José Fernando Díaz

Low-Resolution Structures of Proteins in Solution Retrieved from X-Ray Scattering with a Genetic Algorithm

Small-angle x-ray solution scattering (SAXS) is analyzed with a new method to retrieve convergent model structures that fit the scattering profiles. An arbitrary hexagonal packing of several hundred beads containing the problem object is defined. Instead of attempting to compute the Debye formula for all of the possible mass distributions, a genetic algorithm is employed that efficiently searches the configurational space and evolves best-fit bead models. Models from different runs of the algorithm have similar or identical structures. The modeling resolution is increased by reducing the bead radius together with the search space in successive cycles of refinement. The method has been tested with protein SAXS (0.001 < S < 0.06 A(-1)) calculated from x-ray crystal structures, adding noise to the profiles. The models obtained closely approach the volumes and radii of gyration of the known structures, and faithfully reproduce the dimensions and shape of each of them. This includes finding the active site cavity of lysozyme, the bilobed structure of gamma-crystallin, two domains connected by a stalk in betab2-crystallin, and the horseshoe shape of pancreatic ribonuclease inhibitor. The low-resolution solution structure of lysozyme has been directly modeled from its experimental SAXS profile (0.003 < S < 0.03 A(-1)). The model describes lysozyme size and shape to the resolution of the measurement. The method may be applied to other proteins, to the analysis of domain movements, to the comparison of solution and crystal structures, as well as to large macromolecular assemblies.

The susceptibility of pure tubulin to high magnetic fields: a magnetic birefringence and x-ray fiber diffraction study

The orientational behavior of microtubules assembled in strong magnetic fields has been studied. It is shown that when microtubules are assembled in a magnetic field, they align with their long axis parallel to the magnetic field. The effect of several parameters known to affect the microtubule assembly are investigated with respect to their effect on the final degree of alignment. Aligned samples of hydrated microtubules suitable for low-resolution x-ray fiber diffraction experiments have been produced, and the results obtained from the fiber diffraction experiments have been compared with the magnetic birefringence experiments. Comparisons with earlier fiber diffraction work and small-angle x-ray solution scattering experiments have been made.

Calculation of pathways for the conformational transition between the GTP- and GDP-bound states of the Ha-ras-p21 protein: Calculations with explicit solvent simulations and comparison with calculations in vacuum

The transitions between the water‐equilibrated structures of the GTP and GDP forms of Ha‐ras‐p21 have been calculated by using the targeted molecular dynamics (TMD) method (Schlitter et al., Mol. Sim. 10:291–309, 1993) both in vacuo and with explicit solvent simulation. These constrained molecular dynamics calculations result in different pathways, depending on the nucleotide bound. Each pathway consists in a sequence of transitions affecting six segments of the protein, four of them forming a hydrophilic cleft around the nucleotide. The transitions are initiated by the removal or introduction of the γ‐phosphate of the nucleotide and proceed sequentially, crossing several low‐energy transition states. The movements are transmitted either by direct interactions between the segments or through the nucleotide. The GTP to GDP pathway is initiated by the removal of the nucleotide γ‐phosphate. This gives some space to Gly12, Gly13, and Val14. Their movement is transmitted to the target recognition domain and the switch II region, forcing these segments to adopt another position. In a second step the target recognition domain and the switch II region undergo conformational transitions to reach an intermediate conformation. Finally, there is a relaxation of the target recognition domain to its final state that forces the switch II region to reach its target conformation. The calculated pathways allow the identification of many residues that play an important role in the conformational changes, explain the altered transformation properties of some, and suggest mutations to alter the pathway. Proteins 28:434–451, 1997.

Molecular mechanisms of pressure induced conformational changes in BPTI

We have performed a 800 ps molecular dynamics simulation of bovine pancreatic trypsin inhibitor (BPTI) in water coupled to a pressure bath at 1, 10,000, 15,000, and 20,000 bar. The simulation reproduces quite well the experimental behavior of the protein under high pressure. The protein keeps its globular form, but adopts a different conformation with a very small reduction in volume. Some residues in the hydrophobic core become exposed to water and a large part of the secondary structure of the protein, (60% of the sheet structure and 40% of the helical structure) is denatured between 10 and 15 kbar. This is in good agreement with experimental data (Goossens, K., et al. Eur. J. Biochem, 236:254–262, 1996) that show denaturation of BPTI between 8 and 14 kbar. A further Increase of the pressure results in a freezing of the protein as deduced from the large decrease of the mobility of the residues. During the simulation, the normal structure of water changes from an ice Ih‐like to an ice VI‐like structure, while keeping the liquid state. The driving force of the high pressure induced conformational transition seems be the higher compressibility of the water compared with the protein. This produces a change in the solvent properties and leads to penetration of the solvent into the hydrophobic core.

Equilibrium and kinetic study of the conformational transition toward the active state of p21Ha-ras, induced by the binding of BeF3- to the GDP-bound state, in the absence of GTPase-activating proteins.

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595–15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115–117). In this work, we have used a fluorescent active mutant (Y32W) of p21Ha- ras to demonstrate that BeF3 − binds to the GDP·p21Ha-ras complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (K d at 25u2009°C = 8.1 ± 0.3 mm) and is endothermic (ΔH = 22.3 ± 1.6 kJ mol−1). The similarity between the GTP-bound form and the GDP·BeF3 −-bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP·BeF3 −·p21Ha- ras complex to its final state. Both steps are endothermic (ΔH 1 = 15 ± 2 kJ mol−1 and ΔH 2 = 8 ± 2 kJ mol−1). The kinetically determined enthalpy change of 23 ± 4 kJ mol−1 is in excellent agreement with the equilibrium analysis.

Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase)

Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme.

Fast mixing device for time-resolved synchrotron x-ray scattering studies of radiation sensitive proteins

A fast mixing device apparatus has been constructed to study the kinetic structural reactions of radiation sensitive proteins by time-resolved small angle x-ray scattering. The sample cell has a volume of 0.75 ml and is translated through the synchrotron x-ray beam as the experiment progresses. The dead time of the device is 50 ms, and it is able to mix viscous liquids with ratios ranging from 1:1 to 1:250 with a measured precision of 0.1 μl. The device uses two motor driven syringes that are individually controlled. The whole instrument is thermostated and is remotely controlled. Cleaning of the cell is carried out remotely, removing the need to enter the x-ray hutch between each measurement. Commissioning of the apparatus was carried out by following the assembly of the radiation sensitive protein tubulin into microtubules induced by two different chemical jumps.

Mechanism of pressure denaturation of BPTI.

Abstract We have performed a 800 ps molecular dynamics (MD) simulation of bovine pancreatic trypsin inhibitor (BPTI) in water coupled to a pressure bath at 1, 10000, 15000 and 20000 bar, which reproduces quite well the experimental behaviour [1]. The protein keeps its globular form, but adopts different conformations with only small reductions in volume. Some residues in the hydrophobic core become exposed and parts of the secondary structure is denatured between 10 and 15 kbar.