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Biomedical subjects

G T DeTitta

Publications and source records attributed to G T DeTitta.

At least 19 recordsLinked to original sources
PubMed1999

A method to produce microseed stock for use in the crystallization of biological macromolecules.

A method is presented for producing a seed-stock mixture for macromolecular crystallization. A PTFE bead and micro-centrifuge tube act as mortar and pestle for pulverizing seed crystals of macromolecules. Energy for the bead's motion is supplied by a vortex mixer or an ultrasonic bath. The crushed crystal is serially diluted to prepare a seed-stock mixture of the desired concentration for crystallization. Crystals produced using both hanging-drop vapor diffusion and a capillary microbatch method show expected dilution behavior. This technique of producing seed stock is compared with traditional means and advantages over the standard protocol are demonstrated.

Biochemistry↗
PubMed1996

The rate of water equilibration in vapor-diffusion crystallizations: dependence on the distance from the droplet to the reservoir.

The rate of water equilibration in hanging-drop vapor-diffusion experiments was studied as a function of the distance separating the hanging drop from the surface of the reservoir solution. Hanging drops of 1.00 M NaCl were allowed to partially equilibrate with reservoirs of 2.00 M NaCl at room temperature. Over the range of droplet-reservoir distances examined, 7.6-119.4 mm, the larger the distance that separated the droplet and reservoir, the slower the droplet equilibrated with the reservoir. The variation of the rate of equilibration with droplet-reservoir distance was non-linear; the rate was most sensitive to variations in the droplet-reservoir separation at short separations. A mathematical model of the equilibration kinetics was developed that fits the experimental data. The model is based on the assumption that the rate-limiting step in vapor-diffusion equilibration is transit of water across the vapor space. A simple device to vary the rate of water equilibration, and thereby optimize macromolecular crystal growth conditions, is described.

Journal Article↗
PubMed1995

Non-ideality of aqueous solutions of polyethylene glycol: consequences for its use as a macromolecular crystallizing agent in vapor-diffusion experiments.

Microisopiestic measurements of the concentrations of polyethylene glycol (PEG 8000) paired with the salts sodium chloride, ammonium sulfate and magnesium sulfate heptahydrate have been made in a sitting-drop arrangement with PEG in the droplet and salt in the reservoir. Resulting graphs of the concentrations of PEG and salt that are equivalent with respect to the vapor pressure of water are non-linear, do not intersect their origins, and demonstrate that relatively low (mM) salt concentrations are equivalent to relatively high PEG concentrations. The consequences of each of these observations for macromolecular crystallization by the vapor-diffusion technique when PEG is employed as the crystallizing agent are discussed.

Journal Article↗
PubMed1995

Chaperone salts, polyethylene glycol and rates of equilibration in vapor-diffusion crystallization.

The kinetics of water-vapor equilibration in macromolecular crystallization were investigated for sitting droplets of aqueous polyethylene glycol (PEG) 8000 as a function of concentration. Equilibrations, set up with initial concentrations of PEG in the droplet at half those in the reservoir, were very slow for concentrations of relevance to the macromolecular crystal growth problem. At 301 K, 24 micro l droplets at initial concentrations of 2.5, 5.0 and 7.5%(w/v) PEG require 12, 5, and 3 weeks to reach equilibrium, respectively. On the other hand, the addition of modest quantities of sodium chloride to both droplet and reservoir increases the rate of equilibration for aqueous PEG sitting droplets significantly. At 293 K, droplets with initial volumes of 24 micro l and PEG concentrations of 5%(w/v) require 12 weeks to reach equilibrium, while droplets of the same volume and initial concentrations of 5%(w/v) PEG and 200 mM NaCI require less than two weeks to reach equilibrium. The slow vapor-diffusion equilibrations of pure PEG solutions, and the subsequent increase in these rates with colligative agents such as salt, are a consequence of the non-ideality of aqueous PEG solutions. These results are of interest both from a practical and a theoretical viewpoint. They underscore the importance of kinetic factors in macromolecular crystal growth, help to explain apparent inconsistencies of outcome in PEG-mediated crystallizations, and yield another methodology for the optimization of crystal growth conditions, namely the control of the kinetics of equilibration using colligative agents.

Journal Article↗
PubMed1995

Rate of water equilibration in vapor-diffusion crystallization: dependence on the residual pressure of air in the vapor space.

