, with the macromolecule and with one another. Three cases are considered: (1) A and B are ligands that bind stoichiometrically to the macromolecule. This case reduces to Wyman's binding polynomial analysis. (2) A and B are two substances at high concentration that interact selectively with the macromolecule. (3) A is a species that binds stoichiometrically to the macromolecule, while B is a component at high concentration that interacts weakly with the macromolecule.

Chemical Phenomena↗
PubMed1989

Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies.

The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986). This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium. The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions. The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form. The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride. These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper.

Calorimetry↗
PubMed1989

Low-temperature unfolding of a mutant of phage T4 lysozyme. 2. Kinetic investigations.

A disulfide-bridged variant of bacteriophage T4 lysozyme has been found to undergo a low- as well as high-temperature unfolding transition in guanidinium chloride [see Chen and Schellman (1989)]. The kinetics for this process have been followed for several temperatures, a range of guanidinium chloride concentrations, and a number of values of pH. Microscopic rate constants for protein unfolding and refolding were extracted from these data to explore the nature of the cold unfolding transition. The data were interpreted using transition-state theory. It was found that the Arrhenius energy is temperature dependent. The transition state is characterized by (1) a high energy and low entropy compared to the native state, (2) a heat capacity which is closer to the native state than to the unfolded state, and (3) a low exposure to solvent compared to the unfolded state, as judged by its interaction with guanidinium chloride. With increasing concentration of guanidinium chloride, the low-temperature unfolding rate increases strongly, and the refolding rate decreases very strongly.

Escherichia coli↗
PubMed1984

X-ray diffraction studies on cation-collapsed DNA.

The polyamines spermidine, spermine and putrescine are now known to induce tertiary collapse of DNA. In this collapsed state DNA assumes a compact toroidal conformation. However, the structural details of DNA in these compact particles and the forces that stabilize the collapsed state are not clear. We show here that the structural arrangement of DNA in this tertiary conformation is determined by the chemical structure of the agent used to collapse. We have used aliphatic triamines (NH+3--(CH2)3--NH+2--(CH2)n--NH+3 with n = 3, 4, 5 and 8) and diamines (NH+3--(CH2)x--NH+3 with x = 2, 3, 4 and 6) to collapse DNA. We find that the Bragg spacing and the calculated interhelical spacing for a hexagonal packing model vary systematically with the length of the methylene bridge. We also find that the ionic strength of the solution has no effect on the Bragg spacing. This observation suggests that the arrangement of DNA strands in the complexes is determined by the structure of the polycation, and argues against suggestions that the structure of the collapsed state is maintained by the balance of long-range electrostatic repulsive and attractive forces. Instead we propose that DNA helices form a hexagonal array with counterions in the interstices between the helices resulting in a stable three-dimensional phase with high structural order. Arguments are presented favoring such a model in terms of stabilizing and destabilizing thermodynamic forces.

Animals↗
PubMed1979

Stability of phage T4 lysozymes. II. Unfolding with guanidinium chloride.

The denaturation by guanidinium chloride of three phage lysozymes (wild type and two mutants) was investigated. The study of solvent denaturation permitted the investigation of the relative stabilities of the proteins at neutral pH, in contrast to thermal denaturation studies reported earlier which could only be performed in acid pH. The results were interpreted assuming that the free energy of solution of proteins is a linear function of denaturant concentration. Using standard thermodynamic formulas this permits the calculation of the stabilities of the three proteins in the absence of guanidinium chloride. The single point mutation Trp 138 leads to Tyr leads to relatively large changes in stability and the interaction of the protein with guanidinium chloride. The changes associated with the subsequent double mutation, Trp 126 leads to Tyr, Trp 158 leads to Tyr, are much smaller indicating a relatively smooth adjustment of the protein structure to the changed side chains. Models of the structural effects of point mutations are discussed. It is found that the mutation at position 138 does not fit a model in which the effect of a substitution is to introduce an energetic strain in the structure. It does fit a model in which there is a partial unravelling of the structure as a result of the mutation. However, there are no changes in the backbone circular dichroism spectra associated with the mutation. The two observations are not necessarily in conflict. Further physical studies are required for the resolution of the problem.

Guanidines↗