De Novo Design And Engineering Of Functional Metal And Porphyrin-Binding Protein Domains

In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two \u27next-generation\u27 sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II

The proteolytic enzymes of proteus vulgaris.

Thesis (Ph.D.)--Boston UniversityTwo variations of the formol titration were developed to determine proteolytic activity. The first used phenolphthalein as an end-point indicator while the second was a potentiometric titration with an end point of pH 7.8. The standard error of the mean for the two methods were -+ 0.3 and -+ 0.13 microeqs. respectively. The substrate used was a 3% gelatin solution with a tris (hydroxymethyl) aminomethane buffer at pH 7.4, the optimum pH of enzyme activity. [TRUNCATED] The production and purification of the proteolytic enzymes of Proteus vulgaris are discussed. Kaolin was used as an adsorbent in the purification. Subsequent elution yielded a preparation containing two proteolytic enzymes and an inactive traction. The active traction consists of a relatively heat-stable enzyme and a heat-labile enzyme. The kinetics of these two enzymes was, studied to determine the Michaelis-Menten constants, effects of substrate, effect of end products, the molar energy of activation, and the inactivation by heat. A unit of activity based on reaction rates is proposed

Thermodynamics of an ideal generalized gas: I. Thermodynamic Laws

The equations of state for an ideal relativistic, or generalized, gas, like an ideal quantum gas, are expressed in terms of power laws of the temperature. In contrast to an ideal classical gas, the internal energy is a function of volume at constant temperature, implying that the ideal generalized gas will show either attractive or repulsive interactions. This is a necessary condition in order that the third law be obeyed and for matter to have an electromagnetic origin. The transition from an ideal generalized to a classical gas occurs when the two independent solutions of the subsidiary equation to Lagrange's equation coalesce. The equation of state relating the pressure to the internal energy encompasses the full range of cosmological scenarios, from the radiation to the matter dominated universes and finally to the vacuum energy, enabling the coefficient of proportionality, analogous to the Grüeisen ratio, to be interpreted in terms of the degrees of freedom related to the temperature exponents of the internal energy and the absolute temperature expressed in terms of a power of the empirical temperature. The limit where these exponents merge is shown to be the ideal classical gas limit. A corollary to Carnot's theorem is proved, asserting that the ratio of the work done over a cycle to the heat absorbed to increase the temperature at constant volume is the same for all bodies at the same volume. As power means, the energy and entropy are incomparable, and a new adiabatic potential is introduced by showing that the volume raised to a characteristic exponent is also the integrating factor for the quantity of heat so that the second law can be based on the property that power means are monotonically increasing functions of their order. The vanishing of the chemical potential in extensive systems implies that energy cannot be transported without matter and is equivalent to the condition that Clapeyron's equation be satisfied

B_s Mesons using Staggered Light Quarks

Last year we proposed using staggered fermions as the light quarks, combined with nonrelativistic heavy quarks, in simulations of heavy-light mesons. A first round of tests which focuses on the B_s meson has been completed using quenched lattices, and results are presented here for the kinetic B_s mass, the B_s^* - B_s splitting, and f_{B_s}. The next project, already underway, is to compute the B and B_s decay constants and spectra on the n_f = 2+1 and 3 MILC lattices. We report on progress with one set of these configurations.Comment: Talk presented by M.W. at Lattice2002(heavyquark