The kinetics of water equilibration in vapor-diffusion crystallization experiments are sensitive to the residual pressure of air in the vapor chamber. Experiments with sitting droplets of 10%(w/v) PEG, allowed to equilibrate with reservoirs of 20%(w/v) PEG, were conducted at pressures ranging from 80 to 760 mm Hg. Equilibrations were interrupted after one, four, five and seven days to assess their progress. Even down to the lowest pressures examined it was found that a decrease in pressure leads to an increase in the rate of equilibration. The residual pressure of air in the vapor chamber can be varied to tailor the time course of equilibration in macromolecular crystal growth experiments.

Journal Article↗
PubMed1994

Time courses of equilibration for ammonium sulfate, sodium chloride and magnesium sulfate heptahydrate in the Z/3 crystallization plate.

Time courses of equilibration for three salts, sodium chloride, ammonium sulfate and magnesium sulfate heptahydrate have been measured in the Z/3 crystallization plate. It is shown that by varying both the diffusant and the reservoir depth the time taken to equilibrate can be as short as 200 or as long as 1400 h. Thus, the present design of the plate should accommodate a wide variety of desired crystallization kinetics.

Journal Article↗
PubMed1994

Structure solution by minimal-function phase refinement and Fourier filtering. I. Theoretical basis.

Eliminating the N atomic position vectors rj, j = 1, 2, ..., N, from the system of equations defining the normalized structure factors EH yields a system of identities that the EH's must satisfy, provided that the set of EH's is sufficiently large. Clearly, for fixed N and specified space group, this system of identities depends only on the set [H], consisting of n reciprocal-lattice vectors H, and is independent of the crystal structure, which is assumed for simplicity to consist of N identical atoms per unit cell. However, for a fixed crystal structure, the magnitudes magnitude of /EH/ are uniquely determined so that a system of identities is obtained among the corresponding phases psi H alone, which depends on the presumed known magnitudes magnitude of /EH/ and which must of necessity be satisfied. The known conditional probability distributions of triplets and quartets, given the values of certain magnitudes magnitude of /E/, lead to a function R(psi) of phases, uniquely determined by magnitudes magnitude of /E/ and having the property that RT < 1/2 < RR, where RT is the value of R(psi) when the phases are equal to their true values, no matter what the choice of origin and enantiomorph, and RR is the value of R(psi) when the phases are chosen at random. The following conjecture is therefore plausible: the global minimum of R(psi), where the phases are constrained to satisfy all identities among them that are known to exist, is attained when the phases are equal to their true values and is thus equal to RT.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallography, X-Ray↗
PubMed1994

Structure solution by minimal-function phase refinement and Fourier filtering. II. Implementation and applications.

The minimal function, R(psi), has been used to provide the basis for a new computer-intensive direct-methods procedure that shows potential for providing fully automatic routine solutions for structures in the 200-400 atom range. This procedure, which has been called shake-and-bake, is an iterative process in which real-space filtering is alternated with phase refinement using a technique that reduces the value of R(psi). It has been successfully tested using experimental data for a dozen known structures ranging in size from 25 to 317 atoms and crystallizing in a variety of space groups. The details of this procedure, the parameters used and the results of these applications are described.

Computers↗
PubMed1993

On the application of the minimal principle to solve unknown structures.

The Shake-and-Bake method of structure determination is a new direct methods phasing algorithm based on a minimum-variance, phase invariant residual, which is referred to as the minimal principle. Previously, the algorithm had been applied only to known structures. This algorithm has now been applied to two previously unknown structures that contain 105 and 110 non-hydrogen atoms, respectively. This report focuses on (i) algorithmic and parametric optimizations of Shake-and-Bake and (ii) the determination of two previously unknown structures. Traditional tangent formula phasing techniques were unable to unravel these two new structures.

Algorithms↗
PubMed1993

Application of the minimal principle to peptide structures.

A new direct-methods procedure has been devised which consists of phase refinement via the minimal function, R(phi), alternated with Fourier summation and real space filtering. All phases are initially assigned values by computing structure factors for a randomly positioned set of atoms. These phases are then refined by using a parameter shift method to minimize R(phi). The refined phases are Fourier transformed, and a specified number of the largest peaks in the electron-density function are found and used as a new trial structure. The probability of a trial structure converging to a solution appears to depend on structural complexity and a number of refinement parameters. This procedure shows potential for providing fully automatic routine solutions for structures in the 200-400 atom range.

Journal Article↗
PubMed1980

Carboxybiotin translocation mechanisms suggested by diffraction studies of biotin and its vitamers.

Biotin is a coenzyme that fixes CO2 for transfer in a family of carboxylase, decarboxylase, and transcarboxylase enzymes. Their enzyme reactions involve two basic steps during which a carboxybiotinyl intermediate forms at one site and translocates to a second (distinct) site for CO2 transfer. Our diffraction studies of biotin and its vitamers suggest that translocation involves rotation about one, or at most two, bonds in biotin's valeryl chain. The rotations are energetically economical gauche in equilibrium trans rotations about the two valeryl bonds nearest the biotin bicyclic ring. They move a carbon atom of a CO2 moiety bound at N-1' approximately 7 A, a distance in accord with spectroscopic measurements of one of the biotin enzymes. From our studies we infer that sulfur in biotin imparts to the valeryl chain a conformational variability necessary for bond rotation and, hence, translocation between catalytic sites.

Biotin↗
PubMed1980

Molecular structure and intermolecular interactions of N1'-methoxycarbonylbiotin methyl ester: a model for carboxybiotin.

The crystal structure of N1'-methoxycarbonylbiotin methyl ester, a model for N1'-carboxybiotin, has been determined. The ureido carbonyl bond has more double bond (keto) character than does the corresponding bound in free biotin, which has single bond (enolate) character. In addition, there is an interesting intermolecular interaction between the ureido carbonyl oxygen and a methyl group. Comparison of the molecular structure and crystal packing with those of free biotin suggests that the coenzyme may have evolved with the incorporation of the ureido moiety because the electronic configuration of this region of the molecule is sensitive to N1' carboxylation. On decarboxylation, the ureido carbonyl bond becomes more polarized (C-O-), thereby facilitating the deprotonation of N1' and increasing its nucleophilicity. As a result, carboxylation can occur readily. On carboxylation, the carbonyl bond is depolarized (C = O), allowing the carboxylated coenzyme to interact with nonpolar groups and carboxylate them. Thus, the carboxylation and decarboxylation of biotin appear to act as a mechanistic switch, turning off and on the polarization of the ureido carbonyl bond as well as modulating the nucleophilicity of N1'.

Biotin↗
PubMed1979

Thromboxane molecules do not adopt the prostaglandin hairpin conformation.

The hairpin conformational hypothesis has been proposed to rationalise much of the structure-activity and receptor-binding data which have accumulated for the prostaglandin (PG) hormones. The hairpin conformation, thought to be necessary for PG activity, requires that the alpha- and omega-chains of the molecule be extended and in parallel alignment, separated by a van der Waals contact distance for the full length of the chains, with the ends of the chains approximately 5.5 A apart. The similarity between the structures of the thromboxanes (TXs) and the PGs suggests that the profile of activity of TXs, like that of PGs, centres on subtle conformational variation of the hairpin geometry. Thromboxane B2 (TXB2) is a stable hydrolysis product of a highly reactive, short-lived intermediate, thromboxane A2 (TXA2), which is formed from the prostaglandin endoperoxide (PGH2) as indicated in Fig. 1. An examination of molecular models of TXA2 and TXB2 suggests that the structural differences between the ring moieties may have much less influence in altering the side-chain conformation of TXs than do substitutents on the relatively more flexible cyclopentane ring of a PG molecule. We report here the first diffraction analysis of a thromboxane structure and note that the molecular conformation is not hairpin shaped.

Molecular Conformation↗
PubMed1977

Conformations of prostaglandin F 2alpha and recognition of prostaglandins by their receptors.

The conformation of prostaglandin F 2alpha (PGF 2alpha) has been determined by x-ray diffraction techniques. Two independent conformers of PGF 2alpha, studied as the tris(hydroxymethyl)methylamine salt, are observed to adopt the familiar "hairpin" conformation with the alpha and omega chains aligned roughly parallel. The conformers differ in ring conformation and at the C(17)-C(18) bond, one adopting a C(9) envelope ring conformation and a trans geometry at the C(17)-C(18) bond, while the other adopts a C(8) envelope ring conformation and a novel gauche geometry about C(17)-C(18). Comparison of the conformation of PGF 2alpha with that of prostaglandin E2 suggests a recognition mechanism which would permit PGF 2alpha and prostaglandin E receptors to distinguish between the two potent prostaglandins. The recognition model explains much of the binding data for the PGF 2alpha receptor in the corpus luteum and predicts the existence of an interesting PGF 2alpha analog.

Molecular Conformation↗